Note: Descriptions are shown in the official language in which they were submitted.
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
TITLE OF THE INVENTION
METHODS OF IDENTIFYING GABAB RECEPTOR SUBTYPE-SPECIFIC
AGONISTS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
60/ 285,969, filed April 24, 2001, which is a Continuation-In-Part of U.S.
Provisional
Application No. 60/212,426, filed June 19, 2000, which is a Continuation-In-
Part of
U.S. Provisional Application No. 60/212,152, filed June 16, 2000, the contents
of
which are incorporated herein by reference in their entirety.
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
The present invention provides methods for identifying substances that
are agonists of GABAB receptors that are heteromers of gbla and gb2 subunits
where
the substances are not agonists of GABAB receptors that are heteromers of gblb
and
gb2 subunits or gblc and gb2 subunits. Nor are the substances agonists or
GABAg
receptors comprising other alternative gbl isoforms with gb2 subunits.
BACKGROUND OF THE INVENTION
GABA (~y-amino-butyric acid) is the most widely distributed amino
acid inhibitory neurotransmitter in the vertebrate central nervous system. The
principal physiological role of GABA in the neural axis is synaptic
inhibition.
The biological activities of GABA are mediated by three types of
GABA receptors: ionotropic GABAA receptors, metabotropic GABAB receptors, and
ionotropic GABAC receptors. GABAA receptors convey fast synaptic inhibition by
activating a postsynaptic chloride conductance that is allosterically
modulated by
benzodiazepines, barbituates, and neurosteroids (Mody et al., 1994, Trends
Neurosci.
17:517-525). GABAB receptors mediate the slower, longer lasting synaptic
inhibitory actions implicated in hippocampal long term potentiation, slow-wave
sleep,
-1-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
J. Pharmacol. Exp. Ther. 292:2-7 and references therein). GABAB receptors play
a
role in the mediation of late inhibitory postsynaptic potentials (IPSPs) by
mediating
slow synaptic inhibition by GABA via G-proteins. This is thought to result
from the
activation of K+ channels (Mody et al., 1994, Trends Neurosci. 17:517-525).
Presynaptic GABAB receptor activation has generally been reported to result in
the
inhibition of Ca2+ conductance, leading to a decrease in the evoked release of
neurotransmitters (Andrade et al., 1986, Science 234:1261-1265; Takahashi et
al.,
1998, J. Neurosci. 18:3138-3146). Many of the physiological roles of GABAB
receptors can be attributed to the modulation of P/Q (cxlA, a2s, (31
subunits), and N-
type (alB, a28, X31 subunits) voltage-dependent calcium channels (VD-CCs) by
presynaptic receptors and modulation of inwardly rectifying K+ channels
(GIRKs) by
postsynaptic GABAB receptors (Bowery & Enna, 2000, J. Pharmacol. Exp. Ther.
292:2-7 and references therein). It has been suggested that pharmacologically
distinct
receptor subtypes mediate pre- and postsynaptic actions (Wojcik & Neff, 1984,
Mol.
Pharmacol. 25:24-28; Bonanno et al., 1997, Br. J. Pharmacol. 120:60-64:
Bonanno
~ Raiteri, 1993, J. Pharmacol. Exp. Ther. 265:765-768; for a review see Kerr &
Ong,
1995), but this has not been revealed by molecular studies to date.
GABAB receptor regulation of VD-CC function is thought to be
mediated by G-protein (3~y subunits via a membrane delimited mechanism
(Herlitze et
al., 1996, Nature 380:258-262; Ikeda et al., 1996, Nature 380:255-258)
resulting in
the inhibition of membrane Ca2+ conductance and a decrease in neurotransmitter
release (Doze et al., 1995, J. Neurophysiol. 74:43-53; Wu & Saggau, 1997,
Trends
Neurosci. 20:204-212). Activation of presynaptic GABAB receptors negatively
coupled to VD-CCs is likely the mechanism underlying the anti-nociceptive
effects of
GABA and the prototypic nonselective GABAg receptor agonist baclofen which
have
been reported to inhibit the release of pain transmitters such as calcitonin
gene-related
peptide and substance P in spinal cord slices (Malcangio & Bowery, 1993, J.
Pharmacol. Exp. Ther. 266:1490-1496). Baclofen has also been reported to be
efficacious when given intrathecally for the treatment of central pain
following stroke
or spinal cord injury (Loubser & Akman, 1996, Pain Sympt. Mgmt. 12:241-247).
However, its wider clinical use has been limited because doses (p.o.) needed
for
efficacy are associated with flaccidity and hypotonia.
GABAg receptors belong to the superfamily of seven transmembrane-
spanning G-protein coupled receptors that are coupled to neuronal K+ or Ca2+
channels. GABAB receptor activation increases K+ or decreases Ca2+ conductance
-2-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
and also inhibits or potentiates stimulated adenylyl cyclase activity. The
expression
of GABAB receptors is widely distributed in the mammalian neural axis (e.g.,
frontal
cortex, hippocampus, cerebellum, thalamus, spinal cord, dorsal root ganglia
and has
been observed in many peripheral organs as well (Belley et al., 1999, Biorg.
Med.
Chem.7:2697-2704).
A large number of pharmacological activities have been attributed to
GABAB receptor activation, e.g., analgesia; hypothermia; catatonia;
hypotension;
reduction of memory consolidation and retention; and stimulation of insulin,
growth
hormone, and glucagon release (see Bowery, 1989, Trends Pharmacol. Sci. 10:401-
407 for a review). It is well accepted that GABAB receptor agonists and
antagonists
are pharmacologically useful in indications such as stiff man syndrome,
gastroesophogeal reflux, neuropathic pain, incontinence and treatment of cough
and
cocaine addiction. For example, the GABAB receptor agonist (-)baclofen, as
part of a
racemic mixture with (+)baclofen, a structural analog of GABA, is a clinically
effective muscle relaxant (Bowery & Pratt, 1992, Arzneim.-Forsch./Drug Res.
42:215-223). -(~)baclofen, has been sold in the United States as a muscle
relaxant
under the name LIORESAL~ since 1972.
Functional GABAB receptors are formed following the co-expression
of two protein subunits having characteristics similar to those of the
metabotropic
glutamate receptors, viz., a signal peptide sequence followed by a large N-
terminal
domain believed to represent a ligand binding pocket that shares structural
similarity
to bacterial perisplasmic leucine, isoleucine, valine binding LIV-BP proteins.
This
putative extracellular ligand binding domain precedes seven transmembrane
spanning
domains. The hallmark seven transmembrane spanning domains are typical of G-
protein coupled receptors (GPCRs), although metabotropic glutamate receptors
and
GABAB receptor proteins are considerably larger than most GPCRs. Recombinant
expression of the two GABAB receptor gbl and gb2 subunit proteins, either
together
or separately, demonstrated that functional GABAB receptors were formed only
when
both proteins were expressed in the same cell, most likely as heterodimers
(Jones et
al., 1998, Nature 396:674-679; White et al., 1998, Nature 396:679-682;
Kaupmann et
al., 1998, Nature 396:683-687; Kuner et al., 1999, Science 283:74-77; Ng et
al., 1999,
J. Biol. Chem. 274:7607-7610; and International Patent Publication WO
99/40114).
The GABAB receptor heterodimer is composed of a subunit known as
GABABRla (or a splice variant known as GABABRlb) (Kaupmann et al., 1997,
Nature 386:239-246) together with a subunit known variously as GABABR2 (White
-3-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
et al., 1998, Nature 396:679-682; Jones et al., 1998, Nature 396:674-679),
GBR2
(Kuner et al., 1999, Science 283:74-77), gb2 (Ng et al., 1999, J. Biol. Chem.
274;7607-7610; or HG20 (International Patent Publication WO 99140114). A third
splice variant of GABABRla has been reported by Ng et al., 2001, Mol Pharm.
59:144-152. This third variant is known as gblc, in keeping with the
terminology
used by Ng et al. in which GABABRla is referred to as gbla and GABABRlb is
referred to as gblb. The GABAB receptor heterodimer is generally accepted to
be the
functional GABAB receptor. However, it remains possible that GABAB receptor
monomers or homodimers are functional when in certain cellular environments
(Kuner et al., 1999, Science 283:74-77; Kaupmann et al., 1997, Nature 386:239-
246;
Kaupmann et al., 1998, Nature 396:683-687).
Gabapentin (NEURONTIN~, 1-(aminomethyl)cyclohexaneacetic
acid) was developed as a brain penetrant structural analog of GABA to treat
spasticity
and to reduce polysynaptic spinal reflexes (reviewed by Bryans & Wustrow,
1999,
Med. Res. Rev. 19:149-177 and references therein). Gabapentin is an
anticonvulsant
used for the treatment of refractory partial seizures and secondary
generalized tonic-
clonic seizures. It has been proposed to have mood-stabilizing properties and
may be
useful in certain neuropathies such as diabetic neuropathy or post-herpetic
neuralgia.
Gabapentin monotherapy appears to be efficacious for the treatment of pain and
sleep
interference associated with diabetic peripheral neuropathy and exhibits
positive
effects on mood and quality of life (Rowbotham et al., 1998, J. Am. Med. Assn.
280:1837-1842). Gabapentin is also effective in the treatment of post-herpetic
neuralgia (PHN), a syndrome of often intractable neuropathic pain following
herpes
zoster (shingles) that eludes effective treatment in many patients. Mood and
quality
of life of PHN patients also improve with gabapentin therapy (Rowbotham et
al.,
1998, J. Am. Med. Assn. 280:1831-1836).
Gabapentin has been shown to be effective in reducing the number of
partial seizures in patients with drug-resistant partial epilepsy (U.K.
Gabapentin Study
Group, 1990, Lancet, 335:1114-1117). Gabapentin has been studied for use in
neurodegenerative disorders such arnyotrophic lateral sclerosis, cocaine
addiction and
in various psychiatric disorders such as bipolar disorder and may be of use as
an
anxiolytic (reviewed by Bryans & Wustrow, 1999, Med. Res. Rev. 19:149-177 and
references therein). It has been shown to have antihyperalgesic action in an
inflammatory pain model (Field et al., 1997, Br. J. Pharmacol. 121:1519-1522).
-4-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
Gabapentin has been reported to inhibit K+-evoked Ca2+ rises in
neocortical synaptosomes via inhibition of VD-CCs (Fink et al., 2000, Br. J.
Pharmacol. 130:900-906; Stefani et al., 1998, Neuropharmacology 37:83-91) and
reduce K+-evoked glutamate release from neocortical and hippoccampal slices
(Dooley et al., 2000, Neurosci. Letts. 280:107-110). Gabapentin has also been
reported to inhibit excitatory neurotransmitter release in the spinal cord
dorsal horn
(Shimoyama et al., 2000, Pain 85:405-414; Patel et al., 2000, Br. J.
Pharmacol.
130:1731-1734). Gabapentin has recently been reported to be a selective
agonist at
the recombinant gb 1 a-gb2 heterodimer and neuronal GABAB receptor coupled to
GIRKs with no partial agonist or antagonist activity at gblb-gb2 or gblc-gb2
subtypes (Ng et al., 2001, Mol Pharm. 59:144-152).
Gabapentin's mechanism of action has been the object of much study,
but no consensus has arisen. Various hypotheses have been proposed. For
example,
Taylor et al., 1998, Epilepsy Res. 29:233-249 list the following
possibilities: (1)
gabapentin crosses several membrane barriers in the body via a specific amino
acid
transporter (system L) and competes with leucine, isoleucine, valine, and
phenylalanine for transport; (2) gabapentin increases the concentration and
probably
the rate of synthesis of GABA in the brain; (3) gabapentin binds with high
affinity to
a binding site in brain tissues that is associated with an auxiliary subunit
of voltage-
sensitive calcium channels; (4) gabapentin reduces the release of several
monoamine
neurotransmitters; (5) gabapentin inhibits voltage-activated sodium channels;
(6)
gabapentin increases serotonin concentrations in human whole blood, which may
be
relevant to neurobehavioral actions; and (7) gabapentin prevents neuronal
death. See
also Taylor, 1997, Rev. Neurol. (Paris) 153 (Supply 1:539-S45 and Brown & Gee,
1998, J. Biol. Chem. 273:25458-25465 for other references discussing possible
mechanisms of action for gabapentin.
Gabapentin has been reported to bind with nanomolar affinity to the
auxiliary aas subunit of voltage dependent-calcium channels (VD-CCs) (Gee et
al.,
1996, J. Biol. Chem. 271:5768-5776). However no direct functional correlation
to
this binding has been reported to date, and it is unknown whether this
accounts for the
anti-convulsant, anti-hyperalgesic and anti-nociceptive actions of gabapentin
(Taylor
et al., 1998, Epilepsy Res. 29:233-249). Gabapentin has been reported to have
no
effect on VD-CCs in cultured rodent neurons (Rock et al., 1993, Epilepsy Res.
16:89-
98) and in acutely dissociated human dentate gyros granule cells from patients
with
temporal lobe epilepsy (Schumacher et al., 1998, Epilepsia 39:355-363). Yet,
-5-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
gabapentin has been reported to inhibit predominantly L-type calcium currents
in
isolated rat neocortical, striatal, and pallidal neurons (Stefani et al.,
1998,
Neuropharmacology 37:83-91). More recently, gabapentin has been found to
inhibit
K+-evoked glutamate release from rat neocortical and hippocampal slices (Fink
et al.,
2000, Br. J. Pharmacol. 130:900-906; Dooley et al., 2000, Neurosci. Letts.
280:107
110). However, the mechanisms underlying these gabapentin actions were not
elucidated.
It is noteworthy that gabapentin is believed not to act through GABAB
receptors. See The Compendium of Pharmaceuticals. 'and Specialties, Thirty-
third
edition, 1988, pp. 1101-1102, Canadian Pharmacists Association, Ottawa, ON,
CA,
where it is stated that gabapentin "does not interact with GABA receptors."
See also
Rowbotharn. et al., 1998, J. Am. Med. Assn. 280:1837-1842, at page 1838: "Its
[i.e.,
gabapentin's] mechanism of action has not yet been fully elucidated, but
appears not,.
to involve binding to GABA receptors [citing Goa & Sorkin, 1993, Drugs 46:409-
427]." Field et al., 1997, Br. J. Pharmacol. 121:1513-1522 state: "Although
gabapentin was originally designed as a GABA analogue which would penetrate
into
the CNS, it does not interact with either GABAA or GABAB receptors ..."
SUMMARY OF THE INVENTION
The present invention is directed to methods for identifying substances
that are agonists of GABAB receptors that are heteromers of gbla and gb2
subunits
where the substances are not agonists of GABAB receptors that are heteromers
of
gblb and gb2 subunits, gblc and gb2 subunits, or other gb1-gb2 heteromer
subytpes.
The substances inhibit presynaptic calcium currents, activate post-synaptic
potassium
currents, and inhibit somatic calcium currents. The substances are agonists of
GABAB receptors that are coupled to inwardly rectifying K+ channels in xenopus
oocytes or to GABAg receptors negatively coupled to voltage dependent-calcium
channels in heterologous expression systems such as HEK-293 cells and
melanotroph
cell lines derived from mouse intermediary lobe pituitary tumors. The
substances are
also agonists of GABAB receptors that are negatively coupled to voltage
dependent-
calcium channels in rat hippocampal neurons and spinal cord neurons. The
substances are not agonists of GABAA receptors and exhibit more selectivity
for
effector pathways and a distinct mechanism of activation (e.g., rapid
desensitization at
the GABAB receptor) as compared to baclofen.
-6-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
The combination of characteristics outlined above is possessed by
gabapentin and indicates that the substances identified by the methods of the
present
invention represent a class of substances that, like gabapentin, are expected
to be
useful in the treatment of such conditions as psychiatric disorders, e.g.,
bipolar
disorders, social phobias, and anxiety; epilepsy and other convulsant
disorders;
gastroesophogeal reflux; cocaine addiction; neurodegenerative disorders such
as
amyotrohic lateral sclerosis; and multiple chronic pain states such as
diabetic
neuropathy or post-herpetic neuralgia.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A shows an amino acid alignment of the extracellular N-
terminal domains of the human gbla, gblb, gblc isoforms. The proposed signal
peptide cleavage site of gbl is marked with scissors. Putative N-glycosylation
sites
(1) are indicated and arrows (~) delimit the Sushi domains (SU). An arrow (->)
marks the start of the LIV-BP-like domain. Gaps (...) were introduced to
maximize
alignment. The gbla sequence shown is a portion of SEQ.ll~.N0.:2. The gblc
sequence shown is a portion of SEQ.ID.NO.:B. The gblb sequence shown is a
portion of SEQ.ID.N0.:6. Figure 1B shows the structure of the GABAB heteromer
pan agonists GABA and baclofen and the gbla subtype-specific agonist
gabapentin.
Figure 2A-F shows modulation of Kir 3.1/3.2 in Xenopus oocytes by
different gbl/gb2 heteromers. Currents were measured by holding oocytes at -80
mV.
The dark bar in each trace denotes changing from perfusion of oocytes with KD-
98
solutions to solution containing 100 ~.M GABA. The light bar denotes the
beginning
of perfusion with 100 ~tM gabapentin. Co-expression of human gb2 with mouse
gbla
(Figure 2A), human gbla (Figure 2B), gblb (Figure 2C) or gblc (Figure 2D).
Control traces: M2 muscarinic receptor (Figure 2E) or (32AR (Figure 2F) co-
expressed with Kir 3.2. In the case of the (32 adrenergic receptor it was
necessary to
co-express the bovine Gsa subunit as well. Arrows denote different patterns of
desensitization for GABA- or gabapentin-mediated receptor activation. Each
trace is
representative of at least four separate experiments performed on different
oocytes.
Figure 3A-B shows that gabapentin modulates Kir3.1/3.2 only via
gbla receptor heteromers. Figure 3A shows the fold stimulation of Kir 3.1/3.2
current by gabapentin or GABA over basal current (set to 1.0). Fold
stimulation was
calculated by dividing maximal ligand-stimulated current by basal current
measured
in KD-98 solution. Note that the effects of GABA (at an identical
concentration) are
-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
always significantly larger than for gabapentin. Figure 3B shows the dose-
response
relation for GABAB ligands at gbla/gb2. Co-expression of murine gbla and human
gb2 results in the modulation of Kir 3.113.2 in a dose-dependent manner.
Currents
were measured at various doses of GABA, gabapentin or baclofen and normalized
relative to the basal current. Averaged data from many experiments (minimum of
n=4 for each point on the three dose-response curves) was pooled and fit to
the Hill
equation using GraphPad Prism v2Ø As positive controls, the response of Kir
3.2
(expressed without Kir 3.1) to M2 muscarinic receptors (1 ~.M carbachol, n=6)
or to
(32-adrenergic receptors (co-expressed with Gsa, 1 ~t.M isoproterenol, n=17)
was
measured.
Figure 4 shows that gabapentin activates potassium currents via
GABAB receptors in CA1 pyramidal cells ire situ. (A) Micrograph .of biocytin
filled
CA1 pyramidal neurons exposed to gabapentin. Calibration mark, 100 hum. (B) I-
V
relations were obtained during voltage ramps from -60 to -160 mV in control
ACSF
and in the presence of 1 mM gabapentin (Gp, Bl). Gabapentin currents (B2) were
isolated by subtracting currents from I-V relations in control ACSF from those
in the
presence of gabapentin. The current evoked by 1mM gabapentin in B2 was
obtained
from the traces shown in B 1. Gabapentin evoked outward currents at membrane
potentials between -60 and -100 mV. In the same cell, gabapentin currents
increased
in magnitude with increasing doses (0.1-1 mM). (C) Bath application of 2-20
~,M
baclofen.elicited in CA1 pyramidal cells similar potassium currents with a
comparable reversal potential. (D) The mean chord conductance (measured at -80
mV) of baclofen and gabapentin currents increased in dose-dependent fashion
(the
number above each bar indicates the number of cells tested). The dose-response
relationship for gabapentin was shifted approximately ten-fold higher relative
to that
of baclofen. The mean chord conductance of gabapentin (l mM) and baclofen (20
p,M) potassium currents were blockedyby the GABAB antagonist CGP55845 (at 4
and
1 ~M concentration, respectively).
Figure 5 shows presynaptic GABAB inhibition of GABA synaptic
transmission in hippocampus: inefficacy of gabapentin and efficacy of
baclofen. (A)
Experimental arrangement for evoking monosynaptic fast GABAA IPSCs by
electrical stimulation of inhibitory fibers in stratum radiatum in the
presence of
Mockers of glutamate synaptic transmission (20 ~M CNQX and 50 ~uM AP5) during
whole cell voltage clamp recording from pyramidal cells. (B) At resting
membrane
potential, stimulation evoked fast outward IPSCs in control ACSF (with CNQX
and
_g_
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
AP5). IPSCs were depressed during bath application of 20 [uM baclofen. (C) In
contrast, during bath application of 1 mM gabapentin, IPSCs were minimally
affected
relative to control. (D) The mean reduction of IPSCs by baclofen was dose-
dependent (2-20 ~uM) and the presynaptic actions of 20 p.M baclofen were
completely
antagonized by 1 p,M of the GABAB antagonist CGP55845. Gabapentin, at
concentrations between 0.1-1 mM, did not produce such reductions in IPSC
amplitude. The numbers above the bars indicate the number of cells tested in
each
condition. The small mean reduction in the presence of gabapentin was not
significatly different from that seen with repeated application of control
ACSF (with
CNQX and APS; diagonal bars) or during application of 4 ~u,M CGP55845.
Figure 6A-B shows the cDNA sequence (SEQ.~.N0.:1) and Figure
6C shows the amino acid sequence (SEQ.ID.N0.:2) of human gbla ~(GenBank
accession no. AJ225028).
Figure 7A-B shows the cDNA sequence (SEQ.ID.NO.:3) and Figure
7C shows the amino acid sequence (SEQ.)~.N0.:4) of murine gbla (GenBank
accession no. AF114168).
Figure 8A shows the cDNA sequence (SEQ.ID.N0.:5) and Figure 8B
shows the amino acid sequence (SEQ.ID.N0.:6) of human gblb (GenBank accession
no. AJ225029).
Figure 9A shows the cDNA sequence (SEQ.ID.N0.:7) and Figure 9B
shows the amino acid sequence (SEQ.ID.N0.:8) of human gblc (GenBank accession
no. AJ012187).
Figure l0A-B shows the cDNA sequence (SEQ.ID.N0.:9) and Figure
lOC shows the amino acid sequence (SEQ.ID.NO.:10) of human gb2 (GenBank
accession no. AF058795).
Figure 11 shows the pharmacological actions of GABAB ligands on
high K+-evoked activation of VD-CCs in mlL cells endogenously expressing gbla
heteromers. Panel A shows there is a sharp increase in [Ca2+] followed by a
slower
second peak or shoulder in responses to depolarization by high extracellular
K+
concentrations. Panel B shows that baclofen (1 p,M) reduces the primary
response.
Panel C shows that baclofen effects (1 ~,M) are completely reversed by
addition of 3
p,M CGP55845. CPG55845 (3 p,M) has no significant effect on the response of
mIL
cells to depolarization (Panel D). Panel E shows that 1 p.M gabapentin reduces
the
primary Ca2+responses of cells similar to 1 ~M baclofen (top middle panel) and
that
-9-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
this reduction is also blocked by 1 ~M CGP55845 (Panel F). In each panel, the
individual Ca2+ responses of 6-8 cells are shown.
Figure 12 shows a dose response study of the ability of gabapentin to
inhibit influx of Ca2+ after high K+ depolarization. mIL cells were treated 1-
5 min
with the indicated doses of gabapentin, then depolarized with high K+ as
described in
Example 6. Bars represent the standard error for the number of cells indicated
in
brackets. The EC50 was calculated after fitting the data using GraphPad.
Figure 13 shows that inhibition of K+-evoked calcium mobilization by
1 ~M gabapentin is blocked in a dose-dependent manner with 30 nM - 3 ~.~.M
CGP55845. CGP55845 and gabapentin are abbreviated CGP and GBP in the graphs
respectively. Each error bar represents 1 SEM. Number of cells analyzed is
noted in
brackets.
Figure 14 shows that antisense knockdown of the endogenous GABAB ,
gbla subunit in mIL cells result in the block of gabapentin-induced inhibition
of the
primary increase in [Ca2+]i following K+ depolarization and activation of VD-
CCs.
There was no significant difference among any of the control values and the
values
for the various ADN treatments when compared by analysis of variance;
therefore the
results are presented as normalized to the value for high K+ depolarization
(Column
A) set to 10010. Both 10 p.M and 30 ~M gabapentin inhibited the depolarization-
induced rise in intracellular [Ca2+] (compare A to B and C). None of the
deoxynucleotide treatments in themselves affected the depolarization-induced
rise in
intracellular [Ca2+] (compare A, D, G and J). gbla ADN treatment completely
blocked the ability of gabapentin to inhibit the rise in [Ca2+]i (compare B
and C to
D, E and F), whereas gblb ADN at two concentrations tested had no such effect
on
gabapentin, being indistinguishable from control values (compare A, B and C
vs. G,
H and I). Treatment with the gbla mis-sense probe was also without effect when
compared to control values (compare A. B and C vs J. K and Ll and like the
~hllL
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
sub- (C) and supra-threshold (D) evoked somatic Ca2+ responses (traces 2 vs
1), but
did not prevent the generation of Ca2+ spikes when somatic current injection
was
increased (traces 3). Baclofen similarly reduced Ca2+ responses; subthreshold
(E),
suprathreshold (F).
Figure 16 shows that gabapentin inhibits Ca2+ responses in a dose-
dependent manner. Dose/response histograms of gabapentin (100 ~,M to 1 mM)
actions on both membrane depolarizations (A, C) and Ca2+ responses (B, D) for
sub-
(A, B) and supra-threshold (C, D) current injections. In the presence of 1 mM
gabapentin, cells were still able to generate Ca2+ spikes when somatic current
injection was increased. In these conditions, both membrane depolarizations
and
Ca2+ responses were not significantly different from control. (C, D) Summary
histograms of baclofen (40 ~,M) effects on sub- (E, G) and supra-threshold (F,
H)
responses. Bars on histograms represent SEM.
Figure 17 shows gabapentin and baclofen inhibition of Ca2+ responses
via GABAB receptor activation. In the presence of the GABAB receptor
antagonist
CGP55845 (4 ~,M), gabapentin (2 mM; A, B) and baclofen (40 ,uM; C, D) failed
to
depress responses evoked by either sub- (A, C) or supra-threshold (B, D)
current
injection. Summary histograms of gabapentin (2 mM) and baclofen (40 ,uM)
effects
on responses evoked by sub- (E) and supra-threshold (F) stimulation, in the
absence
and presence of CGP55845.
DETAILED DESCRIPTION OF THE INVENTION
For the purposes of describing the present invention:
"gbla" refers to the human GABAB receptor subunit known as
GABABRla in Kaupmann et al., 1998, Proc. Natl. Acad. Sci. USA 95:14991-14996,
the amino acid sequence (SEQ.ID.N0.:2) of which can be found at GenBank
accession no. AJ225028 (see also GenBank accession no. AJ012185), as well as
to its
mammalian orthologs. The amino acid sequence (SEQ.ID.N0.:4) of the mouse
ortholog of gbla is found at GenBank accession no. AF114168. gbla also refers
to
other GABAB receptor subunits that have minor changes in amino acid sequence
from those described in the previous two sentences as long as those other
GABAB
receptor subunits have substantially the same biological activity as the
subunits
described in the previous two sentences.
"gblb" refers to the human GABAB receptor subunit known as
GABABRlb in Kaupmann et al., 1998, Proc. Natl. Acad. Sci. USA 95:14991-14996,
-11-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
the amino acid sequence (SEQ.)D.NO.:6) of which can be found at GenBank
accession no. AJ225029, as well as to its mammalian orthologs. gblb also
refers to
other GABAB receptor subunits that have minor changes in amino acid sequence
from those described in the previous sentence as long as those other GABAB
receptor
subunits have substantially the same biological activity as the subunits
described in
the previous sentence.
"gb 1 c" refers to a human GABAB receptor subunit having the amino
acid sequence SEQ.)D.N0.:8, encoded by DNA having the nucleotide sequence
SEQ.ID.N0.:7, as well as to its mammalian orthologs. .The GenBank accession
no.
for human gblc is AJ012187. gblc also refers to other GABAB receptor subunits
that have minor changes in amino acid sequence from those described in the
previous
two sentences as long as those other GABAB receptor subunits have
substantially the
same biological activity as the subunits described in the previous two
sentences.
"gb2" refers to a human GABAB receptor subunit having the amino
acid sequence SEQ.)D.N0.:9 encoded by DNA having the nucleotide sequence
SEQ.ID.NO.:10, as well as to its mammalian orthologs. The amino acid sequence
of
the rat ortholog of gb2 is found at GenBank accession no. AF058795. gb2 also
refers
to other GABAB receptor gb2 subunits that have minor changes in amino acid
sequence from those described in the previous two sentences as long as those
other
GABAB receptor subunits have substantially the same biological activity as the
subunits described in the previous two sentences. For example, Clark et al.,
2000,
Brain Res. 860:41-52 disclosed two additional gb2 c-terminal variants in the
rat. A
human gb2 sequence is also found at GenBank accession no. AF056085.
"gbla heteromer" refers to a GABAB receptor that comprises a gbla
subunit and a gb2 subunit and does not comprise a gblb or gblc subunit.
Preferably,
the gbla heteromer is a heterodimer of a gbla subunit and a gb2 subunit.
"gblb heteromer" refers to a GABAB receptor that comprises a gblb
subunit and a gb2 subunit and does not comprise a gbla or gblc subunit.
Preferably,
the gblb heteromer is a heterodimer of a gblb subunit and a gb2 subunit.
"gblc heteromer" refers to a GABAB receptor that comprises a gblc
subunit and a gb2 subunit and does not comprise a gbla or gblb subunit.
Preferably,
the gblc heteromer is a heterodimer of a gblc subunit and a gb2 subunit.
"gbla cells" refers to cells that express gbla heteromers but not gblb
or gb 1 c heteromers;
-12-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
"gblb cells" refers to cells that express gblb heteromers but not gbla
or gb 1 c heteromers;
"gblc cells" refers to cells that express gblc heteromers but not gbla
or gblb heteromers;
A gb2 polypeptide has "substantially the same biological activity" as
native gb2 (i.e., SEQ.ID.NO.:10) if that polypeptide has an amino acid
sequence that
is at least about 80% identical to, preferably at least about 95% identical
to, more
preferably at least about 97% identical to, and most preferably at least about
99%
identical to SEQ.ID.NO.:10 and can form heteromers with either a gbla, gblb,
or
gblc polypeptide, thus forming a functional GABAB receptor.
"Functional GABAB receptor" refers to a GABAB receptor formed by
co-expression of gb2 and gbla, gblb, or gblc, most preferably resulting in a
heterodimer of gb2 and either gbla, gblb, or gblc, where the functional GABAB
receptor mediates at Ieast one functional response when exposed to the GABAB
receptor agonist GABA. Examples of functional responses are: pigment
aggregation
in Xenopus melanophores, negative modulation of cAMP levels, coupling to
inwardly
rectifying potassium channels, mediation of late inhibitory postsynaptic
potentials in
neurons, increases in potassium conductance, decreases in calcium conductance,
MAPKinase activation, extracellular pH acidification, and other functional
responses
typical of G-protein coupled receptors. One skilled in the art would be
familiar with a
variety of methods of measuring the functional responses of G-protein coupled
receptors such as the GABAB receptor (see, e.g., Lerner, 1994, Trends
Neurosci.
17:142-146 [changes in pigment distribution in melanophore cells]; Yokomizo et
al.,
1997, Nature 387:620-624 [changes in cAMP or calcium concentration;
chemotaxis];
Howard et al., 1996, Science 273:974-977 [changes in membrane currents in
Xenopus
oocytes]; McI~ee ~et al., 1997, Mol. Endocrinol. 11:415-423 [changes in
calcium
concentration measured using the aequorin assay]; Offermanns & Simon, 1995, J.
Biol. Chem. 270:15175-15180 [changes in inositol phosphate levels]). Depending
upon the cells in which heteromers of gb2 and either gbla, gblb, or gblc are
expressed, and thus the G-proteins with which the functional GABAB receptor
thus
formed is coupled, certain of such methods may be appropriate for measuring
the
functional responses of such functional GABAB receptors. It is well within the
competence of one skilled in the art to select the appropriate method of
measuring
functional responses for a given experimental system.
-13-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
A gbla polypeptide has "substantially the same biological activity" as
a native gbla polypeptide if that polypeptide has an amino acid sequence that
is at
least about 80% identical to, preferably at least about 95% identical to, more
preferably at least about 97% identical to, and most preferably at least about
99%
identical to SEQ.m.N0.:2 and either (1) has a Kd or EC50 for an amino acid (in
particular neutral and branched chain amino acids, including leucine,
isoleucme,
valine), amino acid analogue (such as 'y-hydroxybutyrate or phosphinic acids),
GABAB receptor agonist (such as (R,S)baclofen, gabapentin or similar 3-
liphophilic
substituted GABA analogues, or (L)-glutamic acid), or GABAg receptor
antagonist
IO (such as CGP71872, saclofen, or phaclofen), that is no more than 5-fold
greater than
the Kd or EC50 of a native gbla polypeptide for the same amino acid, amino
acid
analogue, GABAB receptor agonist, or GABAB receptor antagonist or (2) can form
heteromers with a gb2 polypeptide, thus forming a functional GABAB receptor.
Native gbla polypeptides include the murine gbla sequence shown as
SEQ.ID.N0.:4;
the rat gbla polypeptide disclosed in Kaupmann et al., 1997, Nature 386:239-
246; the
human gbla sequence disclosed in GenBank accession number AJ225028
(SEQ.ID.N0.:2); and the protein encoded by the DNA sequence disclosed in
GenBank accession number Y11044.
A gblb polypeptide has "substantially the same biological activity" as
a native gblb polypeptide if that polypeptide has an amino acid sequence that
is at
least about 80% identical to, preferably at least about 95% identical to, more
preferably at least about 97% identical to, and most preferably at least about
99%
identical to SEQ.JD.N0.:6 and either (1) has a Kd or EC50 for an amino acid
(in
particular neutral and branched chain amino acids, including leucine,
isoleucine,
valine), amino acid analogue (such as 'y-hydroxybutyrate or. phosphinic
acids),
GABAB receptor agonist (such as (R,S)baclofen, gabapentin or similar 3-
liphophilic
substituted GABA analogues, or (L)-glutamic acid), or GABAg receptor
antagonist
(such as CGP71872, saclofen, or phaclofen), that is no more than 5-fold
greater than
the Kd or EC50 of a native gblb polypeptide for the same amino acid, amino
acid
analogue, GABAB receptor agonist, or GABAB receptor antagonist or (2) can form
heteromers with a gb2 polypeptide, thus forming a functional GABAB receptor.
Native gblb polypeptides include the human gblb sequence disclosed in GenBank
accession number AJ225029 (SEQ.ll~,N0.:6) and the rat gblb polypeptide
disclosed
in Kaupmann et al., 1997, Nature 386:239-246.
- 14-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
A gblc polypeptide has "substantially the same biological activity" as
a native gblc polypeptide if that polypeptide has an amino acid sequence that
is at
least about 80% identical to, preferably at least about 95% identical to, more
preferably at least about 97% identical to, and most preferably at least about
99%
identical to SEQ.ID.N0.:8 and either (1) has a Kd or ECSp for an amino acid
(in
particular neutral and branched chain amino acids, including leucine,
isoleucine,
valine), amino acid analogue (such as y-hydroxybutyrate or phosphinic acids),
GABAB receptor agonist (such as (R,S)baclofen, gabapentin or similar 3-
liphophilic
substituted GABA analogues, or (L)-glutamie acid), or GABAB receptor
antagonist
(such as CGP71872, saclofen, or phaclofen), that is no more than 5-fold
greater than
the Kd or ECSp of a native gblc polypeptide for the same amino acid, amino
acid
analogue, GABAB receptor agonist, or GABAB receptor antagonist or (2) can form
heteromers with a gb2 polypeptide, thus forming a functional GABAB receptor.
Native gblc polypeptides include the amino acid sequence shown as
SEQ.ID.N0.:8.
A substance "activates a functional response" by interacting with
functional GABAB receptors on the surface of cells when the cells are exposed
to the
substance, leading to an increase in the level of the functional response. For
example,
if the functional response is the activation of a Kir channel, then a
substance that
activates a "functional response" of a GABAB receptor is a substance that acts
as an
agonist at the GABAB receptor so as to cause increased potassium ion flow
through
the Kir channel.
A "conservative amino acid substitution" refers to the replacement of
one amino acid residue by another, chemically similar, amino acid residue.
Examples
of such conservative substitutions are: substitution of one hydrophobic
residue
(isoleucine, leucine, valine, or methionine) for another; substitution of one
polar
residue for another polar residue of the same charge (e.g., arginine for
lysine;
glutamic acid for aspartic acid).
The present invention provides methods for identifying substances that
are subtype-specific agonists of the GABAB receptor. In particular, the
substances
function as agonists of GABAB receptors that are heteromers of gbla and gb2
subunits. The substances are not agonists of GABAB receptors that are
heteromers of
gblb and gb2 subunits; nor are they agonists of GABAB receptors that are
heteromers
of gblc and gb2 subunits. In CAl pyramidal neurons of rat hippocampal slices,
the
substances activate post-synaptic potassium currents but do not
presynaptically
depress GABA inhibitory postsynaptic currents. The substances are not agonists
of
-15-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
GABAA receptors. The substances are agonists of GABAB receptors that are
negatively coupled to voltage dependent-calcium channels in HEK293 cells,
melanotroph cell lines derived from mouse intermediary lobe pituitary tumors,
and in
rat hippocampal neurons or spinal cord neurons.
There are currently three known isoforms of the GABAB receptor gbl
subunit: gbla, gblb, and gblc. gbla, gblb, and gblc are proteins of 961, 844,
and
899 amino acids, respectively, differing only in that portion of their ligand
binding
extracellular N-termini that precedes a domain that is homologous to the
bacterial
periplasmic leucine-binding protein (Ng et al., 2001, Mol. Pharm. 59:144-152;
Kaupmann et al., 1997, Nature 386:239-246; Galvez et al., 1999, J. Biol. Chem.
274:13362-13369) (Figure 1A). The gbla-specific N-terminal sequence is
comprised
primarily of two protein-protein interacting Sushi Repeat (also known as short
consensus repeat) domains of ~60 amino acids, the first corresponding to T26-
R98
and the second to K102_N160 described by Kaupmann et al., 1998, Proc. Natl.
Acad.
Sci. USA 95:14991-14996. gblb differs from gbla in that the first 164 amino
acids
of gbla are replaced by 47 different amino acids. Thus gblb lacks both N-
terminus
Sushi Repeats. The gblc isoform differs from gbla by an in-frame 62 amino acid
deletion and elimination of one Sushi Repeat, leaving a single Sushi Repeat
interacting module.
Activation of neuronal GABAB receptors leads to increases in K+
membrane conductance which have been associated with a postsynaptic site
(Sodickson & Bean, 1998, J. Neurosci. 18:8153-8162). gbla and gblb have been
reported to exhibit differential post and presynaptic localizations,
respectively (Benke
et al., 1999, J. Biol. Chem. 274:27323-27330; Fritschy et al., 1999, Eur. J.
Neurosci.
11:761-768). These two subunits, as well as the gblc isoform, were tested to
determine whether, when expressed a~ homomers or heteromers with gb2, they
could
couple with Kir3.1/3.2 in Xenopus oocytes, an established model for studying
GPCR-
activated inward rectifiers (Kir channels; Dascal, 1997, Cell Signal. 9:551-
573).
Human or murine gbla, human gblb, and human gblc isoforms are inactive when
expressed individually (data not shown). All three gbl isoforms require co-
expression with gb2 in order to form structurally distinct, functional GABAB
receptors that can couple to Kir 3.1/3.2 channels and that are activated by
GABA
(Figure 2). However, gabapentin (100 ~.M) could only activate Kir 3.1/3.2
channels
through the gbla heteromer (Figure 2). Co-expression of human gbla and gb2
with
Kir 3.113.2 in Xenopus oocytes resulted in a significant stimulation of Kir
current in
-16-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
response to 100 p.M GABA (297 +/- 30.5% increase over control current measured
at
-80 mV, n=6). This was confirmed at the murine gbla heteromer (290 +/- 40%
increase over control current measured at -80 mV, n=6), and maximal
stimulation was
similar with the human gbla heteromer. Gabapentin agonism at the gbla
heteromer
could be blocked by 1 p,M CGP71872, a GABAB antagonist (data not shown). In
contrast to the results obtained with gbla heteromers, no response to
gabapentin was
detected (n=6) after stimulation of gblb (n=9) or gblc (n=4) heteromers,
although
GABA-mediated responses were always detected in the same oocytes (Figure 2,
°
Figure 3A). This demonstrates a gbla subtype-specific effect of gabapentin.
Another difference between the responses to GABA and gabapentin
was manifested by the rapid and consistent desensitization of the response to
gabapentin during its continual presence (Figure 2), essentially resulting in
a return to M,
basal current levels during 1 minute of stimulation. A markedly reduced
desensitization was occasionally detectable in responses to GABA (Figure 2) or
baclofen (data not shown). As for stimulation of GABAB receptors by GABA, only
modest desensitization of current was detected during stimulation by M2- or
(32AR
agonists (Figure 2), consistent with reports in the literature using the
oocyte
expression system (Doupnik et al., 1997, Proc. NatI. Acad. Sci. USA 94:10461-
10466; Fidler-Lim et al., 1995, J. Gen. Physiol. 105:421-439).
Studies of dose dependency at gbla heteromers revealed a rank order
of potency: GABA > baclofen > gabapentin with EC50 values of 1.1, 1.9, and 19
p,M,
respectively (Figure 3B). Consistent with the potency of GABA in this assay,
synaptic concentrations of GABA have been reported to reach up to 5 p,M (Mody
et
al., 1994, Trends. Neurosci. 17:517-525). The approximately 20 p.M potency of
gabapentin at the gb 1 a heteromer is also consistent with its therapeutic
dose as
monotherapy in the treatment of epilepsy or neuropathy (10-100 ~M in brain
following dosing up to 3.6 g/day) (Bryans & Wustrow, 1999, Med. Res. Rev.
19:149-
177; Backonja et al., 1998, J. Am. Med. Assn. 280:1831-1836; Rowbotham et al.,
1998, J. Am. Med. Assn. 280:1837-1842). This suggests that one mechanism by
which gabapentin exerts its therapeutic action is through gbla subtype-
specific
GABAB receptor agonism.
Gabapentin and baclofen at 10 p,M final concentration inhibited
[3H]TBOB specific binding to rat brain cortex GABAA chloride channel site by
18%
and 5%, respectively. However, gabapentin was inactive (up to 100 ~M) in
functional assays at recombinant GABAA receptors (data not shown). Gabapentin
-17-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
and baclofen displaced specific [3H]GABA binding at rat cerebellum GABAB
receptors by 24-26% and 22%, respectively, suggesting that gabapentin, like
baclofen,
interacts with neuronal GABAB receptors.
Gabapentin was tested to determine if it was active at native GABAB
receptors in CA1 pyramidal neurons of rat hippocampal slices (Luscher et al.,
1997,
Neuron 19:687-695) (Figure 4; see Example 4 for experimental details).
Currents
evoked by bath application of baclofen and gabapentin were isolated using
voltage
ramps and a subtraction procedure during whole cell patch clamp recordings
(Nurse
& Lacaille, 1999, Neuropharmacol. 38:1733-1742). Whole cell membrane currents
were measured during voltage ramps from -40 to -140 mV. Currents obtained from
the I-V relation in control ACSF were subtracted from those in the presence of
either
baclofen or gabapentin (Figure 4B) to isolate drug-activated currents. The
current= w
voltage relation for these currents indicated that 1 mM gabapentin activated
outward
currents at membrane potentials near rest (Figure 4B). These gabapentin
currents
reversed and became inward near -100 mV (mean Erev = -101.0 ~ 2.2 mV, n = 7
cells), which is near the equilibrium potential for K+ in this preparation.
These
results indicate that gabapentin activated potassium currents in CA1 pyramidal
neurons. Gabapentin currents were dose-dependent, their mean chord conductance
increasing with doses between 0.01-1 mM (Figure 4B and 4D). Bath application
of 2-
20 ~,M (-)baclofen elicited similar potassium currents that were outward at
membrane
potentials near rest, reversed near -100 mV (mean Erev = -101.5 ~ 2.6 mV, n =
6
cells), and were dose-dependent (Figure 4C and 4D). Potassium currents
activated by
1 mM gabapentin and 20 ,uM baclofen were coupled to GABAB receptors since they
were reduced by 89% and 84%, respectively, by pretreatment with the GABAB
receptor antagonist CGP55845 (4 and 1 ,uM respectively, Figure 4D). These
results
indicate that gabapentin activated potassium currents linked to postsynaptic
GABAB
receptors m CA1 pyramidal cells and these actions of gabapentin were similar
to the
postsynaptic actions of baclofen.
Since neuronal GABAB receptors are also located presynaptically and
such presynaptic GABAB receptors are coupled to inhibition of transmitter
release
(Bowery, 1993, Ann. Rev. Pharmacol. Toxicol. 33 :109-147), it was of interest
to
assess whether gabapentin activated presyntaptic GABAg receptors that inhibit
GABA release in hippocampal neurons ira situ (Davies et al., 1990, J. Physiol
(London) 424:513-531; Thompson, 1994, Prog. Neurobiol. 42:575-609). Fast
monosynaptic GABA inhibitory postsynaptic currents (IPSCs) were evoked in CA1
_18_
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
pyramidal cells by electrical stimulation of stratum radiatum in the presence
of
blockers of glutamate synaptic transmission (antagonists of non-NMDA and NMDA
glutamate receptors, CNQX and APS, respectively) (Figure 5A). These fast
outward
IPSCs recorded near resting membrane potential were mediated by GABAA
receptors
since they were completely antagonized by 25 ,uM bicuculline (n=11 cells, data
not
shown). Fast GABAA IPSCs were reversibly reduced by 75% by bath application of
20 ACM baclofen (Figure 5B). This presynaptic inhibition by baclofen was dose-
dependent (2-20 ~.M) and was antagonized by the GABAB receptor antagonist
CGP55845 (1 p,M; Figure 5D). In contrast, gabapentin (0.01-1 mM) did not
IO significantly depress GABAA lPSCs (Figure 5C and 5D), even during bath
applications that elicited outward currents in the same cells (not shown). The
small
and variable reduction in IPSC amplitude observed in the presence~of
gabapentin (6-
13%) was not significantly different from the reduction observed during
repeated
application of control ACSF (7%) or after pretreatment with the GABAB
antagonist
GP55845 (16%) (Figure 5D). This minor reduction therefore does not result from
gabapentin effects. These results indicate that whereas baclofen activated
presynaptic
GABAB receptors and inhibited GABA release, gabapentin did not. Thus
gabapentin
does not have presynaptic actions like baclofen in CAI hippocampus.
Furthermore,
gabapentin was inactive up to 100 ~,M in functional assays at the recombinant
GABAA al(33~y2, oc3(33y2, and a4(33~y2 receptor subtypes (data not shown).
Gabapentin was also inactive up to 100 ~,M in functional assays at the
recombinant
NMDA NR2B receptor (data not shown).
In addition, further studies with gabapentin demonstrated that GABAB
receptors are negatively coupled to voltage-dependent calcium channels (VD-CCs
) in
rat hippocampal neurons. During combined whole Bell recording and multiphoton
Ca2+ imaging in hippocampal neurons in situ, gabapentin significantly
inhibited, in a
dose-dependent mariner, subthreshold soma depolarizations and Ca2+ responses
evoked by somatic current injection. Further, gabapentin almost completely
blocked
Ca2+ action potentials and Ca2+ responses elicited by suprathreshold current
injection. However, larger current injection overcame this inhibition of Ca2+
action
potentials, suggesting that gabapentin did not predominantly affect L-type
Ca2+
channels. The depressant effect of gabapentin on Ca2+ responses was coupled to
the
activation of neuronal GABAB receptors since they were blocked by CGP55845,
and
baclofen produced similar effects. Thus, gabapentin activation of neuronal
gbla
heteromers negatively coupled to VD-CCs is potentially an important
therapeutic
-19-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
mechanism of action of gabapentin, which may be linked to inhibition of
neurotransmitter release in some systems.
The results disclosed herein further indicate that gabapentin may have
multiple anticonvulsant actions linked to GABAB receptors. In addition to its
selective activation of gb 1 a heteromers coupled to GIRKs that produce
postsynaptic
hyperpolarization, gabapentin may inhibit Ca2+ influx during burst discharges
or
seizures via its activation of postsynaptic gbla heteromers negatively coupled
to VD-
CCs. It is interesting to note that gabapentin actions on hippocampal neurons
are
therefore dictated not only by its selective activity at the gbla heteromer
subtype, but
also by the cellular domain where these receptors are found in the cell.
Gabapentin-
sensitive GABAg receptors present in the soma and dendritic regions couple to
VD-
CCs (Figures 15-17) and GIRKs (Figure 4). In contrast, the gblb and gblc
heteromer
subtypes, which are likely present in glutamate and GABA axon terminals of
hippocampal neurons and also are negatively coupled to VD-CCs, are insensitive
to
gabapentin. This is in agreement with the subtype selective agonist activity
defined
using recombinant receptors (Figures 2 and 3) and the lack of presynaptic
effect of
gabapentin on synaptic transmission in hippocampus (Figure 5).
Gabapentin has been recently reported to depress excitatory amino
acid neurotransmission in spinal cord dorsal horn (Patel et al:, 2000, British
J
Pharmacol. 130:1731-1734; Shimoyama et al., 2000, Pain 85:405-414), and the
effect
of the agonist gabapentin on GABAg receptors coupled to VD-CCs could account
for
these effects since a well established physiological role of presynaptic
neuronal
GABAB receptors is inhibition of P/Q and N-type VD-CCs and transmitter release
(Bowery & Enna, 2000, J. Pharmacol. Exp. Ther. 292:2-7; Wu & Saggau, 1997,
Trends Neurosci. 20:204-212; Menon-Johansson et al., 1993, Pflugers Arch.
425:335-
343). This conclusion is also consistent with the anatomical localization of
the gbla
heteromer to some presynaptic elements in the neural axis (Benke et al., 1999,
J. Biol.
Chem. 274:27323-27330; Billinton et al., 1999, Br. J. Pharmacol. 126:1387-
1392;
Towers et al., 2000, Eur. J. Neurosci. 12:3201-3210). GABAB receptor
distribution
studies in the lumbar spinal cord and dorsal root ganglia showed that the gbla
mRNA
is the predominant species (accounting for ~90°Io) of the total gbl
mRNA in the
afferent fiber cell body. This suggests that gbla subunits together with gb2,
which
exhibit equivalent density to gbla, comprise presynaptic GABAB receptors on
primary afferent terminals (Towers et al., 2000, Eur. J. Neurosci. 12:3201-
3210).
Indeed, in this report, immunocytochemical analysis showed denser labeling of
gbla
-20-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
in the superficial dorsal horn and present in neuropil whereas gblb was more
associated with cell bodies in this region.
The predominant expression of gbla heteromers in the superficial
laminae where nociceptive primary afferent fibers terminate, together with
studies
which suggests the anti-nociceptive effects of baclofen (Hammond & Drower,
1984,
Eur. J. Pharmacol. 103:121-125; Sawynok & Dickson, 1985, Pharmacology 31:248-
259; Henry, 1982, Neuropharmacology 21:1085-1093) and gabapentin (Xiao &
Bennet, 1996, Analgesia 2:267-273; Patel et al., 2000, Br. J. Pharmacol.
130:1731-
1734; Shimoyama et al., 2000, Pain 85:405-414) are mediated presynaptically,
suggest that, at least in part, the anti-hyperalgesic, anti-allodynic and anti-
nociceptive
effects of gabapentin can be attributed to selective activation of presynaptic
gbla
heteromers coupled to VD-CCs in the spinal cord dorsal horn. Additionally, the
selective agonism of presynaptic gbla heteromers in the spinal cord dorsal
horn may
underlie the efficacy of GABAB agonists such as baclofen in the treatment of
urinary
incontinence.
A structural basis for the pharmacological difference among GABAB
receptor subtypes likely owes to gbla-specific sequences which comprise
primarily a
Sushi repeat (K102_N160) which is absent in the gblb and gblc subunits. A
recent
model has been proposed which suggests that agonist binding at GABAB receptors
is
similar to bacterial periplasmic amino acid binding proteins where the
extracellular
domain folds into two lobes separated by a hinge region (Galvez et al., 1999,
J. Biol.
Chem. 274:13362-13369). It may be that the gbla-specific Sushi domain
modulates
the closure of this "Venus Flytrap"-like domain such that it can bind
gabapentin while
other gbl receptor subtypes cannot. Another possible explanation for the
pharmacological difference among GABAB receptor subtypes would be the
existence
of an additional protein or proteins from the cellular environment which may
also be
required for gabapentin activity at GABAB receptors. Such a requirement would
be
analogous to the case of the CGRP receptor where CRLR and RAMPS are required
for the functional CGRP receptor. This accessory protein or proteins would be
present in a gabapentin-sensitive GABAB receptor expressing cells but not in
gabapentin-insensitive GABAB receptor expressing cells.
GABA inhibition in the CNS involves multiple mechanisms. These
include fast postsynaptic inhibition via activation of GABAA receptor chloride
channels, slow postsynaptic inhibition via activation of GABAB receptors and G-
-21-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
protein-regulated inward rectifying potassium channels, and presynaptic
inhibition via
negative modulation of Ca2+ channels in presynaptic terminals, reducing
glutamate
and GABA release (Nicoll et al., 1990, Physiol. Rev. 70:513-565; Sivilotti et
al.,
1991, Prog. Neurobiol. 36:35-92; Thompson et al., 1994, Prog. Neurol. 42:575-
609).
Fast postsynaptic GABAA responses result from activity at single synapses,
whereas
slower GABAB responses necessitate the synchronous activation of multiple
presynaptic fibers (Dutar et al., 1988, Nature 332:156-158; Otis et al., 1992,
J.
Neurophysiol. 67:227-235). In addition, postsynaptic GABAB receptors appear
important for curtailing epileptiform activity in the presence of impaired
GABAA
inhibition (Malouf et al., 1990, Neuroscience 35:53-61; Scanziani et al.,
1991, J.
Physiol. (London) 444:375-396). In hippocampal neurons, postsynaptic GABAB
receptor activation leads to membrane hyperpolarization, mediated by inward
rectifying potassium channels (Luscher et al., 1997, Neuron 19:687-695).
Further,
subcellular localization studies show that gbla is predominantly postsynaptic
whereas
gblb is largely presynaptic (Benke et al., 1999. J. Biol. Chem. 274:27323-
27330;
Fritschy et al., 1999, Eur. J. Neurosci. 11:761- 769) although one study
concludes
differently (Billinton et al., 1999, Br. J. Pharmacol. 126:1387-1392). The
present data
show for the first time that gabapentin is a GABAB gbla heteromer subtype-
specific
agonist and is selective for postsynaptic GABAB receptors in hippocampus,
providing the first in situ evidence of structurally and pharmacologically
distinct pre-
and postsynaptic GABAB receptor subtypes.
The present study thus shows that gabapentin is a member of a class of
pharmacological agents that possess a particular combination of
characteristics:
~ they are agonists of GABAB receptors that are heteromers of
gbla and gb2 subunits
~ they are not agonists of GABAB receptors that are heteromers
of gblb and gb2 subunits
~ they are not agonists of GABAB receptors that are heteromers
of gblc and gb2 subunits
~ they activate post-synaptic potassium currents
~ they do not presynaptically depress GABA inhibitory
postsynaptic currents
they are not agonists of GABAA receptors
they exhibit rapid desensitization and a distinct mechanism of
activation compared to baclofen at the coupled receptor
-22-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
they are agonists of GABAB receptors that are negatively
coupled to voltage dependent-calcium channels in heterologous expression
systems
such as HEK-293 cells, melanotroph cell lines derived from mouse intermediary
lobe
pituitary tumors and in rat hippocampal neurons, dorsal root ganglion neurons,
and
spinal cord dorsal horn neurons
It is likely that this combination of characteristics, or some subset of
this combination, is responsible for the therapeutic effectiveness of
gabapentin.
Therefore, it would be of great interest to identify other substances that
share this
combination of characteristics, or some subset of this combination. The
present
invention provides methods for identifying such substances.
The present invention provides methods of identifying gbla subtype-
specific agonists of the GABAB receptor that comprise (a) determining that a
substance is an agonist of GABAB receptors comprising a gbla subunit; and (b)
determining that the substance is not an agonist of GABABreceptors comprising
a
gb 1 b or gb 1 c subunit.
Optionally, the methods comprise determining that the substance
activates post-synaptic potassium currents but does not presynaptically
depress
GABA inhibitory postsynaptic currents. The methods also optionally include
determining that the substance is not an agonist of GABAA receptors and/or
determining that the substances are agonists of GABAg receptors that are
negatively
coupled to voltage dependent-calcium channels in heterologous expression
systems
such as HEK-293 cells, melanotroph cell lines derived from mouse intermediary
lobe
pituitary tumors, and in .rat hippocampal neurons, dorsal root ganglion
neurons and
spinal cord dorsal horn neurons.
One method of identifying substances that are likely to be gbla
subtype-specific agonists is to identify those substances that are capable of
binding to
gbla heteromers but that are not also capable of binding to gblb or gblc
heteromers.
This can be done by screening a collection of compounds against three types of
cells,
with each type of cell expressing either a gbla, gblb, or gblc heteromer and
determining the amount of each compound that binds to each type of cell. Those
compounds for which at least 3 times, preferably at least 10 times, and even
more
preferably at least 50 times more compound is bound to cells expressing the
gbla
heteromer (as compared to cells expressing either gblb or gblc heteromers) are
likely
to be gbla subtype-specific agonists. Of course, one would go on to test such
compounds in the functional assays that are described herein to make sure that
they
- 23 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
are indeed agonists rather than antagonists or compounds that simply bind
without
affecting function.
Accordingly, the present invention includes a method of identifying a
substance that is a gbla subtype-specific agonist comprising:
(a) exposing a substance, separately, to gbla cells, gblb cells, and
gblc cells;
(b) quantitating the binding of the substance to the gbla cells, gblb
cells, and gblc cells;
where, if the amount of binding of the substance to the gbla cells is at
least 3 times greater than the amount of binding of the substance to either
gblb or
gblc cells, then;
(c) determining whether the substance activates a functional
response of a gbla heteromer;
where if the substance activates a functional response of a gbla
heteromer then the substance is a gbla subtype-specific agonist.
The identification of gbla subtype-specific agonists can be facilitated
by the use of gabapentin. New gbla subtype-specific agonists are likely to be
able to
compete with gabapentin for binding to gbla heteromers. This allows for the
development of assays to identify gbla subtype-specific agonists based on such
competition. Moreover, pregabalin ((S)-3-isobutylgaba) is a compound that is
structurally related to gabapentin and has been found to inhibit the binding
of
[3H]gabapentin to brain membranes (Taylor et al., 1993, Epilepsy Res. 14:11-
15) as
well as to have similar pain relieving effects in animal models (Field et al.,
1999, Pain
80:391-398). In view of this similarity between the two compounds, it is
within the
scope of the present invention to utilize pregabalin instead of gabapentin in
certain
embodiments of the invention. Also within the scope of the invention is the
utilization of other 3'substituted GABA analogues.
Accordingly, the present invention includes a method for identifying
gbla subtype-specific agonists that comprises:
(a) providing gbla cells;
(b) exposing the gbla cells to gabapentin or pregabalin in the
presence and in the absence of a substance;
(c) measuring the binding of gabapentin or pregabalin to the gbla
cells in the presence and in the absence of the substance;
-24-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
where, if the amount of binding of gabapentin or pregabalin is less in
the presence of the substance than in the absence of the substance, then;
(d) determining whether the substance binds to gblb cells and
gblc cells;
where, if the substance does not bind to gblb cells and gblc cells,
then;
(e) determining whether the substance activates a functional
response of a gbla receptor;
where if the substance activates a functional response of a gbla
receptor then the substance is a gbla subtype-specific agonist.
The skilled person will recognize that it is generally beneficial to run
controls at various points in the methods described herein. For example, in
the
method described immediately above, it will usually be helpful to have a
control for
step (c) in which the binding of gabapentin or pregabalin to the cells is
shown to be
dependent on the presence of gbla heteromers. This can be done by measuring
the
binding of gabapentin or pregabalin to cells that are substantially the same
as the cells
of step (c) except for the lack of gbla heteromers. One way to do this is to
use cells
that recombinantly express gbla heteromers. The non-recombinant parent cells
would then serve as controls. Another control would be to take the substances
identified by the methods described herein and confirm that the substances do
not
activate a functional response at GABAB receptors that are gblb heteromers or
gblc
heteromers.
One skilled in the art would also recognize that the phrase "does not
bind to" in the methods described herein has a relative meaning. This phrase
does not
exclude some low level, insignificant binding that is non-specific, z.e., that
is not due
to the presence of gblb or gblc. Such non-specific binding can be assessed by
running various controls. This phrase may even apply to situations where the
substance does bind to gblb or gblc, but at an insignificant amount as
compared to its
binding to gbla. In this context, an insignificant amount would be, e.g., 5%,
1%, or
0.1 % or less.
The present invention includes a method of identifying a substance that
is a gbla subtype-specific agonist comprising:
(a) providing cells expressing gbla but not gblb or gblc;
(b) exposing the cells of step (a) to a substance;
(c) quantitating the binding of the substance to the cells of step (a);
- 25 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
(d) providing cells expressing gblb but not gbla or gblc;
(e) exposing the cells of step (d) to the substance;
(f) quantitating the binding of the substance to the cells of step (d);
(g) providing cells expressing gblc but not gbla or gblb;
(h) exposing the cells of step (g) to the substance;
(i) quantitating the binding of the substance to the cells of step (g);
where, if the amount of binding of the substance to the cells of step (a)
is at least 3 times greater than the amount of binding of the substance to the
cells of
step (d) and the cells of step (g), then;
(j) determining whether the substance activates a functional
response of a gbla heteromer;
where if the substance activates a functional response of a gbla
heteromer then the substance is a gbla subtype-specific agonist.
The cells of steps (a), (d), and (g) should be substantially identical
except for their differences in expression of gbla, gblb, and gblc. Qne method
of
obtaining such cells is to recombinantly express gbla, gblb, or gblc in a cell
line that
does not naturally express gbla, gblb, or gblc.
Preferred embodiments of the methods described herein make use of
recombinant cells containing expression vectors that direct the expression of
the
various GABAB receptor subunits. Thus, the present invention includes a method
for
identifying gbla subtype-specific agonists that comprises:
(a) providing cells comprising an expression vector encoding gb2
and an expression vector encoding gbla;
(b) culturing the cells under conditions such that gb2 and gbla are
expressed and gbla heteromers are formed;
(c) exposing the cells to gabapentin or pregabalin in the presence
and in the absence of a substance;
(d) measuring the binding of gabapentin or pregabalin to the gbla
heteromers in the presence and in the absence of the substance;
where if the amount of binding of gabapentin or pregabalin is less in
the presence of the substance than in the absence of the substance, then;
(e) determining whether the substance binds to gblb cells and
gblc cells;
where, if the substance does not bind to gblb cells and gblc cells,
then;
-26-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
(f) determining whether the substance activates a functional
response of a gbla heteromer;
where if the substance activates a functional response of a gbla
heteromer then the substance is a gbla subtype-specific agonist.
The gblb cells and gblc cells of step (e) should be substantially
identical to each other as well as to the cells of step (a) except for the
differences in
expression of gbla, gblb, and gblc in the three types of cells. One method of
producing such cells is to begin with a parental cell line that does not
express either
gbla, gblb, or gblc and to separately transfect expression vectors encoding
gbla,
gblb, and gblc into the parental cells, thereby producing three cell lines,
each cell
line expressing only one of gbla, gblb, and gblc.
One skilled in the art would recognize that the phrase "where if the
amount of binding of gabapentin or pregabalin is less in the presence of the
substance
than in the absence of the substance" in the methods described herein has a
relative,
not an absolute, meaning. Substances of interest identified by the methods
described
herein cause the binding of gabapentin or pregabalin to be less in a non-
trivial
manner. Such substances would, e.g., decrease the binding of gabapentin or
pregabalin by at least about 50%, preferably by about 75%, more preferably by
about
95%, and even more preferably by about 99%.
One skilled in the art would understand that when practicing such
competition assays as those described herein, it is important that the
relative amounts
of the various compounds and substances used be appropriate. For example, in
the
method described immediately above, one would not attempt to detect
competitive
displacement of the binding or gabapentin or pregabalin by the substance by
using a
vast excess (e.g., a 1,000-fold molar excess) of substance as compared to
gabapentin
or pregabablin. One would preferably use the substance and the gabapentin or
pregabalin at more or less equal molar concentrations.
The present invention includes a method for identifying a gbla
subtype-specific agonist that comprises:
(a) determining whether a substance activates a GABAB receptor
functional response in gb 1 a cells;
(b) determining whether the substance activates a GABAB
receptor functional response in gblb cells;
(c) determining whether the substance activates a GABAB
receptor functional response in gblc cells;
-27-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
where if the substance activates a GABAB receptor functional
response in gbla cells, but not in gblb or gblc cells, then the substance is a
gbla
subtype-specific agonist.
In particular embodiments of the methods described herein, the
functional response is selected from the group consisting of: modulation of
the
activity of an ion channel; changes in calcium concentration; changes in a
signal from
a reporter gene whose expression is controlled by a promoter that is induced
by
interaction of an agonist with the GABAB receptor; and changes in membrane
currents. In particular embodiments, the change in membrane current is
measured in
Xenopus oocytes. In other embodiments, the change in membrane current is
caused
by the modulation of the activity of an inwardly rectifying potassium current.
In other embodiments, the change in membrane current is caused by
the modulation of the activity of a voltage dependent-calcium channel (VD-CC).
It is
known that activation of the GABAB receptor inhibits ion flow through VD-CCs
(Dolphin, 1995, Exp. Physiol. 80:1-36; Filippov et al., 2000, J. Neurosci.
20:2867-
2874). Therefore, it is possible to use changes in intracellular calcium
levels arising
from such inhibition of VD-CCs as a surrogate measure of the activity of
agonists at
the GABAB receptor. When the functional response is a change in intracellular
calcium concentration, such a change can be monitored by the use of
appropriate
indicator dyes (e.g., furs-2, fluo-3, indo-1, Calcium Green; see Veli~elebi et
al., 1999,
Meth. Enzymol. 294:20-47) and suitable detection instruments.
Electrophysiological
measures could also be used to detect activity at GABAB receptors coupled to
VD-
CCs.
Indicator dyes are substances which show a change in a fluorescent
characteristic upon binding calcium, e.g., greatly increased intensity of
fluorescence
and/or a change in fluorescent spectra (i.e., a change in emission or
excitation
maxima). Fluo-3, furs-2, and indo-1 are commonly used calcium indicator dyes
that
were designed as structural analogs of the highly selective calcium chelators
ethylene
glycol-bis((3-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) and 1,2-
bis(2-
aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). The fluorescence
intensity from fluo-3 increases by more than 100-fold upon binding of calcium.
While the unbound dye exhibits very.little fluorescence, calcium-bound fluo-3
shows
strong fluorescence emission at 526 nm. Furs-2 is an example of a dye that
exhibits a
change in its fluorescence spectrum upon calcium binding. In the unbound
state,
furs-2 has an excitation maximum of 362 nm. This excitation maximum shifts to
335
_~8_
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
nm upon calcium binding, although there is no change in emission maximum.
Binding of calcium to fura-2 can be monitored by excitation at the two
excitation
maxima and determining the ration of the amount of fluorescence emission
following
excitation at 362 nm compared to the amount of fluorescence emission following
excitation at 335 nm. A smaller ratio (i.e., less emission following
excitation at 362
nm) indicates that more fura-2 is bound to calcium, and thus a higher internal
calcium
concentration in the cell.
The use of calcium indicator dyes entails loading cells with the dye, a
process which can be accomplished by exposing cells to the membrane-permeable
acetoxymethyl esters of the dyes. Once inside the plasma membrane of the
cells,
intracellular esterases cleave off the esters, exposing negative charges in
the free dyes.
This prevents the free dyes from crossing the plasma membrane and thus leaves
the
free dyes trapped in the Bells. Measurements of fluorescence from the dyes are
then
made, the cells are treated in such a way that the internal calcium
concentration is
changed (e.g., by exposing gbla cells to a gbla subtype-specific agonist), and
fluorescence measurements are again taken.
Fluorescence from the indicator dyes can be measured with a
luminometer or a fluorescence imager. One preferred detection instrument is
the
Fluorometric Imaging Plate Reader (FLIPR) (Molecular Devices, Sunnyvale, CA).
The FLIPR is well suited to high throughput screening using the methods of the
present invention as it incorporates integrated liquid handling capable of
simultaneously pipetting to 96 or 384 wells of a microtiter plate and rapid
kinetic
detection using a argon laser coupled to a charge-coupled device imaging
camera.
Using this approach, it may be desirable to engineer the cells employed so as
to
recombinantly express calcium channels that are coupled to the GABAB receptor
as
well as promiscuous G-proteins.
A typical protocol for use of calcium indicator dyes would entail
plating gbla, gblb, and gblc cells into clear, flat-bottom, black-wall 96 well
plates
(e.g., those made by Costar or Vue-plates from Packard) and allowing the cells
to
30. grow overnight in standard tissue culture conditions (e.g., 5% C02,
37°C). The cells
are generally plated at a density of about 10,000 to 100,000 cells per well in
appropriate growth medium. On the day of the assay, growth medium is removed
and
dye loading medium is added to the wells.
If the calcium indicator dye is fluo-3, e.g., dye loading medium could
be prepared by solubilizing 50 pg of fluo-3-AM ester (Molecular Probes F-1242)
in
-29-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
22 ~l DMSO to give a 2 mM dye stock. Immediately before loading the cells, 22
~l
20% pluronic acid (Molecular Probes P-3000) is added to the dye. The tube
containing the dye is mixed with a vortex mixer and 42 ml of the dye/pluronic
acid
solution is added to 10.5 ml of Hanks Balanced Salt Solution (GibcoBRL Cat #
14025-076) with 20 mM HEPES (GibcoBRL Cat # 1560-080), 2.5 mM probenecid
(Sigma Cat # P-8761), and 1% fetal bovine serum (GibcoBRL Cat # 26140-087; not
BSA)). The dye and the loading medium are mixed by repeated inversion (final
dye
concentration about 4 ~.M).
Growth medium can be removed from the cells by washing with the
Denley Cellwash (wash medium is Hanks Balanced Salt Solution (GibcoBRL Cat #
14025-076) with 20 mM HEPES (GibcoBRL Cat # 1560-080), 2.5 mM probenecid
(Sigma Cat # P-8761), and 0.1% bovine serum albumin (Sigma Cat # A-9647; not
FBS) three times at volume setting "F" and one last time at volume setting
"0,"
leaving 100 ~1 residual medium in the wells after the fourth wash. Then 100
~.l of the
dye in the loading medium is added to each well with a 12 channel pipetter.
The cell
plate is placed back in the C02 incubator to load for 60 minutes.
Following dye loading, fluorescent measurements of the cells are taken
prior to exposure of the cells to substances that are to be tested for gbla
subtype-
specific agonist activity. The cells are then exposed to the substances and
those
substances that cause a change in a fluorescent characteristic of the dye are
identified.
The measuring instrument can be a fluorescent plate reader such as the FLIPR
(Molecular Devices). Substances that cause a change in a fluorescent
characteristic in
the gbla cells but not in the gblb or gblc cells are gbla subtype-specific
agonists.
The exact details of the procedure outlined above are meant to be
illustrative. One skilled in the art would be able to optimize experimental
parameters
(cell number, dye concentration, dye loading time, temperature of incubations,
cell
washing conditions, and instrument settings) by routine experimentation
depending
on the particular relevant experimental variables (e.g., type of cell used,
identity of
dye used). Several examples of experimental protocols that can be used are
described
in Veli~elebi et al., 1999, Meth. Enzymol. 294:20-47.
The present invention provides a method for identifying gbla subtype-
specific agonists comprising:
(a) providing gbla cells;
(b) loading the gbla cells with a calcium indicator dye;
-30-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
(c) measuring a fluorescence characteristic of the calcium indicator
dye in the gbla cells in the presence and in the absence of a substance;
(d) providing gblb cells;
(e) loading the gblb cells with a calcium indicator dye;
(f) measuring a fluorescence characteristic of the calcium indicator
dye in the gblb cells in the presence and in the absence of the substance;
(g) providing gblc cells;
(h) loading the gblc cells with a calcium indicator dye;
(i) measuring a fluorescence characteristic of the calcium indicator
dye in the gblc cells in the presence and in the absence of the substance;
where if a change in fluorescent characteristic in the presence as
compared to the absence of the substance is measured in step (c) but not in
step (f)
and step (i) then the substance is a gbla subtype-specific agonist.
In particular embodiments, the calcium indicator dye is selected from
the group consisting of: fluo-3, fura-2, fluo-4, fluo-5, aequorin, calcium
green-I,
Oregon green, 488 BAPTA, SNARE-1, and indo-1.
In particular embodiments, the change in fluorescent characteristic is
an increase in intensity of a fluorescence emission maximum. In other
embodiments,
the change in fluorescent characteristic is a shift in the wavelength of an
absorption
maximum.
In particular embodiments, the cells naturally express both GABAB
receptors and/or calcium channels. In other embodiments, the cells do not
naturally
express GABAB receptors and/or calcium channels but instead have been
transfected
with expression vectors that encoded GABAB receptors and/or calcium channels
so
that the cells recombinantly express the GABAB receptors and/or calcium
channels.
In particular embodiments, the cells have been transfected with an
expression vector that encodes one particular gbl isoform, either gbla, gblb,
or gblc
so that the transfected cells express one of gbla, gblb, or gblc. In certain
embodiments, the cells are also transfected with an expression vector that
encodes
gb2 so that functional heteromers of gb2 and either gbla, gblb, or gblc are
formed in
the cells.
In particular embodiments, the cells have been transfected with an
expression vector that encodes a volatge dependent-calcium channel (VD-CC)
subunit or subunits. In preferred embodiments, the subunit or subunits form a
functional N-type or a P/Q-type VD-CC. N-type or a P/Q-type VD-CCs are
-31 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
composed of an al subunit, an a28 subunit, and, usually at least one (3
subunit.
Therefore, it may be advantageous to transfect the cells with an expression
vector or
vectors that encode an N-type or a P/Q-type al subunit, a28 subunit, and (3
subunit.
Cell lines expressing VD-CCs and GABAB receptors can be used in
the methods of the present invention. VD-CCs are activated by depolarization
of the
plasma membrane. This depolarization can be brought about by raising the level
of
extracellular K+ by, e.g., the addition of KCl to the medium bathing the
cells. This
addition of KCl causes activation of the VD-CCs, influx of extracellular Ca2+
into the
cells, and a consequent rise in [Ca2+]i. This rise in [Ca2+]i can be measured
by the
use of suitable calcium indicator dyes. Readings from the indicator dyes are
generally
taken within the first 10 seconds or so after activation of the VD-CCs since
this is the
time period when the influx of Ca2+ through the VD-CCs peaks. ~ At later
times,
[Ca2+]i rises due to the release of Ca2+ from intracellular stores and it is
desirable to N
minimize interference from this release of intracellular Ca2+
DNA encoding VD-CCs for use in constructing expression vectors
encoding the VD-CCs can be obtained by methods well known in the art. For
example, a cDNA fragment encoding a VD-CC can be isolated from a suitable cDNA
library by using the polymerase chain reaction (PCR) employing suitable primer
pairs. Such primer pairs can be selected based upon the known DNA sequence of
the
VD-CC it is desired to obtain. Suitable cDNA libraries can be made from
cellular or
tissue sources known to contain mRNA encoding the VD-CC. One skilled in the
art
could use published VD-CC sequences to design PCR primers and published
studies
of VD-CC expression to select the appropriate sources from which to make cDNA
libraries in order to obtain DNA encoding the VD-CC. The following
publications
may be of use in this regard:
U.S. Patent No. 5,874,236 and U.S. Patent No. 5,429,921 describe
various al and (3 subunits of human voltage-gated calcium channels;
U.S. Patent No. 5,407,820 and U.S. Patent No. 5,710,250 describe a2
subunits of human voltage-gated calcium channels;
International Patent Publication WO 98/13490 describes a brain-
specific P/Q-type human voltage-gated calcium channel.
An alternative to the use of calcium indicator dyes such as those
discussed above is the use of the aequorin system to monitor GABAB receptor
mediated inhibition of VD-CCs. The aequorin system makes use of the protein
apoaequorin, which binds to the lipophilic chromophore coelenterazine forming
a
-32-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
combination of apoaequorin and coelenterazine that is known as aequorin.
Apoaequorin has three calcium binding sites and, upon calcium binding, the
apoaequorin portion of aequorin changes its conformation. This change in
conformation causes coelenterazine to be oxidized into coelenteramide, C02,
and a
photon of blue light (466 nm). This photon can be detected with suitable
instrumentation.
Since the gene encoding apoaequorin has been cloned (U.S. Patent No.
5,541,309; U.S. Patent No. 5,422,266; U.S. Patent No. 5,744,579; Inouye et
al., 1985,
Proc. Natl. Acad. Sci. USA 82:3154-3158; Prasher et al., 1985, Biochem.
Biophys.
Res. Comm. 126:1259-1268), apoaequorin can be recombinantly expressed in cells
in
which it is desired to measure the intracellular calcium concentration.
Alternatively,
existing cells that stably express recombinant apoaequorin can be-used. Such
cells
derived from HEK-293 cells and CHO-K1 cells are described in Button &
Brownstein, 1993, Cell Calcium 14:663-671. For example, the HEK293/aeql7 cell
line can be used as follows.
The HEK293/aeql7 cells are grown in Dulbecco's Modified Medium
(DMEM, GIBCO-BRL, Gaithersburg, MD, USA) with 10% fetal bovine serum (heat
inactivated), 1 mM sodium pyruvate, 500 ~,g/ml Geneticin, 100 ~g/ml
streptomycin,
100 units/ml penicillin. Expression vectors encoding the desired combination
of
GABAB receptor subunits (gbla, gblb, gblc (or rat gbla-1e), gb2) as well as
the
desired calcium channels subunits (oc 1A, a 1B~ a 1C~ a 1D~ a lEa a2s~ ala
(~2~ ~3~
(34) and perhaps G protein subunits (Gi, Go) can be transfected into the
HEK293/aeql7 cells by standard methods in order to express the desired GABAB
receptor subunits, calcium channels subunits, and G protein subunits in the
HEK293/aeql7 cells. The cells are washed once with DMEM plus 0.1 % fetal
bovine
serum, and then charged for one hour at 37°C /5% C02 in DMEM containing
8 ~M
coelenterazine cp (Molecular Probes, Eugene, OR, USA) and 30 ~.M glutathione.
The cells are then washed once with Versene (GIBCO-BRL, Gaithersburg, MD,
USA), detached using Enzyme-free cellissociation buffer (GIBCO-BRL,
Gaithersburg, MD, USA), diluted into ECB (Ham's F12 nutrient mixture (GIBCO-
BRL) with 0.3 mM CaCl2, 25 mM HEPES, pH7.3, 0.1 % fetal bovine serum). The
cell suspension is centrifuged at 500 x g for 5 min. The supernatant is
removed, and
the pellet was is resuspended in 10 ml ECB. The cell density is determined by
counting with a hemacytometer and adjusted to 500,000 cells/ml in ECB. The
substances to be tested are diluted to the desired concentrations in ECB and
aliquoted
- 33 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
into assay plates, preferably in triplicate, at 0.1 ml/well. The cell
suspension is
injected at 0.1 ml/well, read and integrated for a total of 400 readings using
a
luminometer (Luminoskan Ascent, Labsystems Oy, Helsinki, Finland). Data are
analyzed using the software GraphPad Prism Version 3.0 (GraphPad Software,
Inc.,
San Diego, CA, USA).
It will be understood by those skilled in the art that the procedure
outlined above is a general guide in which the various steps and variables can
be
modified somewhat to take into account the specific details of the particular
experiment that is desired to be run. For example, one could use semisynthetic
coelenterazine (Shimomura, 1989, Biochem. J. 261:913-920; Shimomura et al.,
1993,
Cell Calcium 14:373-378); the time of incubation of the cells with
coelenterazine can
be varied somewhat; somewhat greater or lesser numbers of cells per well can
be
used; and so forth.
For reviews on the use of aequorin, see Creton et al., 1999,
Microscopy Research and Technique 46:390-397; Brini et al., 1995, J. Biol.
Chem.
270:9896-9903; Knight & Knight, 1995, Meth. Cell. Biol. 49:201-216. Also of
interest may be U.S. Patent No. 5,714,666 which describes methods of measuring
intracellular calcium in mammalian cells by the addition of coelenterazine co-
factors
to mammalian cells that express apoaequorin.
Another type of assay provided by the present invention makes use of
Xe~copus laevis oocytes that have been microinjected with RNA encoding GABAB
receptor subunits as well as inwardly rectifying potassium channels (Kirs).
The
oocytes are voltage clamped and then exposed to substances while membrane
currents
are monitored. If the substances are agonists of GABAB receptors, changes in
potassium ion flow across the oocytes' membranes will be seen as a result of
activation of GABAB receptors and coupling of the GABAB receptors to Kirs. If
the
substances are able to cause altered potassium currents in oocytes that
express gbla
heteromers but not also in oocytes that express gblb or gblc heteromers, then
the
substances are gbla subtype-specific agonists.
In an alternative embodiment, the GABAB receptor subunits and the
Kirs are expressed in the oocytes by means of an oocytes expression vector
(e.g.,
PT7TS) rather than by microinjection.
Accordingly, the present invention includes a method for identifying a
gbla subtype-specific agonist of the GABAB receptor comprising:
-34-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
(a) providing a Xefzopus laevis oocyte expressing gbla and gb2 so
as to form a functional gbla heteromer in the oocyte where the oocyte also
expresses
a Kir;
(b) exposing the oocyte of step (a) to a substance while monitoring
potassium ion flow across the oocyte membrane;
(c) providing a Xenopus laevis oocyte expressing gblb and gb2 so
as to form a functional gblb heteromer in the oocyte where the oocyte also
expresses
a Kir;
(d) exposing the oocyte of step (c) tb the substance while
monitoring potassium ion flow across the oocyte membrane;
(e) providing aXenopus laevis oocyte expressing gblc and gb2 so
as to form a functional gblc heteromer in the oocyte where the oocyte also
expresses
a Kir;
(f) exposing the oocyte of step (e) to the substance while
monitoring potassium ion flow across the oocyte membrane;
where if the exposure of the oocytes to the substance results in
increased potassium ion flow in step (b), but not in steps (d) and (f), then
the
substance is a gbla subtype-specific agonist of the GABAB receptor.
Particular types of functional assays that can be used to identify gbla
subtype-specific agonists include transcription-based assays. Transcription-
based
assays involve the use of a reporter gene whose transcription is driven by an
inducible
promoter whose activity is regulated by a particular intracellular event such
as, e.g.,
changes in intracellular calcium levels, that are caused by the interaction of
a receptor
with a ligand. Transcription-based assays are reviewed in Rutter et al., 1998,
Chemistry & Biology 5:8285-8290. Transcription-based assays of the present
invention rely on the expression of reporter genes whose transcription is
activated or
repressed as a result of intracellular events that are caused by the
interaction of a gbla
subtype-specific agonist such as gabapentin with a heteromer of gb2 and gbla
where
the heteromer forms a functional GABAB receptor.
An extremely sensitive transcription-based assay is disclosed in
Zlokarnik et al., 1998, Science 279:84-88 (Zlokarnik) and also in U.S. Patent
No.
5,741,657. The assay disclosed in Zlokarnik and U.S. Patent No. 5,741,657
employs
a plasmid encoding ~3-lactamase under the control of an inducible promoter.
This
plasmid is transfected into cells together with a plasmid encoding a receptor
for which
it is desired to identify agonists. The inducible promoter on the (3-lactamase
is chosen
-35-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
so that it responds to at least one intracellular signal that is generated
when an agonist
binds to the receptor. Thus, following such binding of agonist to receptor,
the level of
(3-lactamase in the transfected cells increases. This increase in (3-lactamase
is
measured by treating the cells with a cell-permeable dye that is a substrate
for
cleavage by ~3-lactamase. The dye contains two fluorescent moieties. In the
intact
dye, the two fluorescent moieties are physically linked, and thus close enough
to one
another that fluorescence resonance energy transfer (FRET) can take place
between
them. Following cleavage of the dye into two parts by (3-lactamase, the two
fluorescent moieties are located on different parts, and 'thus can diffuse
apart. This
increases the distance between the fluorescent moieties, thus decreasing the
amount of
FRET that can occur between them. It is this decrease in FRET that is measured
in
the assay.
The assay described in Zlokarnik and U.S. Patent No. 5,741,657 can be~~
modified to form an assay for identifying agonists of GABAB receptors by using
an
inducible promoter to drive ~3-lactamase where the promoter is activated by an
intracellular signal generated by the interaction of agonists and the GABAB
receptor.
In an alternative version of this assay, cells are treated with a substance
that results in
the activation of the promoter driving the (3-lactamase. This activation is
inhibited by
a signal generated the interaction of agonists and the GABAB receptor. An
example
of this alternative version of the assay could employ (3-lactamase driven by
the CRE
promoter where forskolin stimulation of adenyIyl cyclase activates the CRE
promoter,
thus increasing the concentration of ~i-lactamase in the cells, and this
activation of the
CRE promoter is inhibited by the interaction of agonists and the GABAB
receptor.
To produce the GABAB receptor, a plasmid encoding gb2 and a
~5 plasmid encoding either gbla, gblb, or gblc are transfected into the cells.
The cells
are exposed to the cell-permeable dye and then exposed to substances suspected
of
being agonists of the GABAB receptor. Those substances that cause a decrease
in
FRET are likely to actually be agonists of the GABAB receptor.
By testing the substances against gbla cells and then against gblb and
gblc cells, those substances that are agonists only in gbla cells can be
identified.
Such substances are gbla subtype-specific agonists.
Accordingly, the present invention includes a method for identifying
gbla subtype-specific agonists of the GABAB receptor comprising:
(a) providing gbla cells comprising:
-36-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
(1) an expression vector that directs the expression of gb2
in the gbla cells;
(2) an expression vector that directs the expression of gbla
in the gbla cells;
(3) an expression vector that directs the expression in the
gbla cells of (3-lactamase under the control of an inducible promoter that is
activated
by an intracellular signal generated by the interaction of agonists with the
GABAB
receptor;
(b) exposing the gb 1 a cells to a substrate of ~3-lactamase that is a
cell-permeable dye that contains two fluorescent moieties where the two
fluorescent
moieties are on different parts of the dye and cleavage of the dye by (3-
lactamase
allows the two fluorescent moieties to diffuse apart;
(c) measuring the amount of fluorescence resonance energy
transfer (FRET) in the gbla cells in the absence and in the presence of a
substance to
determine a ratio of the amount of FRET in the absence of the substance to the
amount of FRET in the presence of the substance for the gbla cells;
(d) providing gblb cells comprising:
(4) an expression vector that directs the expression of gb2
in the gblb cells;
(5) an expression vector that directs the expression of gblb
in the gblb cells;
(6) an expression vector that directs the expression in the
gblb cells of (3-lactamase under the control of an inducible promoter that is
activated
by an intracellular signal generated by the interaction of agonists with the
GABAB
receptor;
(e) exposing the gblb cells to a substrate of (3-lactamase that is a
cell-permeable dye that contains two fluorescent moieties where the two
fluorescent
moieties are on different parts of the dye and cleavage of the dye by (3-
lactamase
allows the two fluorescent moieties to diffuse apart;
(f) measuring the amount of FRET in the gblb cells in the absence
and in the presence of the substance to determine a ratio of the amount of
FRET in the
absence of the substance to the amount of FRET in the presence of the
substance for
the gblb cells;
(g) providing gblc cells comprising:
-37-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
(7) an expression vector that directs the expression of gb2
in the gblc cells;
(8) an expression vector that directs the expression of gblc
in the gblc cells;
(9) an expression vector that directs the expression in the
gblc cells of (3-lactamase under the control of an inducible promoter that is
activated
by an intracellular signal generated by the interaction of agonists with the
GABAB
receptor;
(h) exposing the gblc cells to a substrate of (3-lactamase that is a
cell-permeable dye that contains two fluorescent moieties where the two
fluorescent
moieties are on different parts of the dye and cleavage of the dye by (3-
lactamase
allows the two fluorescent moieties to diffuse apart;
(i) measuring the amount of FRET in the gblc cells in the absence
and in the presence of the substance to determine a ratio of the amount of
FRET in the
absence of the substance to the amount of FRET in the presence of the
substance for
the gblc cells;
where if the ratio of the amount of FRET in the absence of the
substance to the amount of FRET in the presence of the substance for the gbla
cells is
greater than the ratio of the amount of FRET in the absence of the substance
to the
amount of FRET in the presence of the substance for the gblb cells and for the
gblc
cells then the substance is a gbla subtype-specific agonist.
Substeps (1)-(3) of step (a), (4)-(6) of step (d), and (7)-(9) of step (g)
can be practiced in any order. The groups of three steps (a)-(c), (d)-(f), and
(g)-(i) can
be practiced in any order. That is, the method can be practiced, e.g., by
first carrying
out steps (d)-(f), then (g)-(i), and then (a)-(e).
In particular embodiments, the ratio of the amount of FRET in the
absence of the substance to the amount of FRET in the presence of the
substance for
the gbla cells is at least about 50%, preferably about 100%, more preferably
about
200%, and even more preferably about 500% greater than the ratio of the amount
of
FRET in the absence of the substance to the amount of FRET in the presence of
the
substance for the gblb cells and for the gblc cells.
In a particular embodiment of the above-described method, the
inducible promoter is a promoter that is activated by changes in membrane
currents,
e.g., changes in potassium currents. In other embodiments, the inducible
promoter is
activated by the transcription factor NEAT, or is activated by a signal
transduced by a
-38-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
chimeric Gq protein, or a signal generated by protein kinase C activity, or by
changes
in intracellular calcium levels. In particular embodiments, the inducible
promoter is a
promoter that is activated by NF-ycB or NEAT, e.g., the interleukin 2 promoter
(Mattila et al., 1990, EMBO J. 9:4425-4433), a MAPKINASE-inducible promoter,
or
a promoter that is regulated by CAMP levels, e.g., the CRE promoter (Chen et
al.,
1995, Anal. Biochem. 226:349-354).
When the intracellular signal generated by the interaction of agonists
with the GABAB receptor is an increase in intracellular calcium levels, the
cells can
also be transfected with a vector encoding a promiscu~aus G-protein such as
615/16 or
GqiS or GqoS.
In particular embodiments of the methods described herein, cells are
transfected, either stably or transiently, with expression vectors that direct
the
expression of gb2, gbla, gblb, gblc, (3-lactamase under the control of an
inducible
promoter that is activated by at least one intracellular signal generated by
interaction
of an agonist with the GABAB receptor, and/or reporter genes. In other
embodiments, the cells are also transfected with a vector encoding a
promiscuous G-
protein such as 615/16 or Gqi5 or GqoS.
A variety of (3-lactamases are known in the art and are suitable for use
in the present methods. One particularly well-studied form of (3-lactamase is
the
product of the Ampr gene of E. coli, TEM-1 ~i-lactamase (Sutcliffe, 1978,
Proc. Natl.
Acad. Sci. USA 75:3737-3741). A version of TEM-l, with its signal sequence
deleted so that it accumulates in the cytoplasm, is disclosed in Kadonaga et
al., 1984,
J. Biol. Chem. 259:2149-2154. ~3-lactamases are produced by a variety of
bacteria
and many (3-lactamases have been well studied. For example, Staphlyococcus
aureus
produces PC 1 (3-lactamase; Bacillus cereus produces a (3-lactamase known as
(3-
lactamase I; Escherichia coli produces RTEM (3-lactamase (Christensen et al.,
1990,
Biochem J. 266:853-861. All that is necessary for a particular ~i-lactamase to
be
suitable for use in the present invention is that it be capable of cleaving
the
fluorescent substrate in such a way that the two fluorescent moieties of the
substrate
can diffuse away from each other following cleavage. This can be easily tested
and
thus the suitability of a particular (3-lactamase can be easily determined.
The amino acid sequences of a variety of suitable (3-lactamases are
disclosed in Ambler, 1980, Phil. Trans. R. Soc. Lond. (Ser. B.) 289:321-331.
One of
skill in the art can readily synthesize synthetic DNA sequences that encode
these (3-
lactamases. Alternatively, these (3-lactamases can be cloned from natural
sources.
-39-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
DNA sequences encoding [3-lactamases can be placed into suitable expression
vectors
and transfected into cells for use in the methods of the present invention. A
DNA
sequence encoding a particular ~3-lactamase that can be used in the methods of
the
present invention is shown in SEQ.ID.N0.:1 of U.S. Patent No. 5,741,657 while
the
corresponding amino acid sequence is shown as SEQ.ID.N0.:2 of U.S. Patent No.
5,741,657. A plasmid containing this DNA (pTG2de11) is described in Kadonaga
et
al., 1984, J. Biol. Chem. 259:2149-2154.
Moore et al., 1997, Anal. Biochem. 247:203-209 describes a method
for engineering a form of RTEMl (3-lactamase that is maintained
intracellularly by
eukaryotic cells. DNA encoding the native signal sequence of RTEM1 (3-
lactamase is
removed and replaced with a~methionine codon. Sequences that provide for
optimal
translational efficiency in eukaryotes are placed immediately upstream of this
methionine by PCR. This modified (3-lactamase coding sequence is then cloned
into
expression vector pRc-CMV (Invitrogen, San Diego, CA). This places the coding
sequences under the control of the human intermediate early cytomegalovirus
promoter and provides a bovine growth hormone polyadenylation sequence. This
construct, known as pCMV-BL, was able to direct the expression of active (3-
lactamase in the cytoplasm of mammalian cells.
A preferred embodiment of the present invention makes use of the
fluorescent (3-lactamase substrate used in the assays for transcriptional
activation
described by Zlokarnik et al., 1998, Science 279:84-88. This substrate is
known as
CCF2/AM and has the following structure
Bt0 " - ~ " OAc
O O
H
/ / N
CI
O O fVl
C02AM
where Ac = acetyl; Bt = butyryl; and AM = acetoxymethyl.
CCF2/AM contains several ester functionalities. These esters make
CCF2/AM membrane-permeant. Because of this membrane-permeant property,
CCF2/AM will be taken up by cells growing in tissue culture following addition
to
-40-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
the media. After uptake, intracellular esterases cleave the esters, giving
rise to CCF2,
which is trapped intracellularly due to its many negative charges. The
structure of
CCF2 is
-O 0-
O O O
H
N
CI
O O NH
N~S
v0
C02
CCF2 contains 7-hydoxycoumarin as the FRET donor at the 7 position..
of the cephalosporin moiety. The 7-hydroxycoumarin has a 6-chloro substituent
to
lower the pKa of CCF2 to 5.1, thus making fluorescence independent of pH at pH
values above 6, as well as a glycine spacer betweent the coumarin and the
cephalosporin moiety. The fluorescent acceptor is fluorescein, which is
attached to
the 7' position of the cephalosporin moiety via a thioether linkage.
Excitation of the coumarin donor of intact CCF2 at 409 nm gives rise
to FRET emission from the fluorescein acceptor having a peak at 520 nm. After
cleavage of CCF2, and the separation of the coumarin and fluorescein,
excitation of
the coumarin donor gives rise to fluorescent emission from the coumarin having
a
peak at 447 nm. Of course, excitation need not be done at arid emission need
not be
measured at precisely the wavelengths mentioned above. For example, one could
excite at 395 nm and measure emission at 530 nm and 460 nm.
In principle, one could measure the amount of FRET by monitoring
either (a) a decrease in the emission-of the donor fluorescent reagent
following
stimulation at the donor's absorption wavelength and/or (b) an increase in the
emission of the acceptor reagent following stimulation at the donor's
absorption
wavelength. In practice, FRET is most effectively measured by emission
ratioing.
Emission ratioing refers to measuring the ratio of emission by the acceptor
and
emission by the donor. In one embodiment, it is the ratio of donor emission to
acceptor emission that is determined in order to measure the amount of FRET
that is
occurring. A low ratio indicates an intact CCF2 structure; this means that
little (3-
lactamase is present and therefore a large amount of FRET is occurring. A high
ratio
-41 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
indicates that CCF2 has been cleaved by (3-lactamase; this means that
relatively more
enzyme is present and therefore a small amount of FRET is occurring.
In another embodiment, it is the ratio of acceptor emission to donor
emission that is determined in order to measure the amount of FRET that is
occurring.
A low ratio indicates that CCF2 has been cleaved by [3-lactamase; this means
that
relatively more enzyme is present and therefore a small amount of FRET is
occurring.
A high ratio indicates an intact CCF2 structure; this means that little (3-
lactamase is
present and therefore a large amount of FRET is occurring.
Emission ratioing can be measured by employing a laser-scanning
confocal microscope. Emission ratioing is preferably done by splitting the
emitted
light from a sample with a dichroic mirror and measuring two wavelength bands
(corresponding to the donor and the acceptor emission wavelengths)
simultaneously
with two detectors. Alternatively, the emitted light can be sampled
consecutively at
each wavelength (by using appropriate filters) with a single detector. In any
case,
these and other methods of measuring FRET are well known in the art.
The use of emission ratioing in the present methods eliminates many
variables that might otherwise confound accurate quantitation such as cell
size, cell
number, probe concentration, and light intensity. The methods of the present
invention are easily monitored with a fluorescence microscope or a plate
reader. The
present invention can be readily adapted for use in 96 well microtiter plates
or even in
higher density well plates, allowing for its use in high throughput screening
programs.
CCF2 is meant to be illustrative of certain preferred substrates for use
in the invention. The invention can also be practiced with other fluorescent
substrates. A general formula for fluorescent substrates of (3-lactamase that
are
suitable for use in the present invention is:
R~ H
~Z, ~ N A
O O N Z"-~y
C02R"
where:
one of X and Y is a fluorescent donor moiety or an ester derivative of said
fluorescent
donor moiety, and the other is a fluorescent acceptor or an ester derivative
of said
-42-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
fluorescent acceptor moiety; where fluorescence resonance energy transfer can
occur
between said fluorescent donor moiety and said fluorescent acceptor moiety;
R' is selected from the group consisting of H, lower alkyl and (CH~)nOH, in
which n
is 0 or an integer from 1 to 5;
R" is selected from the group consisting of H, physiologically acceptable
metal and
ammonium cations, --CHR20C0(CHZ)nCH3, --CHR20COC(CH3)3,
-acylthiomethyl, -acyloxy-alpha-benzyl, -delta-butyrolactonyl, -
methoxycarbonyloxymethyl, -phenyl, -methylsulphinylmethyl, -beta-
morpholinoethyl, -dialkylaminoethyl, -acyloxyalkyl,
-dialkylaminocarbonyloxymethyl and -alkyl, in which R2 is selected from the
group
consisting of H and lower alkyl and in which n is 0 or an integer from 1 to 5;
A is ~~
selected from the group consisting of S, O, SO, SO~ and CH2; and Z' and Z" are
linkers for the fluorescent donor and acceptor moieties.
Preferably, Z' is selected from the group consisting of a direct bond
__(CH~)nCONR~(CH2)m--, __(CH~)nNR~CO(CH~)m--,
__(CH2)nNR2CONR2(CH2)m--, __(CH~)nNR3CSNR~(CH2)m--,
--(CH2)nCONR3(CH2)pCONR2(CH2)m--, __(CH2)n-_~
__(CH2)n~3C0(CH~)pS(CH~)m--~ __(CHZ)nS(CH2)m=-
__(CH2)n0(CH2)m--, __(CH~,)nNR~(CH2)m__~ __(CH~)nS02NR~(CH~)m--~
__(CH2)nC02(CH2)m--
/ ' ~ / ' ~ / and
O
-(CH2)mN
S(CH2)~
O
in which R2 is selected from the group consisting of H and lower alkyl; R3 is
selected
from the group consisting of hydrogen and lower alkyl; and each of n, m and p
is
independently selected from the group consisting of 0 and integers from 1 to
4.
Preferably, Z" is selected from the group consisting of a direct bond to
a heteroatom in Y, --O(CH2)n--, --S(CH2)n--, --NR2(CH2)n--,
- 43 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
__N+R22(CH2)n--~ __pCONR2(CH2)n--~ __02C(CH2)n--~
--SCSNR2(CH2)n--, --SCSO(CH2)n--, and
O
N(CH2)m
-S
O
in which R2 is selected from the group consisting of H and lower alkyl; and
each of n
and m is independently selected from the group consisting of 0 and integers
from 1 to
4..
Suitable fluorescent moieties for the (3-lactamase substrates described
herein, as well as methods of making the (3-lactamase substrates disclosed
herein, are
disclosed in U.S. Patent No. 5,741,657, the disclosures of which are
incorporated by w
reference herein.
The linker in the fluorescent substrate that is cleaved by (3-lactamase is
preferably a cephalosporin. This is because any molecule (such as a
fluorescent
moiety) that can be chemically attached to the 3' substituent of a
cephalosporin is
released upon cleavage of the (3-lactam ring of the cephalosporin by (3-
lactamase
(Albrecht et al., 1991, J. Med. Chem. 34:669-675). Thus, a fluorescent moiety
attached to the 3' substituent will be released upon cleavage and will diffuse
away
from another fluorescent moiety that remains attached to the rest of the
substrate.
In particular embodiments of the above-described methods, the cells
express a promiscuous G-protein, e.g., Gal5 or Gal6. In other embodiments, the
cells have been transfected with an expression vector that directs the
expression of a
G-protein subunit or subunits.
Other transcription-based assays can be used to identify gbla subtype-
specific agonists of the GABAB receptor. Such other assays rely on the use of
reporter genes (other than (3-lactamase) that are under the control of
inducible
promoters. The inducible promoter is activated by an intracellular signal
generated
by the interaction of agonists with the GABAB receptor. gbla cells containing
the
reporter gene are exposed to a suspected agonist and the amount of signal from
the
reporter gene is measured. If the suspected agonist causes an increase in
signal
(relative to a suitable control), then the suspected agonist is further tested
against
gblb cells and gblc cells containing the reporter gene. Those suspected
agonists that
cause an increase in reporter signal that is at least about three times,
preferably about
-44-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
times, and more preferably about 10 times, greater in gbla cells than in
either gblb
or gblc cells are gbla subtype-specific agonists.
Accordingly, the present invention provides a method of identifying
gbla subtype-specific agonists comprising:
5 (a) providing gb 1 a cells containing a reporter gene under the
control of an inducible promoter that is activated by an intracellular signal
generated
by the interaction of agonists with the GABAB receptor;
(b) exposing the gbla cells to a substance;
(c) determining the amount of signal from the reporter gene after
exposing the gbla cells to the substance;
(d) providing gblb cells containing a reporter gene under the
control of an inducible promoter that is activated by an intracellular signal
generated
by the interaction of agonists with the GABAB receptor;
(e) exposing the gblb cells to the substance;
(f) determining the amount of signal from the reporter gene after
exposing the gblb cells to the substance;
(g) providing gblc cells containing a reporter gene under the
control of an inducible promoter that is activated by an intracellular signal
generated
by the interaction of agonists with the GABAB receptor;
(h) exposing the gblc cells to the substance;
(i) determining the amount of signal from the reporter gene after
exposing the gblc cells to the substance;
where if the amount of signal in step (c) is at Ieast three times the
amount of signal in step (f) and in step (i) then the substance is a gb 1 a
subtype-
specific agonist.
Examples of suitable reporter genes are green fluorescent proteins
(GFPs), chloramphenicol acetyl transferase, J3-galactosidase, and luciferase.
The present invention also includes assays for the identification of
gbla subtype-specific agonists where the assays are based upon FRET between a
first
and a second fluorescent dye where the first dye is bound to one side of the
plasma
membrane of a cell expressing either a gbla, gblb, or gblc heteromer and the
second
dye is free to move from one face of the membrane to the other face in
response to
changes in membrane potential. In certain embodiments, the first dye is
impenetrable
to the plasma membrane of the cells and is bound predominately to the
extracellular
surface of the plasma membrane. The second dye is trapped within the plasma
-45-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
membrane but is free to diffuse within the membrane. At normal (i.e.,
negative)
resting potentials of the membrane, the second dye is bound predominately to
the
inner surface of the extracellular face of the plasma membrane, thus placing
the
second dye in close proximity to the first dye. This close proximity allows
for the
generation of a large amount of FRET between the two dyes. Following membrane
depolarization, the second dye moves from the extracellular face of the
membrane to
the intracellular face, thus increasing the distance between the dyes. This
increased
distance results in a decrease in FRET, with a corresponding increase in
fluorescent
emission derived from the. first dye and a corresponding decrease in the
fluorescent
emission from the second dye. See figure 1 of Gonzalez & Tsien, 1997,
Chemistry &
Biology 4:269-277. See also ~Gonzalez & Tsien, 1995, Biophys. J. 69:1272-1280
and
U.S. Patent No. 5,661,035.
In certain embodiments, the first dye is a fluorescent lectin or a
fluorescent phospholipid that acts as the fluorescent donor. Examples of such
a first
dye are: a coumarin-labeled phosphatidylethanolamine (e.g., N-(6-chloro-7-
hydroxy-
2-oxo-2H--1-benzopyran-3-carboxamidoacetyl)-dimyristoylphosphatidyl-
ethanolamine) or N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-
dipalmitoylphosphatidylethanolamine); a fluorescently-labeled lectin (e.g.,
fluorescein-labeled wheat germ agglutinin). In certain embodiments, the second
dye
is an oxonol that acts as the fluorescent acceptor. Examples of such a second
dye are:
bis(1,3-dialkyl-2-thiobarbiturate)trimethineoxonols (e.g., bis(1,3-dihexyl-2-
thiobarbiturate)trimethineoxonol) or pentamethineoxonol analogues (e.g.,
bis(1,3-
dihexyl-2-thiobarbiturate)pentamethineoxonol; or bis(1,3-dibutyl-2-
thiobarbiturate)pentamethineoxonol). See Gonzalez & Tsien, 1997, Chemistry &
Biology 4:269-277 for methods of synthesizing various dyes suitable for use in
the
present invention. In certain embodiments, the assay may comprise a natural
carotenoid, e.g., astaxanthin, in order to reduce photodynamic damage due to
singlet
oxygen.
The present invention includes methods in which the activation of
GABAB receptors is coupled to inwardly rectifying potassium channels.
Activation
of the GABAg receptors results in increased potassium current flow across the
plasma membrane of cells expressing potassium channels (e.g., Kir channels).
This
increased current flow results in a hyperpolarization of the cell membrane
that can be
detected electrophysiologically via voltage or patch clamp techniques or by
use of the
- 46 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
membrane potential or channel dyes or FRET-based dyes as described above since
such hyperpolarization will result in greater FRET.
Accordingly, the present invention provides a method of identifying
gbla subtype-specific agonists comprising:
(a) providing gbla cells comprising:
(1) an expression vector that directs the expression of gb2;
(2) an expression vector that directs the expression of gbla;
(3) an expression vector that directs the expression of an
inwardly rectifying potassium channel;
(4) a first fluorescent dye, where the first dye is bound to
one side of the plasma membrane; and
(5) a second fluorescent dye, where the second fluorescent
dye is free to move from one face of the plasma membrane to the other face in
response to changes in membrane potential;
(b) measuring the amount of fluorescence resonance energy
transfer (FRET) in the gbla cells in the presence and in the absence of a
substance to
determine a ratio of FRET in the absence over FRET in the presence of the
substance
for the gbla cells;
(c) providing gblb cells comprising:
(6) an expression vector that directs the expression of gb2;
(7) an expression vector that directs the expression of gblb;
(8) an expression vector that directs the expression of an
inwardly rectifying potassium channel;
(9) a first fluorescent dye, where the first dye is bound to
one side of the plasma membrane; and
(10) a second fluorescent dye, where the second fluorescent
dye is free to move from one face of the plasma membrane to the other face in
response to changes in membrane potential;
(d) measuring the amount of FRET in the gblb cells in the
presence and in the absence of the substance to determine a ratio of FRET in
the
absence over FRET in the presence of the substance for the gblb cells;
(e) providing gblc cells comprising:
(11) an expression vector that directs the expression of gb2;
(12) an expression vector that directs the expression of gblc;
- 47 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
(13) an expression vector that directs the expression of an
inwardly rectifying potassium channel;
(14) a first fluorescent dye, where the first dye is bound to
one side of the plasma membrane; and
(15) a second fluorescent dye, where the second fluorescent
dye is free to move from one face of the plasma membrane to the other face in
response to changes in membrane potential;
(f) measuring the amount of FRET in the gblc cells in the
presence and in the absence of the substance to deterrriine a ratio of FRET in
the
absence over FRET in the presence of the substance for the gblc cells;
where if the ratio of FRET in the absence of the substance over FRET
in the presence of the substance for the gbla cells is at least about 50%
greater than
the ratio of FRET in the absence of the substance over FRET in the presence of
the .
substance for the gblb and the gblc cells then the substance is a gbla subtype-
specific agonist.
In particular embodiments, the ratio of FRET in the absence of the
substance over FRET in the presence of the substance for the gbla cells is at
least
about 75%, preferably at least about 100%, and even more preferably at least
about
200% greater than the ratio of FRET in the absence of the substance over FRET
in the
presence of the substance for the gblb and the gblc cells.
Of course, one of skill in the art would realize that control assays
should be run where cells that lack at least one of the items recited in
substeps (a) (1)-
(2) are exposed to the substance and FRET is measured. The amount of FRET so
measured in these control assays should be less than the amount of FRET
measured in
the presence of the substance in step (b) above. This will ensure that the
substance is
not acting through a mechanism that has nothing to do with the gbla heteromer.
In
general, one of skill in the art would understand that control assays may be
desirable
for the assays described herein, in order to ensure that the effects measured
come
about through interaction of substances with the gbla heterodimer. Another
type of
control assay that will generally be desirable is to test gblb cells and gblc
cells for
the presence of functional GABAB receptors that are gblb or gblc heteromers by
determining whether GABA can increase a functional GABAB receptor response in
those cells. One of skill in the art would understand how to set up such
control assays
based on the teachings herein combined with knowledge generally known in the
art.
- 48 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
Inwardly rectifying potassium channels that are suitable for use in the
methods of the present invention are disclosed in, e.g., Misgeld et al., 1995,
Prog.
Neurobiol. 46:423-462; North, 1989, Br.~J. Pharmacol. 98:13-23; Gahwiler et
a1.,1985, Proc. Natl. Acad. Sci USA 82:1558-1562; Andrade et al., 1986,
Science
234:1261-1265.
In particular embodiments of the above-described methods, the first
fluorescent dye is selected from the group consisting of: a fluorescent
lectin; a
fluorescent phospholipid; a coumarin-labeled phosphatidylethanolamine; N-(6-
chloro-7-hydroxy-2-oxo-2H--1-benzopyran-3-carboxamidoacetyl)-
I0 dimyristoylphosphatidyl-ethanolamine); N-(7-nitrobenz-2-oxa-1,3-diazol-4-
yl)-
dipalmitoylphosphatidylethanolamine); and fluorescein-labeled wheat germ
agglutinin.
In particular embodiments of the above-described methods, the second
fluorescent dye is selected from the group consisting of: an oxonol that acts
as the
fluorescent acceptor; bis(1,3-dialkyl-2-thiobarbiturate)trimethineoxonols;
bis(1,3-
dihexyl-2-thiobarbiturate)trimethineoxonol; bis(1,3-dialkyl-2-thiobarbiturate)
quatramethineoxonols; bis(1,3-dialkyl-2-thiobarbiturate)pentamethineoxonols;
bis(1,3-dihexyl-2-thiobarbiturate)pentamethineoxonol; bis(1,3-dibutyl-2-
thiobarbiturate)pentamethineoxonol); and bis(1,3-dialkyl-2-
thiobarbiturate)hexamethineoxonols.
The GABAB receptor belongs to the class of proteins known as G-
protein coupled receptors (GPCRs). GPCRs transmit signals across cell
membranes
upon the binding of ligand. The ligand-bound GPCR interacts with a
heterotrimeric
G-protein, causing the Ga subunit of the G-protein to disassociate from the
G(3 and
Gy subunits. The Gcc subunit can then go on to activate a variety of second
messenger systems. In some cases it is the G(3 or Gy subunit that activates
the second
messenger systems.
Generally, a particular GPCR is only coupled to a particular type of G-
protein. Thus, to observe a functional response from the GPCR, it is necessary
to
ensure that the proper G-protein is present in the system containing the GPCR.
It has
been found, however, that there are certain G-proteins that are "promiscuous."
These
promiscuous G-proteins will couple to, and thus transduce a functional signal
from,
virtually any GPCR. See Offermanns & Simon, 1995, J. Biol. Chem. 270:15175-
15180 (Offermanns). Offermanns described a system in which cells are
transfected
with expression vectors that result in the expression of one of a large number
of
- 49 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
GPCRs as well as the expression of one of the promiscuous G-proteins Gal5 or
Goc
16. Upon the addition of an agonist of the GPCR to the transfected cells, the
GPCR
was activated and was able, via Gal5 or Gocl6, to activate the (3 isoform of
phospholipase C, leading to an increase in inositol phosphate levels in the
cells. In
addition to the G-protein described by Offermans, chimeric G-proteins, such as
GqiS,
also exhibit promiscuous coupling of GPCRs to the phospholipase C pathway.
Therefore, the present invention includes assays that are essentially the same
as the
assays described herein using promiscuous G-proteins except that chimeric G-
proteins are used instead of promiscuous G-proteins. Chimeric G-proteins are
described in, e.g., Joshi et al, 1999, Eur. J. Neurosci. 11:383-388.
By making use of these promiscuous G-proteins, it is possible to set up
functional assays for the identification of gbla subtype-specific agonists,
even in the
absence of knowledge of the G-protein with which the GABAB receptor is coupled
in
vivo. One possibility for utilizing promiscuous G-proteins in connection with
the
GABAB receptor includes a method of identifying a gbla subtype-specific
agonist
comprising:
(a) providing gb 1 a cells that express gb2, gb 1 a, and a promiscuous
G-protein, where gb2 and gbla form a heteromer representing a functional GABAB
receptor;
(b) measuring the level of inositol phosphates in the gbla cells;
(c) exposing a portion of the gbla cells to a substance;
(d) measuring the level of inositol phosphates in the gbla cells that
have been exposed to the substance;
(e) determining the ratio of the level of inositol phosphates
measured in step (d) over the level of inositol phosphates measured in step
(b);
(f) providing gblb cells that express gb2, gblb, and a promiscuous
G-protein, where gb2 and gblb form a heteromer representing a functional GABAB
receptor;
(g) measuring the level of inositol phosphates in the gblb cells;
(h) exposing a portion of the gblb cells to the substance;
(i) measuring the level of inositol phosphates in the gblb cells that
have been exposed to the substance;
(j) determining the ratio of the level of inositol phosphates
measured in step (i) over the level of inositol phosphates measured in step
(g);
-50-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
(k) providing gblc cells that express gb2, gblc, and a promiscuous
G-protein, where gb2 and gblc form a heterorner representing a functional
GABAB
,.
receptor;
(1) measuring the level of inositol phosphates in the gblc cells;
~ (m) exposing a portion of the gblc cells to the substance;
(n) measuring the level of inositol phosphates in the gblc cells that
have been exposed to the substance;
(o) determining the ratio of the level of inositol phosphates
measured in step (n) over the level of inositol phosphates measured in step
(1);
where if the ratio measured in step (e) is at least 50% greater than the
ratio measured in steps (j) and (o) then the substance is a gbla subtype-
specific
agonist.
In particular embodiments, the ratio measured in step (e) is at least ,~
100%, 200%, or 500% greater than the ratio measured in steps (j) and (o).
. Levels of inositol phosphates can be measured by monitoring calcium
mobilization. Intracellular calcium mobilization is typically assayed in whole
cells
under a microscope using fluorescent dyes or in cell suspensions via
luminescence
using the aequorin assay. Alternatively, other assays described herein or
known in
the art for measuring calcium levels may be employed.
In methods related to those described above, rather than using changes
in inositol phosphate levels as an indication of GABAB receptor function,
potassium
currents are measured. This is feasible since the GABAB receptor, like other
metabotropic receptors, is coupled to potassium channels. Thus, one could
measure
GABAB receptor coupling to GIRK1, GIRK2, GIRK3, GIRK4, or to other potassium
channels in oocytes. GIRKs, methods of manipulating oocytes, and methods of
measuring potassium channel activity in oocytes and IiEK 293 cells are
described in
Goldin, 1992, Meth. Enzymol. 207:266-279; Quick & Lester, 1994, Meth.
Neurosci.
19:261-279; Smith et al., 1998, J. Cell Biol. 273:23321-23326; Kubo et al.,
1997,
Nature 364:802-806; Krapivinsky et al., 1995, Nature 374:135-141; Dascal et
al.,
1993, Proc. Natl. Acad. Sci. USA 90:10235-10239; Jones et al., 1998, Nature
396:674-679; White et al., 1998, Nature 396:679-682; Kaupmann et al., 1998,
Nature
396:683-687; Kuner et al., 1999, Science 283:74-77; and references cited
therein.
In a particular embodiment of the above-described method, the
promiscuous G-protein is selected from the group consisting of GoclS, Gocl6,
and
chimeric G-proteins such as GqiS. Expression vectors containing Gals or Gal6
are
-51-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
known in the art. See, e.g., Offermanns & Simon, 1995, J. Biol. Chem.
270:15175-
15180; Buhl et al., 1993, FEBS Lett. 323:132-134; Amatruda et al., 1993, J.
Biol.
Chem. 268:10139-10144.
The present invention employs cells co-expressing gb2 and gbla,
gblb, or gblc, resulting in the formation of GABAB receptors that are gbla,
gblb, or
gblc heteromers. Such cells are generally produced by transfecting cells that
do not
normally express GABAB receptors with expression vectors encoding gb2 and
gbla,
gb 1 b, or gb 1 c and then culturing the cells under conditions such that
functional
GABAB receptor heteromers of gbla/gb2, gblb/gb2, or gblc/gb2 are formed. In
this
way, recombinant host cells expressing functional GABAB receptors are
produced.
In some embodiments, the present invention may also employ cell lines derived
from
cerebellum or cortex which naturally express GABAB receptors. .Also suitable
for
use in the present invention are primary cells that have been derived from
animal
brains, e.g., rat CA1 pyramidal neurons.
Recombinant host cells for use in the present invention may be
prokaryotic or eukaryotic, including but not limited to, bacteria such as E.
coli, fungal
cells such as yeast, mammalian cells including, but not limited to, cell lines
of human,
bovine, porcine, monkey and rodent origin, and insect cells including but not
limited
to Drosophila and silkworm derived cell lines. Cells and cell lines which are
suitable
for recombinant expression, many of which are commercially available, include
but
are not limited to, L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL
1.2),
HEK293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1
(ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3
(ATCC CCL 92), NH3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC
CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), melanotroph cell
lines (e.g., tsA58 [Chronwall et al., 2000, Abstract 622.9 from the 30th
Annual
Neuroscience Meeting (Nov. 2000), New Orleans, LA]; mIL39 [Hnasko et al.,
1997,
Endocrinology 138:5589-5596]), Xenopus melanophores, and Xenopus oocytes.
In preferred embodiments, the host cells do not naturally express
GABAB receptors in order to make it easier to distinguish the effects of the
transfected subunits.
Cells that are particularly suitable for use in the present invention are
Xenopus oocytes co-expressing gb2 and gbla, gblb, or gblc, in which gb2 has
formed a functional heteromer with either gbla, gblb, or gblc. The presence of
functional heteromers in such cells can be determined by the use of assays
that
-52-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
measure coupling of functional GABAB receptors to inwardly rectifying
potassium
channels (especially the Kir3 family). In the case of Xenopus oocytes, co-
expression
of gb2 and gbla, gblb, or gblc is often effected by microinjecting RNA
encoding gli2
and RNA encoding gbla, gblb, or gblc into the oocytes rather than by
transfecting
the oocytes with expression vectors encoding gb2 and gbla, gblb, or gblc.
Microinjection of RNA into Xenopus oocytes in order to express proteins
encoded by
the RNA is well known in the art.
Also suitable for use in the present invention are cell lines that have
been isolated from animals where the cell lines express gbla heteromers but
not gblb
or gblc heteromers. One possibility is to derive such cell lines from mouse
pituitary
tumors. Such cell lines can be obtained from trangenic mice that express the
SV40
large T antigen under the control of the pro-opiomelanocortin promoter (Low et
al,
1993, J. Biol. Chem. 268:24967-24975). The use of this promoter leads to
tissue
specific expression of the large T antigen in the mouse pituitary and to the
development of intermediate lobe pituitary tumors. From such tumors,
melanotroph
cell lines can be isolated (see, e.g., the mIL39 cell line described in Hnasko
et al.,
1997, Endocrinology 138:5589-5596). Many of these melanotroph cell lines
express
gbla receptors but not gblb or gblc receptors. Thus, they are suitable for use
in the
methods of the present invention. In a variation of this method, the SV40
large T
antigen can be a temperature sensitive version known as tsA58 (see, e.g., the
mIL-
tsA58 cell line described in Chronwall et al., 2000, Abstract 622.9 from the
30'i'
Annual Neuroscience Meeting (Nov. 2000), New Orleans, LA).
In order to confirm that the melanotroph cell lines described above
express gbla receptors but not gblb or gblc receptors, one can use antisera
that are
specific for gbla, gblb, or gblc. Such antisera can be raised by standard
methods
utilizing as immunogens peptides that are unique to the gbla, gblb, or gblc
amino
acid sequences described herein. Alternatively, one can use antisera that
recognize
more than one GABAB receptor subunit, relying on the difference in molecular
weights of the subunits to distinguish the subunits on Western blots. For an
example
of such a procedure using an antiserum that recognizes gbla and gblb, see
Kaupmann
et al., 1998, Nature 396:683-687. Western blotting of melanotroph cell lines
that are
suitable for use in the methods described herein should indicate the presence
of a
band corresponding to gbla at a molecular weight of about 130 kDa and no
corresponding bands representing gblb (about 100 kDa) or gblc (about 125 kDa).
-53-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
Data presented herein (see Example 11) show that gabapentin (EC50 =
2 ~,M) inhibited up to 70-80% of the total K+-evoked Ca2+ influx via voltage
dependent-calcium channels (VD-CCs) in the mouse pituitary intermediate
melanotrope clonal mIL-tsA58 cell line. tsA58 mIL cells endogenously express
only
gbla heteromers and not also gblb heteromers or gblc heteromers. Moreover,
activity of the agonist gabapentin in tsA58 cells was dose-dependently and
completely blocked with the GABAg receptor antagonist CGP55845, and was nearly
identical to the activity of the prototypic GABAB receptor agonist baclofen in
both
extent and potency. Antisense knockdown of gbla also completely blocked
gabapentin activity while gblb antisense and control oligonucleotides had no
effect,
indicating that gabapentin inhibition of membrane Ca2+ mobilization in these
mIL
cells was dependent on a functional gbla heterorner receptor.
It has been reported recently that gabapentin is a selective agonist for !
the recombinant and neuronal GABAg gbla-gb2 heteromer subtype coupled to
GIRKs, and that it is not a partial agonist and does not block GABA activity
at gblb-
gb2 and gblc-gb2 heteromers (Ng et al., 2001, Mol. Pharm. 59:144-152).
Selective
gabapentin activation of GABAB receptors negatively coupled to VD-CCs may
account for gabapentin actions in K+ evoked Ca2+-dependent responses and this
notion is also consistent with the depressant action of gabapentin on voltage-
sensitive
calcium currents in some central neurons (Stefani et al., 1998,
Neuropharmacology
37:83-91). Indeed, the data presented herein show that gabapentin is an
agonist at
brain GABAB gbla-gb2 heteromer receptors endogenously expressed in mII. cells
and mediates robust dose-dependent inhibition, similar to that of the
prototypical
GABAB receptor agonist baclofen, of VD-CC function. These effects of
gabapentin
can be attributed to selective activity at the GABAB receptor since they could
be
blocked with GABAB receptor antagonists and selective antisense knockdown of
the
gbla subunit. This agrees with the selective activity reported at the
recombinant
GABAg receptors by Ng et al., 2001, Mol. Pharm. 59:144-152.
Recombinant GABAB receptor heteromers have been also shown to
couple to calcium channels in cultured NG108-15 cells and sympathetic neurons
(Easter et al., 2000, J. Physiol. 523P:192P; Filippov et al., 2000, J.
Neurosci. 20:2867-
2874), and activation of native receptors in rat pituitary melanotropes and
dorsal root
sensory neurons leads to inhibition of calcium currents (Hand et al., 2000,
Neurosci.
Letts. 290:49-52; Morris et al., 1998, J. Neurochem. 71:1329-1332). The
results
presented herein also indicate that, in hippocampal neurons in situ,
gabapentin
-54-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
activates GABAB receptors negatively coupled to N- and/or P/Q-type VD-CCs. But
the data presented herein do not support a predominant action of gabapentin on
L-
type Ca2+ channels since gabapentin inhibited subthreshold Ca2+ responses but
did
not prevent Ca2+ action potentials in the present experiments. Gabapentin
actions on
neuronal GABAB receptors coupled to VD-CCs is consistent with the previously
reported actions of baclofen in hippocampal neurons (Scholz & Miller, 1991, J.
Physiol. (Lond.) 444:669-686; Lambert & Wilson, 1996, J. Physiol. (Lond.)
492:115-
127). The studies cited above and the data presented herein underscore that VD-
CCs
represent a major and physiologically important effector for neuronal GABAB
receptors.
The present invention includes a method for identifying a gbla
subtype-specific agonist that comprises:
(a) determining whether a substance activates a GABAB receptor
functional response in a melanotroph cell line that express gbla receptors but
not
gblb receptors or gblc receptors;
(b) determining whether the substance activates a GABAB
receptor functional response in gblb cells;
(c) determining whether the substance activates a GABAB
receptor functional response in gblc cells;
where if the substance activates a GABAB receptor functional
response in the melanotroph cell line, but not in the gblb or gblc cells, then
the
substance is a gbla subtype-specific agonist.
In particular embodiments, the melanotroph cell line is selected from
the group consisting of mIL39 cells and mIL-tsA58 cells.
In particular embodiments, the functional response is selected from the
group consisting of: modulation of the activity of an ion channel; changes in
calcium
concentration; changes in a signal from a reporter gene whose expression is
controlled
by a promoter that is induced by interaction of an agonist with the GABAB
receptor;
and changes in membrane currents. In particular embodiments, the change in
membrane current is caused by the modulation of the activity of an inwardly
rectifying potassium current. In other embodiments, the change in membrane
current
is caused by the modulation of the activity of a voltage dependent-calcium
channel.
In particular embodiments, the gblb cells and gblc cells are cells that
do not naturally express any GABAB receptor subunits and have been transfected
-55-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
with expression vectors encoding gblb or gblc subunits as well as gb2 subunits
so as
to form functional gblb or gblc receptors.
In particular embodiments,. that the substance activates the functional
response in a melanotroph cell line via GABAB receptors is confirmed by
demonstrating that the functional response is abolished or diminished by
treatment of
the melanotroph cells with a specific inhibitor of GABAg receptors (e.g.,
CGP55845).
Also suitable for use in the present invention are GT1 neuronal cell
lines that have been developed from tumors induced iz~ a trangenic mouse by
SV40
large T antigen expression (Martinez de la Escalera et al., 1994,
Neuroendocrinology
59:420-425; Mellon et al., 1990, Neuron 5:1-10). In these cells, stimulation
of
GABAA receptors (e.g., by treatment of the cells with GABAA receptor-specific
agonists such as muscimol or benzodiazapines) leads to secretion of
gonadotrophin ~~
releasing hormone (GnRH). See Martinez de la Escalera et al., 1994,
Neuroendocrinology 59:420-425. Stimulation of these cells with gbla subtype
specific agonists will lead to an inhibition of GnRH release.
Also suitable for use in the present invention are cells that express
polypeptides that comprise amino acid sequences that are similar to, but not
exactly
the same, as the amino acid sequences disclosed herein for gb2, gbla, gblb,
and gblc.
It is generally accepted that single amino acid substitutions do not usually
alter the
biological activity of a protein (see, e.g., Molecular Biology of the Gene,
Watson et
al., 1987, Fourth Ed., The Benjamin/Cummings Publishing Co., Inc., page 226;
and
Cunningham & Wells, 1989, Science 244:1081-1085). Accordingly, suitable cells
for
the practice of the present invention include cells containing polypeptides
where one
amino acid substitution has been made in the gb2, gbla, gblb, or gblc amino
acid
sequences disclosed herein where the polypeptides still retain substantially
the same
biological activity as native gb2, gbla, gblb, and gblc. The present invention
also
includes the use of polypeptides where two, three, four, five, six, seven,
eight, nine,
ten, or more amino acid substitutions have been made in gb2, gbla, gblb, or
gblc
amino acid sequences disclosed herein where the polypeptides still retain
substantially
the same biological activity as native gb2, gbla, gblb, or gblc. In
particular, the
present invention includes embodiments where the above-described substitutions
are
conservative substitutions. In particular, the present invention includes
embodiments
where the above-described substitutions do not occur in the ligand-binding
domain of
gb2, gbla, gblb, or gblc. In particular, the present invention includes
embodiments
-56-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
where amino acid changes have been made in positions of gb2, gbla, gblb, or
gblc
that have not been evolutionarily conserved. For guidance as to which
positions of
gb2, gbla, or gblb have not been evolutionarily conserved, one of skill in the
art can
turn to disclosures such as Figure 1A of Kuner et al., 1999, Science 283:74-
77; Figure
la of Kaupmann et al., 1998, Nature 396:683-687; Figure la of Jones et al.,
1998,
Nature 396:674-679; or Figure 1 of White et al., 1998, Nature 396:679-682.
Such
figures compare the amino acid sequence of gb2 with the amino acid sequences
of
gbla or gblb. Positions in which gb2 does not share the same amino acid as
gbla or
gblb are positions that have not been evolutionarily conserved. One could
readily
create similar comparisons between gb2 and gblc in order to determine
positions in
the amino acid sequence of gblc that have not been evolutionarily conserved.
In order to produce the above-described cells co-expressing gb2 and
either gbla, gblb, or gblc, expression vectors comprising DNA encoding gb2,
gbla,
gblb, and gblc can be transfected into the cells. gb2, gbla, gblb, and gblc
can be
transfected separately, each on its own expression vector, or, alternatively,
a single
expression vector that encodes both gb2 and one of either gbla, gblb, or gblc
can be
used. Transfection is meant to include any method known in the art for
introducing
expression vectors into the cells. For example, transfection includes calcium
phosphate or calcium chloride mediated transfection, lipofection, infection
with a
retroviral construct, and electroporation. Expression of (3-lactamase,
reporter genes,
and/or promiscuous G-proteins can also be effected by transfection of
expression
vectors comprising DNA encoding these proteins.
A variety of expression vectors can be used to express recombinant
gb2, gbla, gblb, gblc, ~3-lactamase, reporter genes, and/or promiscuous G-
proteins.
Commercially available expression vectors which are suitable include, but are
not
limited to, pMClneo (Stratagene), pSGS (Stratagene), pcDNAI and pcDNAIamp,
pcDNA3, pcDNA3.1, pCR3.1 (Invitrogen, San Diego, CA), EBO-pSV2-neo (ATCC
37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224),
pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pCLneo (Promega), pTRE
(Clontech, Palo Alto, CA), pIRESneo (Clontech, Palo Alto, CA), pCEP4
(Invitrogen,
San Diego, CA), pSCl l, pSV2-dhfr (ATCC 37146), and the PT7TS oocyte
expression vector (or similar expression vectors containing the globin 5' UTR
and the
globin 3' UTR). The choice of vector will depend upon cell type used, level of
expression desired, and the like.
-57-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
For convenience of detection in the methods described herein, the
substances may be labeled, e.g., radioactively, enzymatically, fluorescently,
etc.
In particular embodiments of the methods described herein, the
binding affinity (KD) of the gbla subtype-specific agonist for heteromers of
gb2 and
gbla is determined. In particular embodiments, such binding affinity is
between 1nM
and 200 mM; preferably between 5 nM and 1 mM; more preferably between 10 nM
and 100 ~M; and even more preferably between 10 nM and 100 nM.
The conditions under which cells are exposed to substances in the
methods described herein are conditions that are typically used in the art for
the study
of protein-ligand interactions: e.g., physiological pH; salt conditions such
as those
represented by such commonly used buffers as PBS or in tissue culture media; a
temperature of about 4°C to about 55°C; incubation times of from
several minutes to
several hours. Generally, the cells are grown in suspension or tissue culture
and the p~
substances are added directly to the cells, optionally after first washing
away the
media. For embodiments using oocytes, the oocytes are generally isolated and
held in
a bath to which the substances are added at the appropriate times.
DNA encoding subunits of the GABAB receptor can be obtained by
isolating cDNA encoding the subunits from suitable cDNA libraries. A suitable
cDNA library would be, e.g., an adult human cerebellum cDNA library. Such a
library can be prepared by methods well-known in the art. Suitable
oligonucleotides
for use in isolating subtype-specific cDNAs from such a library can be
designed
based upon the DNA sequences encoding gbla, gblb, gblc, and gb2 disclosed
herein,
or based upon the DNA sequences encoding these subunits that are disclosed in
the
scientific literature.
In particular embodiments of the above-described methods, gb2 is a
polypeptide comprising an amino acid sequence selected from the group
consisting
of:
SEQ.ID.NO.:10;
Positions 9-941 of SEQ.ID.NO.:10;
Positions 35-941 of SEQ.ID.NO.:10;
Positions 36-941 of SEQ.ID.NO.:10;
Positions 38-941 of SEQ.ID.NO.:10;
Positions 39-941 of SEQ.m.N0.:10;
Positions 42-941 of SEQ.m.N0.:10;
Positions 44-941 of SEQ.m.N0.:10;
-58-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
Positions 46-941 of SEQ.ID.NO.:10;
Positions 52-941 of SEQ.ID.NO.:10;
Positions 57-941 of SEQ.ID.NO.:10;
the amino acid sequence in GenBank accession no. AF056085;
the amino acid sequence in GenBank accession no. AJ012188;
the amino acid sequence in GenBank accession no. AF058795; and
the amino acid sequence encoded by the DNA sequence deposited in
GenBank accession no. ASF074482.
In particular embodiments of the above=described methods, gb2 is a
chimeric gb2 protein. By chimeric gb2 protein is meant a contiguous
polypeptide
sequence of gb2 fused in frame to a polypeptide sequence of a non-gb2 protein.
For
example, the N-terminal domain and seven transmembrane spanning domains of
.gb2
fused at the C-terminus in frame to a G protein is a chimeric gb2 protein.
Another
example of a chimeric gb2 protein is a polypeptide comprising the FLAG epitope
fused in frame at the amino terminus of amino acids 52-941 of SEQ.ID.NO.:10.
Especially preferred forms of chimeric gb2 proteins are those in which a non-
gb2
polypeptide replaces a portion of the N-terminus of gb2.
Chimeric gbla, gblb, and gblc proteins may also be used in the
present invention. Imparticular embodiments, the chimeric gbla, gblb, or gblc
protein comprises the entire coding region of gbla, gblb, or gblc except for
the
signal sequence fused in frame to a polypeptide sequence of a non-gbla, gblb,
or
gblc protein.
In particular embodiments, the expression vector encoding gb2
comprises a nucleotide sequence selected from the group consisting of:
Positions 293-3,115 of SEQ.ID.N0.:9;
Positions 317-3,115 of SEQ.~.N0.:9;
Positions 395-3,115 of SEQ.ID.N0.:9;
Positions 398-3,115 of SEQ.ID.N0.:9;
Positions 404-3,115 of SEQ.ID.NO.:9;
Positions 407-3,115 of SEQ.ID.NO.:9;
Positions 416-3,115 of SEQ.ID.N0.:9;
Positions 422-3,115 of SEQ.117.N0.:9;
Positions 428-3,115 of SEQ.ID.N0.:9;
Positions 446-3,115 of SEQ.ID.N0.:9; and
Positions 461-3,115 of SEQ.ID.N0.:9.
-59-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
In particular embodiments of the above-described methods, gbla is a
polypeptide comprising an amino acid sequence selected from the group
consisting
of:
SEQ.117.N0.:2;
SEQ.ID.N0.:4; and
the GABABRla amino acid sequence reported in Kaupmann et al.,
1997, Nature 386:239-246;:239-246.
In particular embodiments of the methods described herein, gblb is rat
gblb and has the amino acid sequence known as GABABRlb reported in Kaupmann
et al., 1997, Nature 386: 239-246 or is human gblb and has the amino acid
sequence
encoded by the DNA sequence deposited in GenBank accession no. AJ225029 or
AJ012186 or is SEQ.ID.N0.:6.
The assays described above could be modified to identify gbla
subtype-specific inverse agonists. In such assays, gbla subtype-specific
inverse
agonists would be identified through a change in the signal that is being
assayed that
is the opposite of the change that is observed with an agonist. For example,
in the
assays using (3-lactamase reporter genes, gbla subtype-specific inverse
agonists
would lead to a decrease in (3-lactamase activity under conditions where gbla
subtype-specific agonists lead to an increase. Similarly, gbla subtype-
specific
inverse agonists can be identified by modifying the functional assays that
monitor
decreases in cAMP levels. In the case of assays for gbla subtype-specific
inverse
agonists, increases in cAMP levels would be observed.
Some of the methods described herein can be modified in that, rather
than exposing whole cells to substances, membranes can be prepared from the
cells
and those membranes can be exposed to the substances. Such a modification
utilizing
membranes rather than cells are especially suitable for those methods that
involve
measuring the binding of the substances to cells.
It may occasionally be sufficient to determine that a substance is an
agonist that is specific for the gbla heteromer as opposed to the gblb
heteromer
without also determining the agonist's effect, if any, on the gblc heteromer.
In such
cases, the methods described herein can be modified so that they are performed
essentially the same way as described above for identifying gbla subtype-
specific
agonists, but the steps that determine the substance's effect on the gblc
heteromer are
not carried out.
-60-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
Accordingly, the present invention also includes the following
methods:
A method of identifying substances that are specific for the gbla
heteromer as opposed to the gblb heteromer that comprises
(a) determining that a substance is an agonist of GABAB receptors
comprising a gbla subunit; and
(b) determining that the substance is not an agonist of GABAB
receptors comprising a gblb subunit.
In particular embodiments, the method furthermore comprises one or
more of the following steps:
(c) determining that the substance activates post-synaptic
potassium currents; .
(d) determining that the substance does not presynaptically depress
GABA inhibitory postsynaptic currents;
(e) determining that the substance is not an agonist of GABAA
receptors;
(f) determining that the substance is an agonist of GABAB
receptors that are negatively coupled to voltage dependent-calcium channels.
A method of identifying substances that are specific for the gbla
heteromer as opposed to the gblb heteromer comprising:
gblb cells;
(a) exposing a substance, separately, to gbla cells and gblb cells;
(b) quantitating the binding of the substance to the gbla cells and
where, if the amount of binding of the substance to the gbla cells is at
least 3 times greater than the amount of binding of the substance to the gblb
cells,
then the substance is specific for the gbla heteromer as opposed to the gblb
heteromer.
A method for identifying a substance that is specific for the gbla
heteromer as opposed to the gblb heteromer that comprises:
(a) providing gbla cells;
(b) exposing the gbla cells to gabapentin or pregabalin in the
presence and in the absence of a substance;
(c) measuring the binding of gabapentin or pregabalin to the gbla
heteromers in the presence and in the absence of the substance;
-61 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
where, if the amount of binding of gabapentin or pregabalin in step (c)
is less in the presence of the substance than in the absence of the substance,
then;
(d) determining whether the substance binds to gblb cells;
where, if the substance does not bind to gblb cells, then the substance
is a substance that is specific for the gbla heteromer as opposed to the gblb
heteromer.
In particular embodiments;
the gbla cells comprise an expression vector encoding gb2 and an
expression vector encoding gbla and the gbla cells are cultured under
conditions
such that gb2 and gbla are expressed and gbla heteromers are formed; and
the gblb cells comprise an expression vector encoding gb2 and an
expression vector encoding gblb and the gblb cells are cultured under
conditions
such that gb2 and gblb are expressed and gblb heteromers are formed.
In particular embodiments, the method further comprises determining
whether the substance that is specific for the gbla heteromer as opposed to
the gblb
heteromer activates a functional response of a gbla receptor.
A method for identifying a substance that is specific for the gbla
heteromer as opposed to the gblb heteromer comprising:
(a) providing a Xenopus laevis oocyte expressing gbla and gb2 so
as to form a functional gbla heteromer in the oocyte where the oocyte also
expresses
a Kir;
(b) exposing the oocyte of step (a) to a substance while monitoring
potassium ion flow across the oocyte membrane;
(c) providing a Xenopus laevis oocyte expressing gblb and gb2 so
as to form a functional gblb heteromer in the oocyte where the oocyte also
expresses
a Kir;
(d) exposing the oocyte of step (c) to the substance while
monitoring potassium ion flow across the oocyte membrane;
where if the exposure of the oocytes to the substance results in
increased potassium ion flow in step (b) but riot in step (d) then the
substance is a
substance that is specific for the gbla heteromer as opposed to the gblb
heteromer.
In particular embodiments;
(i) the oocytes of step (a) have been microinjected with RNA
encoding gb 1 a, gb2, and a Kir;
-62-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
(ii) the oocytes of step (c) have been microinjected with RNA
encoding gblb, gb2, and a Kir.
In particular embodiments, the monitoring of steps (b) and (d) is done
by patch clamp recordings.
A method for identifying a substance that is specific for the gbla
heteromer as opposed to the gblb heteromer comprising:
(a) determining whether a substance activates a GABAB receptor
functional response in gb 1 a cells;
(b) determining whether the substance activates a GABAB
receptor functional response in gblb cells;
where if the substance activates a GABAB receptor functional
response in gbla cells, but not in gblb, then the substance is specific for
the gbla
heteromer as opposed to the gblb heteromer.
In particular embodiments, the functional response is a decrease in
intracellular calcium levels.
In particular embodiments, the decrease in intracellular calcium levels
is measured by the use of a calcium indicator dye.
In particular embodiments, the calcium indicator dye is selected from
the group consisting of: fluo-3, fura-2, fluo-4, fluo-5, aequorin, calcium
green-1,
Oregon green, 488 BAPTA, SNARF-l, and indo-1.
A method for identifying a substance that is specific for the gbla
heteromer as opposed to the gblb heteromer comprising:
(a) providing gbla cells;
(b) loading the gbla cells with a calcium indicator dye;
(c) measuring a fluorescence characteristic of the calcium indicator
dye in the gbla cells in the presence and in the absence of a substance;
(d) providing gblb cells;
(e) loading the gblb cells with a calcium indicator dye;
(f) measuring a fluorescence characteristic of the calcium indicator
dye in the gblb cells in the presence and in the absence of the substance;
where if a change in fluorescent characteristic in the presence as
compared to the absence of the substance is measured in step (c) but not in
step (f)
then the substance is a substance that is specific for the gbla heteromer as
opposed to
the gb 1 b heteromer.
- 63 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
In particular embodiments, the calcium indicator dye is selected from
the group consisting of: fluo-3, fura-2, fluo-4, fluo-5, aequorin, calcium
green-1,
Oregon green, 488 BAPTA, SNARE-1, and indo-1.
In particular embodiments, the change in fluorescent characteristic is
an increase in intensity of a fluorescence emission maximum, a shift in the
wavelength of an emission maximum, or a shift in the wavelength of an
absorption
maximum.
A method for identifying a substance that is specific for the gbla
heteromer as opposed to the gblb heteromer that comprises:
(a) determining whether a substance activates a GABAB receptor
functional response in a melanotroph cell line that express gbla receptors but
not
gblb receptors;
(b) determining whether the substance activates a GABAg ~-
receptor functional response in gblb cells;
where if the substance activates a GABAg receptor functional
response in the melanotroph cell line, but not in the gblb, then the substance
is
specific for the gbla heteromer as opposed to the gblb heteromer.
In particular embodiments, the melanotroph cell line is selected from
the group consisting of mIL39 cells and mIL-tsA58 cells.
In particular embodiments, the functional response is selected from the
group consisting of: modulation of the activity of an ion channel; changes in
calcium
concentration; changes in a signal from a reporter gene whose expression is
controlled
by a promoter that is induced by interaction of an agonist with the GABAB
receptor;
and changes, in membrane currents.
In particular embodiments, the change in membrane current is caused
by the modulation of the activity of an inwardly rectifying potassium current
or the
modulation of the activity of a voltage dependent-calcium channel.
In particular embodiments, the gblb cells are cells that do not naturally
express any GABAB receptor subunits and have been transfected with expression
vectors encoding gblb subunits as well as gb2 subunits so as to form
functional gblb
receptors.
A method for identifying a substance that is specific for the gbla
heteromer as opposed to the gblb heteromer comprising:
(a) providing gbla cells that express apoaequorin;
-64-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
(b) loading the gbla cells with coelenterazine so that aequorin is
formed in the gbla cells;
(c) measuring the emission of light caused by the interaction of
calcium and the aequorin in the gbla cells in the presence and in the absence
of a
substance;
(d) providing gblb cells that express apoaequorin;
(e) loading the gblb cells with coelenterazine so that aequorin is
formed in the gblb cells;
(f) measuring the emission of light, caused by the interaction of
calcium and the aequorin in the gblb cells in the presence and in the absence
of the
substance;
where if less light emission in the presence as compared to the absence
of the substance is measured in step (c) but not in step (f) then the
substance is
specific for the gbla heteromer as opposed to the gblb heteromer. It may
occasionally be sufficient to determine that a substance is an agonist that is
specific
for the gbl~ heteromer as opposed to the gblc heteromer without also
determining the
agonist's effect, if any, on the gblb heteromer. In such cases, the methods
described
herein can be modified so that they are performed essentially the same way as
described above for identifying gbla subtype-specific agonists, but the steps
that
determine the substance's effect on the gblb heteromer are not carried out.
Accordingly, the present invention also includes the following
methods:
A method of identifying substances that are specific for the gbla
heteromer as opposed to the gblc heteromer that comprises
(a) determining that a substance is an agonist of GABAB receptors
comprising a gbla subunit; and
(b) determining that the substance is not an agonist of GABAB
receptors comprising a gblc subunit.
In particular embodiments, the method furthermore comprises one or
more of the following steps:
(c) determining that the substance activates post-synaptic
potassium currents;
(d) determining that the substance does not presynaptically depress
GABA inhibitory postsynaptic currents;
- 65 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
(e) determining that the substance is not an agonist of GABAA
receptors;
(f) determining that the substance is an agonist of GABAB
receptors that are negatively coupled to voltage dependent-calcium channels.
A method of identifying substances that are specific for the gbla
heteromer as opposed to the gblc heteromer comprising:
(a) exposing a substance, separately, to gbla cells and gblc cells;
(b) quantitating the binding of the substance to the gbla cells and
gblc cells;
where, if the amount of binding of the substance to the gbla cells is at
least 3 times greater than the amount of binding of the substance to the gblc
cells,
then the substance is specific for the gbla heteromer as opposed to the gblc
heteromer.
A method for identifying a substance that is specific for the gbla
heteromer as opposed to the gblc heteromer that comprises:
(a) providing gbla cells;
(b) exposing the gbla cells to gabapentin or pregabalin in the
presence and in the absence of a substance;
(c) measuring the binding of gabapentin or pregabalin to the gbla
heteromers in the presence and in the absence of the substance;
where, if the amount of binding of gabapentin or pregabalin in step (c)
is less in the presence of the substance than in the absence of the substance,
then;
(d) determining whether the substance binds to gblc cells;
where, if the substance does not bind to gblc cells, then the substance
is a substance that is specific for the gbla heteromer as opposed to the gblc
heteromer.
In particular embodiments;
the gbla cells comprise an expression vector encoding gb2 and an
expression vector encoding gbla and the gbla cells are cultured under
conditions
such that gb2 and gbla are expressed and gbla heteromers are formed; and
the gblc cells comprise an expression vector encoding gb2 and an
expression vector encoding gblc and the gblc cells are cultured under
conditions
such that gb2 and gblc are expressed and gblc heteromers are formed.
-66-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
In particular embodiments, the method further comprises determining
whether the substance that is specific for the gbla heteromer as opposed to
the gblc
heteromer activates a functional response of a gbla receptor.
A method for identifying a substance that is specific for the gb 1 a
heteromer as opposed to the gblc heteromer comprising:
(a) providing aXenopus laevis oocyte expressing gbla and gb2 so
as to form a functional gbla heteromer in the oocyte where the oocyte also
expresses
a Kir;
(b) exposing the oocyte of step (a) to a substance while monitoring
potassium ion flow across the oocyte membrane;
(c) providing a Xenopus laevzs oocyte expressing gblc and gb2 so
as to form a functional gblc heteromer in the oocyte where the oocyte also
expresses
a Kir;
(d) exposing the oocyte of step (c) to the substance while
monitoring potassium ion flow across the oocyte membrane;
where if the exposure of the oocytes to the substance results in
increased potassium ion flow in step (b) but not in step (d) then the
substance is a
substance that is specific for the gbla heteromer as opposed to the gblc
heteromer.
In particular embodiments;
(i) the oocytes of step (a) have been microinjected with RNA
encoding gbla, gb2, and a Kir;
(ii) the oocytes of step.(c) have been microinjected with RNA
encoding gblc, gb2, and a Kir.
In particular embodiments, the monitoring of steps (b) and (d) is done
by patch clamp recordings.
A method for identifying a substance that is specific for the gbla
heteromer as opposed to the gblc heteromer comprising:
(a) determining whether a substance activates a GABAB receptor
functional response in gbla cells;
(b) determining whether the substance activates a GABAB
receptor functional response in gblc cells;
where if the substance activates a GABAB receptor functional
response in gb 1 a cells, but not in gb 1 c, then the substance is specific
for the gb 1 a
heteromer as opposed to the gblc heteromer.
-67-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
In particular embodiments, the functional response is a decrease in
intracellular calcium levels.
In particular embodiments, the decrease in intracellular calcium levels
is measured by the use of a calcium indicator dye.
In particular embodiments, the calcium indicator dye is selected from
the group consisting of: fluo-3, furs-2, fluo-4, fluo-5, aequorin, calcium
green-l,
Oregon green, 488 BAPTA, SNARF-l, and indo-1.
A method for identifying a substance that is specific for the gbla
heteromer as opposed to the gblc heteromer comprising:
(a) providing gbla cells;
(b) loading the gbla cells with a calcium indicator dye;
(c) measuring a fluorescence characteristic of the calcium indicator
dye in the gbla cells in the presence and in the absence of a substance;
(d) providing gblc cells;
(e) loading the gblc cells with a calcium indicator dye;
(f) measuring a fluorescence characteristic of the calcium indicator
dye in the gblc cells in the presence and in the absence of the substance;
where if a change in fluorescent characteristic in the presence as
compared to the absence of the substance is measured in step (c) but not in
step (f)
then the substance is a substance that is specific for the gbla heteromer as
opposed to
the gb 1 c heteromer.
In particular embodiments, the calcium indicator dye is selected from
the group consisting of: fluo-3, furs-2, fluo-4, fluo-5, aequorin, calcium
green-1,
Oregon green, 488 BAPTA, SNARF-1, and indo-1.
In particular embodiments, the change in fluorescent characteristic is
an increase in intensity of a fluorescence emission maximum, a shift in the
wavelength of an emission maximum, or a shift in the wavelength of an
absorption
maximum.
A method for identifying a substance that is specific for the gbla
heteromer as opposed to the gblc heteromer that comprises:
(a) determining whether a substance activates a GABAB receptor
functional response in a melanotroph cell line that express gbla receptors but
not
gblc receptors;
(b) determining whether the substance activates a GABAB
receptor functional response in gblc cells;
-68-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
where if the substance activates a GABAB receptor functional
response in the melanotroph cell line, but not in the gblc cells, then the
substance is
specific for the gb 1 a heteromer as opposed to the gb 1 c heteromer.
In particular embodiments, the melanotroph cell line is selected from
the group consisting of mIL39 cells and mIL-tsA58 cells.
In particular embodiments, the functional response is selected from the
group consisting of: modulation of the activity of an ion channel; changes in
calcium
concentration; changes in a signal from a reporter gene whose expression is
controlled
by a promoter that is induced by interaction of an agonist with the GABAB
receptor;
and changes in membrane currents.
In particular embodiments, the change in membrane current is caused
by the modulation of the activity of an inwardly rectifying potassium current
or the
modulation of the activity of a voltage dependent-calcium channel.
In particular embodiments, the gblc cells are cells that do not naturally
express any GABAB receptor subunits and have been transfected with expression
vectors encoding gblc subunits as well as gb2 subunits so as to form
functional gblc
receptors.
A method for identifying a substance that is specific for the gbla
heteromer as opposed to the gblc heteromer comprising:
(a) providing gbla cells that express apoaequorin;
(b) loading the gbla cells with coelenterazine so that aequorin is
formed in the gb 1 a cells;
(c) measuring the emission of light caused by the interaction of
calcium and the aequorin in the gbla cells in the presence and in the absence
of a
substance;
(d) providing gblc cells that express apoaequorin;
(e) loading the gblc cells with coelenterazine so that aequorin is
formed in the gblc cells;
(f) measuring the emission of light caused by the interaction of
calcium and the aequorin in the gble cells in the presence and in the absence
of the
substance;
where if less light emission in the presence as compared to the absence
of the substance is measured in step (c) but not in step (f) then the
substance is
specific for the gbla heteromer as opposed to the gblc heteromer.
-69-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
It may occasionally be desirable to identify substances that axe gblb
subtype-specific or gblc subtype-specific agonists of the GABAB receptor. In
such
instances, it will be evident to one skilled in the art that the assays
described herein
can be modified to identify such gblb subtype-specific or gblc subtype-
specific
agonists. In general, this can be done by running the same assays but looking
for
agonist activity (e.g., activation of a functional response) when the
substances are
added to gblb cells or gblc cells but not when the substances are added to
gbla cells.
For example, if one were interested in identifying a gblb subtype-
specific agonist, one could run an assay such as the following:
(a) determining whether a substance activates a GABAB receptor
functional response in gbla cells;
(b) determining whether the substance activates a GABAB
receptor functional response in gblb cells;
(c) determining whether the substance activates a GABAB
receptor functional response in gblc cells;
where if the substance activates a GABAB receptor functional
response in the gblb cells, but not in the gbla or gblc cells, then the
substance is a
gblb subtype-specific agonist.
Similarly, if one were interested in identifying a gblc subtype-specific
agonist, one could run an assay such as the following:
(a) determining whether a substance activates a GABAB receptor
functional response in gbla cells;
(b) determining whether the substance activates a GABAB
receptor functional response in gblb cells;
(c) determining whether the substance activates a GABAB
receptor functional response in gblc cells;
where if the substance activates a GABAB receptor functional
response in the gblc cells, but not in the gbla or gblb cells, then the
substance is a
gblc subtype-specific agonist.
Some of the methods described herein can be modified to take
advantage of other ways of assaying for agonist activity at the GABAB
receptor.
Agonists and inverse agonists may affect the internalization or trafficking of
functional GABAB receptors. For example, in the case of the (32-adrenergic
receptor,
agonist exposure results in receptor internalization. It may be that GABAB
receptor
trafficking is modulated by agonists in a similar manner. Therefore, the
measurement
-70-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
of receptor trafficking between intracellular pools and the cytoplasmic
membrane
may be considered an assay of agonist activity. It would then be possible to
identify
agonist activity by monitoring GABAB receptor trafficking. Such trafficking
can be
monitored by whole cell immunohistochemistry and confocal microscopy or by
surface and intracellular receptor labeling and flow cytometry.
Furthermore, because the functional GABAB receptor may be a
heterodimer, then agonists and inverse agonists may be expected to alter the
ratio of
heterodimer to monomer. Hence the disruption or appearance of a heterodimer
may
be considered an additional screening assay. In this case, the monitoring of
receptor
dimerization or disappearance may be done by the appearance or disruption of
FRET.
Each of the monomers are labeled with a fluorophore such that close proximity
would
allow FRET to occur. Upon agonist binding, one might see disruption of FRET, -
indicating disruption of dimers or increase in FRET indicating more
dimerization in
the course of agonist activation.
Another possibility is to use a microphysiometer to monitor agonist
activity. The activation of many receptor pathways is associated with changes
in
extracellular or intracellular pH. Thus, GABAB receptor agonists can likely be
identified by the use of a microphysiometer to detect such changes when cells
expressing GABAB receptors are exposed to suspected agonists. The use of
microphysiometers is described in Ng et al., 1999, J. Cell. Biochem. 72:517-
527 and
Fischer et al., 1999, J. Membr. Biol. 168:39-45.
While the above-described methods are explicitly directed to testing
whether "a" substance is a gbla subtype-specific agonist of the GABAB
receptor, it
will be clear to one skilled in the art that such methods are generally used
to test
collections of substances, e.g., combinatorial libraries, collections of
natural produces,
the products of a medicinal chemistry lead optimization program, etc., to
determine
whether any members of such collections are gbla subtype-specific agonists.
Accordingly, the use of collections of substances, or individual members of
such
collections, as the substance in the above-described methods is within the
scope of the
present invention.
The methods of the present invention are generally described as
making use of "a" first cell and "a" second cell, or "a" gb 1 a cell, "a" gb
1b cell, or "a"
gblc cell. The use of the singular article is for the sake of clarity of
explanation.
Those skilled in the art will understand that the methods will usually be
practiced with
-71-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
a plurality, often thousands or even millions, of cells, as when cells are
grown in
tissue culture and then used in the methods.
"Substances" can be any substances that are generally screened in the
pharmaceutical industry during the drug development process. For example,
substances may be low molecular weight organic compounds (e.g., having a
molecular weight of less than about 1,000 daltons); RNA, DNA, antibodies,
peptides,
or proteins.
In particular embodiments of the herein-described methods, the
substance is a compound that is produced by modifying the structure of
gabapentin by
methods of medicinal chemistry. As is well known in the art, it is common to
modify
a "lead" compound having a particular pharmacological activity (e.g.,
gabapentin) by
sequentially replacing the functional groups of the compound (e.g., amine
groups,
methyl groups, carboxyl groups, phenolic groups, azido groups, etc.) with
different
functional groups and testing the modified compounds to determine what effect
such
replacement has on the compound's pharmacological properties. In such a
manner,
compounds having improved pharmacological properties such as higher target
specificity, more potent agonist or antagonist activity, or lower toxicity can
be
developed. Comparison of the structures of such modified compounds with the
pharmacological properties of the modified compounds can be especially
informative
in suggesting portions of the compounds which should be conserved and portions
which should be varied in order to arrive at a compound with optimal
properties.
Methods of medicinal chemistry such as these can be applied to gabapentin and
the
modified gabapentin-like compounds so produced can be tested in the various
methods described herein to determine if they possess desirable properties
such as,
e.g., the property of being a gbla subtype-specific agonist.
In particular embodiments of the herein-described methods, the
substance is a 3-alkyl substituted GABA analog.
gbla subtype-specific agonists identified by the above-described
methods are useful in the same manner as other gbla subtype-specific agonists,
e.g.,
gabapentin. Gabapentin has been sold since 1994 in the United States as a
treatment
for epilepsy under the name NEURONTIN~ and, in clinical trials, has been shown
to
be useful in the treatment of diabetic neuropathy and post-herpetic neuralgia.
Given
the wide range of utility displayed by gabapentin, it is clear that those
skilled in the
art would consider the gbla subtype-specific agonists identified by the
methods of the
present invention to be pharamacologically useful.
-72-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
In particular, it is expected that the gbla subtype-specific agonists
identified by the methods of the present invention will be useful in the
treatment of
such conditions as psychiatric disorders, e.g., bipolar disorders, social
phobias, and
anxiety; epilepsy and other convulsant disorders; incontinence;
gastroesophogeal
reflux; cocaine addiction; neurodegenerative disorders such as amyotrohic
lateral
sclerosis; and multiple chronic pain states such as diabetic neuropathy or
post-
herpetic neuralgia:
Methods of making gabapentin and gabapentin-like compounds are
described in U.S. Patent No. 4,024,175 and U.S. Patent No. 4,152,326.
The following non-limiting examples are presented to better illustrate
the invention.
EXAMPLE 1
Receptor expression constructs
The human gbla, gblb and gblc isoforms were obtained from human
cerebellum cDNA (Clontech, Palo Alto, CA) by PCR cloning using Advantage-HF~
PCR kit (Clontech, Palo Alto, CA) and primers based on gbla (Genbank accession
no. AJ225028) and gblb (Genbank accession no. A225029) mRNA sequences
deposited in Genbank. The cloning of the human gb2 receptor DNA (Genbank
accession no.AF069755) has been reported elsewhere (Ng et al., 1999, Genomics
56:288-295). A gb2 construct encoding a modified influenza hemagglutinin
signal
sequence (MKTIIALSYIFCLVFA; SEQ,ID.NO.:11) followed by an antigenic FLAG
(DYKDDDDK; SEQ,ID.N0.:12) epitope, herein referred to as FLAG-gb2, or a gb2
construct encoding the bovine GABAA al signal sequence
(MKKSPGLSDYLWAWTLFLSTLTGRSYGQPSLQD; SEQ,ll~.N0.:13) followed
by a c-myc epitope (EQKLISEEDLN; SEQ,ID.NO.:14), herein referred to as cmyc-
gb2, were used for transient expression in Xenopus oocytes. All GABAB receptor
DNAs were subcloned into the pT7TS Xenopus oocyte expression vector (a gift
from
Dr. Paul Krieg). It is believed that the use of other Xefaopus oocyte
expression
vectors would produce similar results. M2 muscarinic receptor and Gsa cDNAs
were
generously supplied by BioSignal (Montreal, Canada).
- 73 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
EXAMPLE 2
Materials
Gabapentin was extracted from NEURONTIN~ capsules (10 capsules,
containing 400 mg of gabapentin) in boiling ethanol. After filtration through
celite,
the solid was triturated in isopropanol (30 ml) to give 3.21 g of a solid
containing
85% gabapentin and 15% dextrose. Pure gabapentin was obtained by extraction of
the celite cake in boiling methanol, filtration of the light suspension at
room
temperature and trituration of the residue in ether to yield 1.00 g of a white
solid. The
white solid was further purified using preparative HPLC with on-line mass
spectrometric detection. The collected peak was evaporated to dryness and
reconstituted for NMR analysis. The mass spectral and NMR data were consistent
with gabapentin. Gabapentin was also obtained commercially from Sigma (St.
Louis,
MO). Gabapentin was stored at -20°C, and freshly prepared and used
immediately in
the functional assays. GABA, the active enantiomer (R)-baclofen, and CGP55845
were purchased from Sigma and Tocris Cookson, respectively. CGP71872 was
synthesized as previously reported (Belley et al., 1999, Bioorganic Med. Chem.
7:2697-2704).
Indo-1 AM, indo-1 pentapotassium salt, carboxy SNARF-1 AM,
carboxy SNARF-1, pluronic F-127, and Ca2+ calibration kits were purchased from
Molecular Probes, Inc (Eugene, OR). PTX, PenlStrep and dimethylsulfoxide were
obtained from Sigma Chemical (St. Louis, MO). The DME and the Gibco BRL
trypsin-free buffer were purchased from Life Technologies (Grand Island, NY).
Other chemicals and reagents were purchased from Fisher Scientific (St. Louis,
MO).
EXAMPLE 3
Xetiopus ooc tie expression
Xenopus oocytes were isolated and recordings performed as described
(Ng et al., 1999, J. Biol. Chem. 274:7607-7611; Hebert et al., 1994, Proc.
Royal Soc.
London, Series B 256:253-261) with the following modifications. After a brief
(10
min.) hypertonic shock with 125 mM potassium phosphate pH 6.5, oocytes were
allowed to recover in Barth's solution for 1-2 hr. cDNA constructs for various
Kir
-74-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
(Kir 3.1 or Kir 3.2) channel isoforms, human gbla, gblb and gblc, human c-
rnyc,
gb2, marine gbla and flag-gb2 constructs, human M2 muscarinic receptor, human
a2-adrenergic receptor and bovine Gsa were linearized by restriction enzymes
and
purified using Geneclean (Bio IOI). Capped mRNA was made using T7 RNA
polymerase and the mMessage mMachine (Ambion). mRNA synthesis for channel
and receptor constructs was confirmed by loading aliquots of synthesis
reactions on
denaturing formaldehyde agarose gels. Individual oocytes were injected with 5-
10 ng
of RNA (in 25-50 nL) encoding the various marine or human GABAB receptor
subunits and human Kir 3.1!3.2 or with the a2AR/Gsoi or M2 muscarinic receptor
co-
expressed with Kir 3.2. Currents were recorded after 48 hr. Standard recording
solution was KD-98, 98 mM KCI, 1 mM MgCl2, 5 mM K-HEPES, pH 7.5 unless
otherwise stated. Microelectrodes were filled with 3 M KCl and had resistances
of 1-
3 MSZ and 0.1-0.5 MSZ for voltage and current electrodes, respectively. In
addition,
current electrodes were backfilled with 1 % agarose (in 3M KCl) to prevent
leakage as
described in Hebert et al., 1994, Proc. Royal Soc. London, Series B 256:253-
261.
Recordings were made at room temperature using a Geneclamp 500 amplifier (Axon
Instruments). Oocytes were voltage clamped and perfused continuously with
different recording solutions. Currents were evoked by 500 msec voltage
commands
from a holding potential of -10 mV, delivered in 20 mV increments from -140 to
60
mV to test for inward rectifying potassium currents. Data were recorded at a
holding
potential of -80 mV and drugs were added to the bath with a fast perfusion
system.
Data collection and analysis were performed' using pCLAMP v6.0 (Axon
Instruments) and Origin v4.0 (MieroCal) software. For subtraction of
endogenous
and leak currents, records were obtained in ND-96, 96 mM NaCI, 2 mM KCI, 1 mM
MgCl2, 5 mM Na-HEPES and these were subtracted from recordings in KD-98
before further analysis.
EXAMPLE 4
Hippocampal slices and whole cell recordings
Transverse hippocampal slices were obtained from male Sprague-
Dawley rats (29-40 days postnatal) as described previously (Ouardouz &
Lacaille,
1997, J. Neurophysiol. 77:1939-1949: Chapman & Lacaille, 1999, J. Neurosci.
19:8637-8645). Animals were anesthetized with halothane prior to decapitation.
The
- 75 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
brain was removed from the skull and submerged in cold ACSF (124 mM NaCI, 2.5
rnM KCI, 1.25 mM NaH2P04, 2 mM MgS04, 2 mM CaCl2, 26 mM NaHC03 and
mM dextrose saturated with 95% 02 and 5% C02. Hippocampal slices (300,um)
were cut with a vibratome as described previously (Ouardouz & Lacaille, 1997,
J.
5 Neurophysiol. 77:1939-1949) and transferred to a holding chamber for at
least 1 hour
prior to recording. For whole cell recordings, individual slice were submerged
in a
chamber mounted on an upright microscope (Zeiss Axioskop) and perfused with
ACSF at a flow rate of 2.5 to 3.0 ml/min. All solutions were applied at room
temperature. CA1 pyramidal neurons were visualized using DIC and an infrared
10 CCD camera (Cohu 6500). Patch pipettes (4 to 8 MS2,) were filled with (in
mM): 140
K-gluconate, 5 NaCI, 2 MgCl2, 10 HEPES, 0.5 EGTA, 2 ATP-Tris, 0.4 GTP-Tris, 1
phosphocreatine, 0.1 % biocytin, pH adjusted to 7.2 to 7.3 with KOH. Whole
cell
voltage clamp recordings were made with an Axopatch 200 amplifier (Axon
instruments) with low-pass filtering at 10 kHz. Currents were digitized and
stored for
future analysis (pClamp, Axon Instruments). Voltage measurements were
corrected
for liquid junction potentials (Neher, 1992, Meth. Enzymol. 207:123-131). All
drugs
were bath applied. Baclofen and gabapentin currents were obtained as described
previously (Nurse & Lacaille, 1999, Neuropharmacol. 38:1733-1742), using
membrane potential ramps and a subtraction procedure. Monosynaptic fast GABA
IPSCs were evoked by placing an ultra-small concentric bipolar electrode
(Frederick
Haer 16-75-3) in stratum radiatum within close proximity to the pyramidal
neuron
and applying constant current pulses (0-200 ~.A, 0.05 ms). Glutamatergic non-
NMDA and NMDA EPSCs were blocked by perfusing the slices with ACSF
containing 20 ~,M CNQX (RBI) and 50 ~.M AP5 (RBI), respectively. (-)Baclofen
(RBI), gabapentin (Sigma), and the GABAB antagonist CGP55845 (Tocris) were
bath
applied.
The GABAB agonist (-)baclofen was bath applied at Vhold = -60 mV.
I-V relations were obtained during membrane potential ramps from -60 to -160
mV
over a 800 ms period, first in control ACSF and then in the presence of drug.
Averaged currents were obtained from 3 successive responses in each condition.
Agonist currents were isolated by subtracting currents in control ACSF from
currents
in the presence of the agonist (Iagonist = II-V,agonist - II-V,acsf). Chord
conductance
measures were obtained at Vm = -80 mV for agonist currents using the formula
Gm =
I / (Vm - Erev) where Erev was the observed mean reversal potential for the
agonist.
The theoretical EK was calculated using the formula EK = RT/F * In [K]o/[K]i .
-76-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
Monosynaptic fast GABAA IPSCs were evoked with ultra-small concentric bipolar
electrodes (Frederick Haer) placed in stratum radiatum within close proximity
to the
pyramidal neuron and using constant current pulses (15-90 ~.A, 0.5 ms) during
blockade of non-NMDA and NMDA synaptic transmission with 20 ~.M CNQX (RBI)
and 50~.M AP5 (RBI), respectively. Differences between two groups were
compared
using Student's t-tests (significance level set at cc = 0.05). Data are
reported as mean
~ standard deviation, unless otherwise noted.
Histological procedures for revealing biocytin-filled cells were as
described previously (Chapman & Lacaille, 1999, J. Neurosci. 19:8637-8645).
Briefly, slices were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer,
rinsed,
and stored at 4°C. Sections were treated with 1% H202, washed in 2.5%
dimethylsulfoxide and 0.1% Triton X in 0.1 M phosphate buffer, and incubated
in
avidin-biotin complex (ABC kit, Vectors lab, Burlingame CA). After rinsing,
sections were incubated in 0.05% 3'3-diaminobenzidine 4 HCI, 0.02% NiS04, 0.1
M
imidazole, and 0.001% H202 in Tris-buffered saline. Sections were then rinsed
and
cleared in xylene. Axonal and dendritic arborizations of well-filled cells
were
examined with a light microscope equipped with a CCD camera. Data are reported
as
mean +_ sem unless otherwise noted.
EXAMPLE S
mIL-tsA58 cells and culture conditions
mIL-tsA58 cells were isolated from a mouse intermediate lobe tumor.
Briefly, a strain of transgenic mice was generated that developed pituitary
tumors
because of the melanotrope-specific expression of a pro-opiomelanocortin
promoter-
Simian Virus 40 Large T antigen (temperature sensitive A58 mutant) fusion
gene.
The phenotype of these mice was similar to those described previously (Low et
al,
1993, J. Biol. Chem. 268: 24967-24975) despite the substitution of the tsA58
mutant
T antigen for wildtype. The mIL-tsA58 cells were isolated from a single tumor
using
procedures analogous to those reported for another melanotrope cell Iine
(Hnasko,
1997, Endocrinology 138: 5589-5596). They express the POMC gene and dopamine
D2 receptors. Growth rate and morphology of the cells were similar at
33°C, the
permissive temperature for tsA58, and at 37°C. These cells display a
normal mouse
karyotype after more than 60 passages. They grow either as free floating
spheres of
_ 77 _
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
tightly associated cells or can be coaxed to adhere to plastic, although they
remain
clustered under this condition as well. Few individual cells are found in
cultures, and
the clusters are difficult to dissociate enzymatically without destroying the
cells.
The mIL cells were maintained in DMEM supplemented with 10%
horse serum, 2.5% fetal bovine serum, 100 U/ml PenStrep (10,000 units
penicillin and
mg streptomycin per ml) 2 mM glutamine and 0.1 mM non-glutamine essential
amino acids under 5% C02 at 37°C.
EXAMPLE 6
10 Measurement oflCa2+~i Kinetics
Intracellular calcium ([Ca2+]i) and pH (pHi)) were measured
simultaneously using a custom-built ultra low light multi-imaging video
microscope
as described previously (Beatty et al., 1993, Endocrinology 133:972-984;
Morris et
al., 1994, In: Nuccitelli R (ed.) A Practical Guide to the Study of Ca2+ in
Living
Cells. Meth. Cell. Biol. 40:183-220). Cells grown on #00 coverslips (Corning,
Corning, NY) were simultaneously loaded with 5 ~tM indo-1/AM and 5 ~uM SNARF-
1/AM (cell permeant acetoxymethyl (AM) esters), in DMEM, 12.5%
dimethylsulfoxide, and 0.04% (w/w) Pluronic F-127 for 30 min at 37°C in
a
humidified incubator under 10% C02. After incubation the cells were washed and
left in complete medium at 37°C under 10% C02 for a 30 min recovery
period to
allow the esterase to cleave the dyes to their active, impermeant forms. Cells
were
examined within 90 min following the recovery period.
Coverslips with dye-loaded cells were placed in 1.0 ml standard
balanced salt solution (138 mM NaCI, 2 mM KCI, 2 mM MgCl2. 10 mM HEPES, 5.5
mM glucose, 2 mM CaCl2, and 50 ~.M EGTA, pH 7.4) in a microscope stage
perfusion chamber maintained at 37°C. Phase contrast and fluorescence
images of
the cells were obtained simultaneously at 405 nm, 475 nm, 575 nm and 640 nm.
Using these images as a guide, up to eight regions of interest (ROIs), each
representing a single cell, were defined for Ca2+ (405 and 475 nm images),
along
with eight corresponding regions for pH (575 and 640 nm images). After 30- to
60-
sec of video recording of the [Ca2+]i and pHi baseline activity, 1.0 ml of an
iso-
osmotic, high K+ depolarizing solution (10 mM NaCI, 130 mM KCI, 2 mM MgCl2,
10 mM HEPES, 5.5 mM glucose and 2 mM CaCl2, pH 7.4) was added for a final
_7g_
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
extracellular [K+] of 66 mM. During recordings, the addition of other
chemicals or
drugs was made directly to the bath in 100-fold excess to achieve the final
concentrations indicated. In all cases, osmolarity changed less than 1% by the
addition of these agents. Ionomycin (1 ~iM), a Ca2+ ionophore, was added at
the end
of some experiments to ensure viability of the cells as well as the
responsiveness of
the fluorescent dyes. In certain cases, baclofen, gabapentin or the GABAB
receptor
agonist CGP55845 was added 5 min prior to, or pertussis toxin IO-14 hours
prior to,
the exposure of the cells to K+ and the measurement of [Ca2+Ji and pHi.
The effects of GABAB receptor agonists and antagonists were tested
as follows: Five minutes before the start of the baseline recording, drugs
were added
to the bath to the final concentrations indicated. If both a GABAB receptor
agonist
and an antagonist were being tested, then the antagonist was added first.
After
baseline recording, the cells were depolarized with high K+ as described
above.
EXAMPLE 7
Antisense oli o~deo-x~nucleotide synthesis and administration
Two antisense deoxynucleotides (ADN) directed against either
GABABRla (gbla) or GABABRlb (gblb) isoforms of the receptor were designed
and synthesized. The gbla ADN is antisense to bases 5'-CAC CAG CAG CAG CAG
CAG -3' (a portion of SEQ.ID.NO.:11) of GABABRla (bases 4-22, accession
number AJ102185) and the gblb ADN is antisense to bases 5' ACA GGG TCC CCC
CGG GCC-3' (a portion of SEQ.II~.N0.:12) of GABABRIb (bases 4-22, accession
number AJ02186). Using the NIH BLAST search engine, these antisense sequences
showed extensive overlaps with GABABRla and GABABRlb receptor sequences
from other species, but had no significant overlaps with sequences of other
cDNAs as
of December, 2000. A mis-sense probe containing the same nucleotide base
content
as the gbla ADN, but with bases randomly assigned, was used as a control. This
mis-
sense probe, 5' CCA GCA GAC ACG CAG CAG -3' (SEQ.ID.NO.:13) has 8
overlaps with the gbla antisense probe and no known complementarity with other
sequences. All nucleotide sequences were synthesized by Integrated DNA
Technologies (Coralville, IA) as phosphorothioated derivatives.
For ADN experiments, the mIL cells were exposed to nucleotide for a
total of four days, and then tested for Ca2+ channel activity by fluorescence
video
-79-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
microscopy. The cells were first cultured in T25 flasks for 1-2 days. The
cultures
were then placed in 5 ml of serum-free medium and treated with 5 ~,l of 1.0 mM
of
the test ADN or mis-sense oligodeoxynucleotide solution (10 ,uM final
concentration). Following a 2 hr incubation at 37°C under 5% C02, 500
p,1 of fetal
horse serum and 125 ,u1 of fetal bovine serum were added to each flask. Five
,u1 of
nucleotide solution were added to each flask at 2 and 3 days of culture. Cells
were
harvested on day 3 in serum-free medium, and then plated onto cover slips in
12 well
plates. The cells were serum-deprived for 30 min at 37°C under 5% C02
to facilitate
adherence to the cover slip, then nucleotide was added to IO ~.M final
concentration.
Serum was added after an additional 30 min and Ca2+channel activity tested 24
hr
later.
EXAMPLE 8
Data analysis of fCa2+li and nHi measurements
Results were analyzed either in real time or from video tape
recordings. Twenty-five consecutive frames were averaged in real time, then
Ca2+
and pH ratio images of the microscope field (uncorrected for background or
shading
error) displayed on the RGB display at one image per second. At the same time
the
integrated gray levels of up to eight regions of interest (ROIs) were
extracted from the
25-frame average image and the data stored on an ASKII file for further
analysis. In
addition, the uncorrected ratio values for Ca2+ and pH were plotted on the VGA
screen, permitting immediate evaluation of cell viability and the effects of
treatments
on [Ca2+]i and pHi. The experiments were also recorded on 3l4 inch U-matic
video
tape as a backup and to allow analysis of other cells in the video field since
the same
data display and data extraction procedures could be applied offline.
Correction of
[Ca2+]i, using the prevailing intracellular pH, standardization, data
reduction
analysis, and statistical methods has been previously described (Moms et al.,
1994,
In: Nuccitelli R (ed.) A Practical Guide to the Study of Ca2+ in Living Cells.
Meth.
Cell. Biol. 40: 183-220).
K+-depolarization of mIL cells results in a two phase increase in
[Ca2+]i. The fast phase, which peaks within about 10 sec, is due to influx
through
high voltage activated Ca2+ channels. A second slower phase, with a lower
amplitude that peaks much later (30-60 sec), is due to release from
intracellular
-80-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
stores. Therefore the change in Ca2+ level due to membrane channel activity
was
measured as:
Percent change in [Ca2+]i = (Max Ca2+ - Min Ca2+ )/(Min Ca2+ x 100), where Max
Ca2+ _ (maximal [Ca2+]i value achieved within 10 sec following depolarization;
Min
Ca2+ = initial resting [Ca2+]i).
EXAMPLE 9
Electrophysiology and calcium ima"i~ng of hippocampal neurons in brain slices
Experiments were performed on CA1 pyramidal neurons in 300 ~.m
thick hippocarnpal slices from 25-28 day old male Sprague-Dawley rats (Nurse &
.
Lacaille, 1999, Neuropharmacol. 38:1733-1742). Slices were allowed to recover
for
at least one hour before use. The recording chamber was continuously perfused
with
oxygenated (95% 02/5°Io CO2) artificial cerebrospinal fluid (aCSF)
containing (in
mM) 124 NaCI, 2.5 KCI, 2.5 CaCl2, 26 NaHC03, 1.25 NaH2P04, 2 MgS04, 10
glucose, pH 7.35-7.4. Experiments were conducted in the presence of 0.5~M
tetrodotoxin (TTX) to block voltage-dependent Na+ channels. To block K+
channels
in the recorded neuron, patch pipettes (4-8 MSZ) were filled with a Cs-based
solution
containing (in mM) 140 CsMeS03, 1 MgCl2, 5 NaCI, 2 ATP, 0.4 GTP, 10 HEPES
and 100 ~M of the Ca2+ indicator Oregon Green BAPTA-I (Molecular Probes,
Eugene OR USA), titrated with CsOH to pH 7.25-7.28. Combined whole cell
current-
clamp recordings and confocal calcium imaging were performed from CAl
pyramidal
neurons using an Axopatch 200B amplifier (Axon Instrument, Foster City, CA)
and a
multi-photon confocal laser scanning microscope (Zeiss, LSM 510) equipped with
a
40x long-range water-immersion objective (numerical aperture 0.8).
After obtaining the whole-cell configuration, at least 20 min were
allowed for intracellular diffusion of the fluorophore. For two-photon
confocal
imaging, a tunable mode-locked Ti:Sapphire laser at 780 nm was used (5W Verdi
argon ion laser and Mira 900, Coherent). Emission was detected through a long-
pass
filter (cut-off 505 nm) and recorded to a PC using the LSM 510 software
(Zeiss). The
confocal aperture was opened fully. Linescans were taken from the soma at a
rate of
3.8 ms per line for a total scan time of 12 s (Figure 5A1). Pyramidal cells
were
activated by somatic depolarizing current injections via the recording pipette
(Figure
-81 -
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
5A2). Series resistance were monitored and compensated throughout the
experiments. For linescans, the fluorescence intensity (Fline) was averaged
over the
region of interest of the line across the cell soma. Changes in fluorescence
were
calculated for each line relative to the averaged baseline fluorescence prior
to
stimulation (Frest) and expressed as: %~F/F = [(Fline - Frest)~ Fresh x 100.
The
values were then processed with a low-pass digital filter to remove fast
transients
(Igor Pro, Wavemetrics, Lake Oswego, OR) and the peak calcium response was
determined for each linescan. Linescans and electrophysiological recordings
were
initiated manually. To compensate for small variations in the start time of
the
linescans, electrophysiological and Ca2+ responses were temporally aligned by
eye
(Figure 5A2).
EXAMPLE 10
Statistical anal, sis
The level of significance for differences between means was measured
by Fisher's test, or analysis of variance (ANOVA) followed by the Bonferroni
post-
test (GraphPad InStat, San Diego, CA).
EXAMPLE 11
Gabapentin inhibits VD-CCs in a melanotr~h cell line
Figure 11A shows typical changes in intracellular Ca2+ levels of
individual mIL cells in response to depolarization by high extracellular X+
concentration. There is a sharp and~major increase in [Ca2+~i attributed to
the
depolarization and activation of the VD-CCs followed by a slower second peak
or
shoulder due to release from intracellular stores consistent with previous
findings in
melanotropes (Morris et al., 1998, J. Neurochem. 71: 1329-1332). Figure I IB
shows
that the prototypical nonselective GABAB receptor agonist baclofen (1 p.M)
reduces
the primary Ca2+ response. Figure 11C shows that baclofen effects are reversed
by
addition of the GABAB receptor antagonist CPG55845 (3 p,M) (compare top middle
and right panels). CPG55845 has no significant effect on the response to
depolarization (compare Figures 11A and 11C). Figure 11D shows that 1 p.M
_82_
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
gabapentin action is nearly identical to 1 ~tM baclofen (compare to Figure
11B). The
gabapentin effect is also completely blocked by 3 p,M CGP55845 (compare
Figures
11D and 11E). Figure 12 shows the dose response curve for gabapentin
inhibition of
K+-evoked calcium mobilization with an ECSp 2 p.M and 70-80% of the total
channel
activity inhibited at 1 mM. Figure 13 shows that the VD-CC dependent rise in
intracellular Ca2+, is blocked by 1 ~M gabapentin, and that the gabapentin
activity
could be blocked completely and in a dose-dependent manner (30 nM-3 ~.M) by
the
GABAB receptor antagonist CGP55845. Gabapentin inhibition of calcium
mobilization was similar in magnitude (70-80%) and potency to baclofen (EC50 1
p.M) suggesting that gabapentin actions were mediated by binding to the native
gbla
heteromer.
To confirm that the inhibition of calcium mobilization owed to
selective activation of the endogenous gbla heteromer and not to an activity
of
gabapentin on the endogenous VD-CC (e.g., binding to the a28 subunit), we
tested
whether ADN treatment could selectively abolish the effect of gabapentin on K+-
evoked increase in intracellular Ca2+.
mIL cells were treated for 4 days with either gbl a or gblb ADNs or a
gbla mis-sense targeting sequence (see Example 7). These conditions were
identical
to the conditions used in previous studies which demonstrated that selective
knockdown of either gbl or gb2 subunits but not mis-sense control antisense
led to a
selective reduction in protein expression of the targeted gene product, and in
both
cases a complete loss of GABAB receptor-initiated reduction in VD-CC function.
In
this series of experiments, 10 and 30 ~M gabapentin mediated 70-80% inhibition
of
the total K+-evoked VD-CC activity (Figure 14, compare A to B and C).
Antisense
knock-down of gbla in mIL cells was accompanied by a complete block of 10 and
30
~M gabapentin activity (Figure 14, compare columns B and C to E and F). In
contrast, treatment with gblb ADN or the gbla mis-sense nucleotide was without
effect on both 10 and 30 p.M concentrations of gabapentin (compare columns B
and C
to H and I, K and L respectively). Taken together this demonstrated that
gabapentin
inhibition of calcium mobilization in mIL cells was due to activation of
gabapentin-
sensitive endogenously expressed gbla-gb2 heteromers.
-83-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
EXAMPLE 12
Gabapentin inhibition of VD-CCs via neuronal GABAB receptors in rat
hippocampal
neurons ih situ
To confirm stimulation by gabapentin at neuronal GABAB receptors
coupled to VD-CCs irz situ, we combined whole cell current clamp recordings of
pyramidal cells with multiphoton confocal calcium imaging (Figure 15A1-2) and
examined the effects of gabapentin on calcium responses evoked by somatic
current
injections in CA1 pyramidal neurons of rat hippocampal slices. In the presence
of
TTX and a K+ channel blocker (intracellular Cs), positive current pulses were
applied
to the pyramidal cell soma via the recording electrode and the evoked calcium
responses were recorded electrophysiologically (membrane potential) and
optically ~~
(fluorescence) (Figure 15A2). The amplitude of the current pulse was varied to
elicit
Ca~+ responses by sub-threshold stimulation (Figure 15B1 and 15C1) and Ca~+
spikes by supra-threshold stimulations (Figure 15B2 and 15C2). Sub-threshold
current injections induced Ca2+ responses of small amplitude and short
duration
(Figure 15B1 and 15C1), whereas supra-threshold current injections triggered
larger
and longer-lasting Ca~+ responses (Figure 15B2, 15C2) at the cell soma. In our
experimental conditions, these responses induced by somatic current injection
were
solely mediated by Ca~+ since they were totally blocked in Caz+-free aCSF (n=2
cells, data not shown).
For a given sub-threshold current injection, the membrane
depolarization and its associated Ca2+ response (traces 1 in Figure 15B1) were
significantly depressed in presence of 1 mIVI gabapentin (traces 2 in Figure
15B1).
These effects of gabapentin were reversible (data not shown). When the current
pulse
amplitude was adjusted to elicit a Ca~+ spike in the pyramidal cell (traces 1
in Figure
15B2), the same current pulse in presence of gabapentin (1mM) failed to induce
a
Ca2+ spike (traces 2 in Figure 15B2) and solely evoked a subthreshold
depolarization
and a very small Ca2+ response. However, cells were still capable of producing
Ca2+
spikes in the presence of gabapentin, since larger current injections induced
a Ca2+
spike and its associated large and long-lasting Ca2+ response at the cell soma
(traces
3 in Figure 15B2). Both the peak amplitude of the Ca2+ spike and Ca~+ response
elicited by the larger stimulation in the presence of gabapentin were not
significantly
different from those in control conditions (76.3~4.7mV and 165.3~27°Io
~F/F in
-84-
CA 02413451 2002-12-18
WO 01/98779 PCT/CA01/00909
gabapentin vs 82.9~3mV and 218.7~24% OF/F in control respectively, n=4, Figure
15B2).
As observed for gabapentin, baclofen (40 ~M) depressed in a
reversible manner the membrane depolarizations and Ca2+ responses induced by
both
sub- and supra-threshold somatic current injections (Figure 15C). As for
gabapentin,
increasing the somatic current injection in the presence of baclofen restored
both
Ca2+ spikes and Ca2+ responses to levels similar to those in control
(71.9~5.6rnV
and 167.9~61.4% ~F/F in baclofen vs 76.9~2.5mV and 254.1~28.6% t1F/F in
control
conditions; n=3, Figures 15C2 and 16D).
The inhibition of Ca2+ responses by gabapentin was dose-dependent.
The graphs on Figure 16A-B show the effects of different concentrations of
gabapentin (100 ~M to 1 mM) on membrane depolarizations and Ca2+ responses
evoked by sub- and supra-threshold current injections. To test if the
inhibitory action.
of gabapentin on Ca2+ responses was mediated by activation of GABAB receptors,
we investigated the effect of the GABAB receptor antagonist CGP55845 on
gabapentin actions. Gabapentin (2 mM) significantly reduced membrane
depolarizations and Ca2+ responses evoked by both sub- and supra-threshold
soma
current injections (Figure 17C and D). This depressant effect of gabapentin
was
blocked in the presence of 4 ~.M CGP55845, for both sub- (Figure 17A1 and C)
and
supra-threshold (Figure 17A2 and D) current injections. Similarly, the
inhibition of
Ca2+ responses by baclofen was also blocked by CGP55845 (Figure 17B-D). These
results indicate that gabapentin negatively couples to VD-CCs via GABAB
receptors
in hippocampal pyramidal neurons in situ. Taken together with the selective
activation demonstrated for gabapentin at the endogenous brain GABAB receptor
in
mTL cells, our results suggest that one possible mechanism by which gabapentin
exerts its CNS therapeutic actions is by a selective activation of neuronal
gbla-gb2
GABAB receptor heterodimers coupled to VD-CCs.
The present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the invention
in
addition to those described herein will become apparent to those skilled in
the art
from the foregoing description. Such modifications are intended to fall within
the
scope of the appended claims.
Various publications are cited herein, the disclosures of which are
incorporated by reference in their entireties.
-85-
