Note: Descriptions are shown in the official language in which they were submitted.
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Modulation of Sodium Channels in Dorsal Root Ganglia
by Snlayman Dib-Hajj and Stephen G. Waxman
FIELD OF THE INVENTION
The present invention relates to a novel tetrodotoxin resistant sodium
5 channel and related nucleotides, as well as screening assays for identifying
agents useful in treating acute or chronic pain or other hyperexcitability
states.
This application is related to U.S. Provisional Application 60/072,990, filed
January 29, 1998, U.S. Provisional Application 60/109,402 entitled
"Modulation of Sodium Channels in Dorsal Root Ganglia", filed November 20,
10 1998 and to U.S. Provision Application 60/109,666, entitled "Dii~erential
Role
of GDNF and NGF in the Maintenance of Two TTX-Resistant Sodium
Channels in Adult DRG Neurons," filed on November 20, 1998, all of which
are herein incorporated by reference.
BACKGROUND
15 A. 1~~]18~~
Voltage-gated sodium channels are a class of specialized protein
molecules that act as molecular batteries permitting excitable cells (neurons
and
muscle fibers) to produce and propagate electrical impulses. Voltage-gated Na+
channels from rat brain are composed of three subunits, the pore-forming a
20 subunit (260 KDa) and two auxiliary subunits, X31 (36 KDa) and ~i2 (33 KDa)
that may modulate the properties of the a-subunit; the a subunit is sufficient
to
form a functional channel that generates a Na current flow across the membrane
[references 1,2 as cited below]. Nine distinct a subunits have been identified
in
vertebrates and are encoded by members of an expanding gene family [3 and
25 references therein, 4-6] and respective orthologues of a number of them
have
been cloned from various mammalian species including humans. Specific a
subunits are expressed in a tissue- and developmentally-specific manner [7,8].
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Aberrant expression patterns or mutations of voltage-gated sodium channel
a-subunits underlie a number of human and animal disorders [9-13].
Voltage-gated sodium channel a-subunits consist of four domains
(D1-4) of varying internal homology but of similar predicted structure,
5 connected by three intracellular loops (L1-3): The four domains fold to form
a
channel that opens to both the cytoplasm and the extracellular space via a
pore.
The pore opens and closes depending upon the physiological state of the cell
membrane.
Each domain consists of six transmembrane segments (Sl-6) that allow
the protein to weave through the membrane with infra- and extracellular
linkers.
The linkers of SS-S6 segments of the four domains contain sequences that line
the pore of the channel, and a highly conserved subset of amino acids that
acts
as a filter to selectively allow sodium ions to traverse the channel pore into
the
cytoplasm, thus generating an electric current. The amphiphatic S4 segment, in
each of the four domains, rich in basic residues repeated every third amino
acid,
acts as a voltage sensor and undergoes a conformational change as a result of
the change in the voltage difference across the cell membrane. This in turn
triggers the conformational change of the protein to open its pore to the
extracellular Na+ ion gradient.
20 In most of the known voltage-gated sodium channel a-subunits, the
channels close and change into an inoperable state quickly (inactivate),
within a
few milliseconds, after opening of the pore (activation); SNS-type channels,
on
the other hand, inactivate slowly and require a greater voltage change to
activate. L3, the loop that links domains D3 and D4, contains a tripeptide
which acts as an intracellular plug that closes the pore after activation,
thus
inducing the channel to enter the inactive state. After inactivation, these
channels further undergo conformational change to restore their resting state
and
become available for activation. This period is referred to as recovery from
inactivation (repriming). Different channels reprime at different rates, and
repriming in SNS is relatively rapid.
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Based on amino acid similarities, the voltage-gated sodium channel
family has been further subdivided into two subfamilies [14J. Eight of the
nine
cloned channels belong to subfamily 1. They share many structural features,
particularly in their S4 hansmembrane segments. However, some of them have
5 been shown to have distinct kinetic properties of inactivation and
repriming.
Only a single channel of subfamily 2, also referred to as atypical channels,
has
been identified in human, rat and mouse tissues. This subfamily is primarily
characterized by reduced numbers of basic residues in its S4 segments, and
thus
is predicted to have different voltage-dependence compared to subfamily 1. The
10 physiological function of subfamily 2 channels is currently unknown because
its
electrophysiological properties have not yet been elucidated.
The blocking of voltage-gated sodium channels by tetrodotoxin, a
neurotoxin, has served to functionally classify these channels into sensitive
(TTX-S) and resistant (TTX-R) phenotypes. Two mammalian TTX-R channels
15 have so far been identified, one specific to the cardiac muscle and to very
limited areas of the central nervous system (CNS) and the second, SNS, is
restricted to peripheral neurons (PNS) of the dorsal root ganglia (DRG) and
trigeminal ganglia. Specific amino acid residues that confer resistance or
sensitivity to TTX have been localized to the ion selectivity filter of the
channel
20 pore. The SNS channel is also described in International Patent Application
WO 97/01577.
B. Role of Sodium Channels in Disease States
Because different Na+ channel a-subunit isotypes exhibit different
kinetics and voltage-dependence, the firing properties of excitable cells
depend
25 on the precise mixture of channel types that they express. Mutants of the
cardiac and skeletal muscle a-subunit have been shown to cause a number of
muscle disorders. Some examples are as follows: A change of a single basic
amino acid residue in the S4 of the skeletal muscle channel is sufficient to
change the kinetic properties of this channel and induce a disease state in
many
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patients. A tripeptide deletion in L3 of the cardiac channel, proximal to the
inactivation gate, induces a cardiac disorder called Long QT syndrome. A
single amino acid change in the SS-S6 linker of domain 1 of Scn8a, the region
lining the pore of the channel, causes the mouse mutant "jolting". The total
loss
5 of this channel by a different mutation causes motor end plate "med" disease
in
mice. This mutation is characterized by loss of motor neuron stimulation of
the
innervated muscle.
C. Sodium Channels and Pain
Axonal injury (injury to nerve fibers, also called axons) can produce
10 chronic pain (termed neuropathic pain). A number of studies have
demonstrated
altered excitability of the neuronal cell body and dendrites after axonal
injury
[15-17], and there is evidence for a change in Na+ channel density over the
neuronal cell body and dendrites following axonal injury [18-20]. The
expression of abnormal mixtures of different types of sodium channels in a
15 neuronal cell can also lead to abnormal firing [13], and can contribute to
hyperexcitability, paresthesia or pain.
Rent studies from our group on rat sensory DRG neurons have
demonstrated a dramatic change in the expression profile of TTX-R and TTX-S
currents and in a number of mRNA transcripts that could encode the channels
20 responsible for these currents in DRG neurons following various insults [21-
23].
We have, for example, shown an attenuation of the slowly inactivating, TTX-R
current and simultaneous enhancement of the rapidly inactivating, TTX-S Na+
currents in identified sensory cutaneous afferent neurons following axotomy
[21]. We also have shown a loss of TTX-S, slowly repriming current and
25 TTX-R current and a gain in TTX-S, rapidly repriming current in nociceptive
(pain) neurons following axotomy [22], down-regulation of SNS transcripts and
a simultaneous up-regulation of a-III Transcripts [23]. Also associated with
axotomy is a moderate elevation in the levels of aI and aII mRNAs [24].
These changes in the sodium channel profile appear to contribute to abnormal
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firing that underlies neuropathic pain that patients suffer following axonal
injury.
Inflammation, which is also associated with pain (termed inflammatory
pain), also causes alteration in the sodium current profile in nociceptive DRG
5 neurons. Inflammatory modulators up-regulate TTX-R current in small C-type
nociceptive DRG neurons in culture [25,26]. The rapid action of these
modulators suggests that their action include posth~anslational modification
of
existing TTX-R channels. We have now determined that inflammation also
increases a TTX-R Na+ current and up-regulates SNS transcripts in C-type DRG
10 neurons [58]. This data suggests that changes in the sodium current profile
contribute to inflammation evoked-pain.
D. Therayies for Chronic Pain:
A variety of classes of drugs (anticonvulsants such as phenytoin and
carbamazepine; anti-arrhythmics such as mexitine; local anesthetics such as
15 lidocaine) act on Na+ channels. Since the various Na+ channels produce
sodium
currents with different properties, selective blockade or activation (or other
modulation) of specific channel subtypes is expected to be of significant
therapeutic value. . Moreover, the selective expression of certain a-subunit
isoforms (PNI, SNS; NaN) in specific types of neurons provides a means for
20 selectively altering their behavior.
Nociceptive neurons of the DRG are the major source of the PNS
TTX-R Na+ current. Thus, the Na+ channels producing TTX-R currents
pmvide a relatively specific target for the manipulation of pain producing
neumns. The molecular structure of one TTX-R channel in these DRG
25 neurons, SNS, has been identified but, prior to our research, it has not
been
determined whether there are other TTX-R channels in these neurons. If such
channels could be identified, they would be ideal candidates as target
molecules
that are preferentially expressed in nociceptive neurons, and whose modulation
would attenuate pain transmission.
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SUMMARY OF THE INVENTION
The present invention includes an isolated nucleic acid which encodes a
voltage gated Na+ channel that is preferentially expressed in dorsal root
ganglia
or trigeminal ganglia (the NaN channel). (In our preceding U.S. Provisional
Application 60/072,990, this NaN channel was referred to by its previous name
"NaX.") In a preferred embodiment, the isolated nucleic acid comprises the
sequence shown in Fig. 1, Fig. 7A, Fig. 8A, allelic variants of said sequences
or
nucleic acids that hybridize to the foregoing sequences under stringent
conditions.
In another embodiment, the invention includes an expression vector
comprising an isolated nucleic acid which encodes the voltage gated Na+
channel that is preferentially expressed in dorsal root ganglia or trigeminal
ganglia either alone or with appropriate regulatory and expression control
elements. In a preferred embodiment, the expression vector comprises an
15 isolated nucleic acid having the sequence shown in Fig. 1, Fig. 7A, Fig.
8A,
allelic variants of said sequences or nucleic acids that hybridize to the
foregoing
sequences under stringent conditions.
The present invention further includes a host cell transformed with an
expression vector comprising an isolated nucleic acid which encodes a voltage
gated Na+ channel that is preferentially expressed in dorsal root ganglia or
trigeminal ganglia with appropriate regulatory and expression control
elements.
In a preferred embodiment, the expression vector comprises an isolated nucleic
acid having the sequence shown in Fig. 1, Fig. 7A, Fig. 8A, allelic variants
of
said sequences or nucleic acids that hybridize to the foregoing sequences
under
stringent conditions.
The present invention also includes an isolated voltage gated Na+
channel that is preferentially expressed in dorsal root ganglia or trigeminal
ganglia. In a preferred embodiment, the channel has the amino acid sequence of
Figs. 2, 7B or 8B or is encoded by a nucleic acid having the sequence shown in
Figs. 1, 7A or 8A, allelic variants of said sequences or nucleic acids that
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hybridize to the foregoing sequences under stringent conditions. Peptide
fragments of the channel are also included.
Another aspect of the invention is a method to identify an agent that
modulates the activity of the NaN channel, comprising the steps of bringing
the
agent into contact with a cell that expresses the Na+ channel on its surface
and
measuring depolarization, or any resultant changes in the sodium current. The
measuring step may be accomplished with voltage clamp measurements, by
measuring depolarization, the level of intracellular sodium or by measuring
sodium influx.
Another aspect of the invention is a method to identify an agent that
modulates the transcription or translation of mRNA encoding the NaN channel.
The method comprises the steps of bringing the agent into contact with a cell
that expresses the Na+ channel on its surface and measuring the resultant
level
of expression of the Na+ channel.
The invention also includes a method to treat pain, paraesthesia and
hyperexcitability phenomena in an animal or human subject by administering an
effective amount of an agent capable of modulating, such as by inhibiting or
enhancing, Na+ current flow through NaN channels in DRG or trigeminal
neurons. The method may include administering an effective amount of an
20 agent capable of modulating the transcription or translation of mRNA
encoding
the NaN channel.
Another aspect of the invention is an isolated nucleic acid that is
antisense to the nucleic acids described above. In a preferred embodiment, the
antisense nucleic acids are of sufficient length to modulate the expression of
NaN channel mRNA in a cell containing the mRNA.
Another aspect of the invention is a scintigraphic method to image the
loci of pain generation or provide a measure the level of pain associated with
DRG or trigeminal neuron mediated hyperexcitability in an animal or human
subject by administering labeled monoclonal antibodies or other labeled
ligands
specific for the NaN Na+ channel.
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Another aspect of the invention is a method to identify tissues, cells and
cell types that express the NaN sodium channel. This method comprises the
step of detecting NaN on the cell surface, or en route to the cell surface, or
the
presence of NaN encoding mRNA.
5 The present invention further includes a method of producing a
transformed cell that expresses an exogenous NaN encoding nucleic acid,
comprising the step of transforming the cell with an expression vector
comprising an isolated nucleic acid having the sequence shown in Figs. 1, 7A
or
8A, allelic variants of said sequences or nucleic acids that hybridize to the
10 foregoing sequences under stringent conditions, together with appropriate
regulatory and expression control elements. The invention also includes a
method of producing recombinant NaN protein, comprising the step of culturing
the transfornned host under conditions in which the NaN sodium channel or
protein is expressed, and recovering the NaN protein.
15 The invention also includes an isolated antibody specific for the NaN
channel or polypeptide fragment thereof. The isolated antibody may be labeled.
Another aspect of the invention includes a therapeutic composition
comprising an effective amount of an agent capable of decreasing rapidly
repriming sodium current flow in axotomized, inflamed or otherwise injured
20 DRG neurons or in normal DRG neurons that are being driven to fire at high
frequency. The invention also includes a method to treat acute pain or acute
or
chronic neuropathic or inflammatory pain and hyperexcitability phenomena in
an animal or a human patient by administering the therapeutic composition.
The present invention also includes a method to screen candidate
25 compounds for use in treating pain and hyperexcitability phenomena by
testing
their ability to alter the expression or activity of an NaN channel mRNA or
protein in axotomized, inflamed or otherwise injured DRG neurons.
BRIEF DESCRIPTION OF THE DRAWING FIGURES
Fig. 1 shows the sequence of the rat NaN cDNA.
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Fig. 2 shows the putative amino acid sequence of the rat NaN cDNA.
Predicted transmembrance segments of domains I - IV are underlined. The
amino acid serine "S" in DI-SS2, implicated in the TTX-R phenotype, is in bold
face type.
5 Fig. 3 presents a schematic diagram of predicted secondary structure of the
NaN a-subunit.
Fig. 4 shows the results of RT-PCR analysis for a-NaN in extracts of various
tissues using NaN specific primers. NaN is abundantly expressed in dorsal root
and trigeminal ganglia. Low levels of NaN are detected in cerebral hemisphere
10 and retina tissues. No detectable NaN signal is seen in cerebellum, optic
nerve,
spinal cord, sciatic nerve, superior cervical ganglia, skeletal muscle,
cardiac
muscle, adrenal gland, uterus, liver and kidney.
Fig. 5 shows the tissue distribution of a-NaNby in situ hybridization. A.
Trigeminal ganglion neurons show moderate-to-high hybridization signal. B.
15 Dorsal root ganglion neurons show moderate-to-high hybridization signal in
small neurons. Hybridization signal is attenuated in large neurons (arrow). C.
Sense probe shows no signal in DRG neurons. D., E., and F. No hybridization
signal is seen in spinal cord, cerebellum and liver. All tissues are from
adult
Sprague-Dawley rat. Scale bars = 50 micrometer.
20 Fig. 6 shows the predicted lengths of domain I amplification products of
rat
a-subunits and their subunit-specific restriction enzyme profile.
Figs. 7A-7B set forth the nucleotide and amino acid sequences of the marine
NaN.
Fig. 8A-Fig.BB. Fig.BA is a partial nucleotide sequence of the human NaN.
25 Fig. 8B is a partial amino acid sequence of the human NaN protein.
Fig. 9 shows cultures of DRG neurons obtained from L4/5 ganglia of adult
rats that were reacted with antibody to NaN and then processed for
immunofluorescent localization. a.,b. NaN immunostaining is prominent within
the cell bodies of DRG neurons. c. NaN is present in the neuritic outgrowths,
as
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well as the cell bodies, of DRG neurons. d., d'. Nomarski (d.) and fluorescent
(d'.) images of a neuron that does not express NaN protein.
Fig. 10 shows the location of Scnl la and related genes on distal mouse
chromosome 9. (A) Haplotypes from the Jackson BSS backcross. Black boxes
5 represent C57BL/6J alleles and white boxes represent SPRET/Ei alleles. The
number of animals with each haplotype is given below each column. Missing
data was inferred finm adjacent data when typing was ambiguous. (B) Map of
distal chromosome 9 based on data in (A). Positions of ScnSa and ScnlOa from
the MGD consensus map and the locations of the human orthologs are
10 indicated. Numbers are cM positions on the consensus map
(http://www.informatics jax.org/bin/ccr/index).
DETAILED DESCRIPTION
The present invention relates to a novel gene that we have discovered,
called NaN. NaN encodes a previously unidentified protein, referred to herein
15 as NaN, that belongs to the a-subunit voltage-gated sodium channel protein
family and that produces a TTX-R sodium current. Such channels underlie the
generation and propagation of impulses in excitable cells like neurons and
muscle fibers. NaN is a novel sodium channel, with a sequence distinct finm
other, previously identified, channels. The preferential expression of NaN on
20 sensory, but not other neurons, makes it a very useful target for
diagnostic
and/or therapeutic uses in relation to acute and/or chronic pain pathologies.
Definitions:
This specification uses several technical terms and phrases which are
intended to have the following meanings:
25 The phrase "modulate" or "alter" refers to up- or down-regulating the
level or activity of a particular receptor, ligand or current flow. For
example an
agent might modulate Na+ current flow by inhibiting (decreasing) or enhancing
(increasing) Na+ current flow. Similarly, an agent might modulate the level of
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expression of the NaN sodium channel or the activity of the NaN channels that
are expressed.
The phrase "sodium current" or "Na+ current" means the flow of sodium
ions across a cell membrane, often through channels (specialized pmtein
S molecules) that are specifically permeable to certain ions, in this case
sodium
ions.
The phrase "voltage gated" means that the ion channel opens when the
cell membrane is in a particular voltage range. Voltage-sensitive sodium
channels open when the membrane is depolarized. They then permit Na* ions to
10 flow into the cell, producing further depolarization. This permits the cell
to
generate electrical impulses (also known as "action potentials'.
The phrase "rapidly repriming" means that the currents recover from
inactivation more rapidly than do such currents in most other voltage gated
sodium channel family members.
15 The terms "TTX-R" and '"TTX-S" means that the flow of current
through a cell membrane is, respectively, resistant or sensitive to
tetrodotoxin (a
neurotoxin produced in certain species) at a concentration of about I00 nM.
The phrase "peripheral nervous system (PNS)" means the part of the
nervous system outside of the brain and spinal cord, i.e., the spinal roots
and
20 associated ganglia such as dorsal root ganglia (DRG) and trigeminal
ganglia,
and the peripheral nerves.
The phrase "inhibits Na+ current flow" means that an agent has
decreased such current flow relative to a control cell not exposed to that
agent.
A preferred inhibitor will selectively inhibit such current flow, without
affecting
25 the current flow of other sodium channels; or it will inhibit Na+ current
in the
channel of interest to a much larger extent than in other channels.
The phrase "enhances Na* current flow" means that an agent has
increased such current flow relative to a control cell not exposed to that
agent.
A preferred agent will selectively increase such current flow, without
affecting
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the current flow of other sodium channels; or it will increase Na+ current in
the
channel of interest to a much larger extent than in other channels.
The phrase "specifically hybridizes" refers to nucleic acids which
hybridize under highly stringent or moderately stringent conditions to the
5 nucleic acids encoding the NaN sodium channel, such as the DNA sequence of
Figs. 1, 7A or 8A.
The phrase "isolated nucleic acid" refers to nucleic acids that have been
separated from or substantially purified relative to contaminant nucleic acids
encoding other polypeptides. "Nucleic acids" refers to all fortes of DNA and
10 RNA, including cDNA molecules and antisense RNA molecules.
The phrase "RT-PCR" refers to the process of reverse transcription of
RNA (RT) using the enzyme reverse transcriptase, followed by the
amplification of certain cDNA templates using the polymerise chain reaction
(PCR); PCR requires generic or gene-specific primers and thermostable DNA
15 polymerise, for example, Taq DNA polymerise.
The phrase "preferentially expressed" means that voltage gated Na+
channel is expressed in the defined tissues in detestably greater quantities
than
in other tissues. For instance, a voltage gated Na+ channel that is
preferentially
expressed in dorsal root ganglia or trigeminal ganglia is found in detestably
20 greater quantities in dorsal root ganglia or trigeminal ganglia when
compared to
other tissues or cell types. The quantity of the voltage gated Na+ channel may
be detected by any available means, including the detection of specific RNA
levels and detection of the channel protein with specific antibodies.
25 The present invention relates to a previously unidentified, voltage-gated
sodium channel a-subunit (NaN), predicted to be TTX-R, voltage-gated, and
preferentially expressed in sensory neurons innervating the body (dorsal root
ganglia or DRG) and the face (trigeminal ganglia). The predicted open reading
frame (ORF), the part of the sequence coding for the NaN protein molecule, has
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been determined with the putative amino acid sequence from different species
(rat, mouse, human) presented in Figs. 2, 7B and 8B.
All of the relevant landmark sequences of voltage-gated sodium
channels are present in NaN at the predicted positions, indicating that NaN
belongs to the sodium channel family. But NaN is distinct from all other
previously identified Na channels, sharing a sequence identity of less than
53%
with each one of them. NaN is distinct from SNS, the anly other TTX-R Na+
channel subunit that has been identified, until our discovery, in PNS. We have
identified and cloned NaN without using any primers or probes that are based
10 upon or specific to SNS. Moreover, NaN and SNS share only 47% similarity of
their predicted open reading fi~ame (ORF), comparable to the limited
similarity
of NaN to all subfamily 1 members.
The low sequence similarity to existing a-subunits clearly identifies
NaN as a novel gene, not simply a variant of an existing channel. Sequence
15 variations compared to the other voltage-gated channels indicate that NaN
may
be the prototype of a novel and previously unidentified, third class of TTX-R
channels that may possess distinct properties compared to SNS. NaN and SNS,
which are present in nociceptive DRG and trigeminal neurons, may respond to
pharmacological interventions in different ways. The preferential expression
of
20 NaN in sensory DRG and trigeminal neurons provides a target for selectively
modifying the behavior of these nerve cells while not affecting other nerve
cells
in the brain and spinal cord. A further elucidation of the properties of NaN
channels will be important to understand more fully the effects of drugs
designed to modulate the function of the "TTX-R" currents which are
25 characteristic of DRG nociceptive neurons and which contribute to the
transmission of pain messages, and to abnormal firing patterns after nerve
injury
and in other painful conditions.
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Nucleic acid molecules of the invention include the nucleotide
sequences set forth in Fig. 1, Fig. 7A, Fig. 8A as well as nucleotide
sequences
that encode the amino acid sequences of Fig. 2, Fig. 7B and Fig. 8B. Nucleic
acids of the claimed invention also include nucleic acids which specifically
hybridize to nucleic acids comprising the nucleotide sequences set forth in
Fig.
1, Fig. 7A or 8A or nucleotide sequences which encode the amino acid
sequences of Fig. 2, Fig. 7B or Fig.BB. A nucleic acid which specifically
hybridizes to a nucleic acid comprising that sequence remains stably bound to
said nucleic acid under highly stringent or moderately stringent conditions.
Stringent and moderately stringent conditions are those commonly defined and
available, such as those defined by Sambrook et al. [59] or Ausubel et al.
[60].
The precise level of stringency is not important, rather, conditions should be
selected that provide a clear, detectable signal when specific hybridization
has
occurred.
Hybridization is a function of sequence identity (homology), G+C
content of the sequence, buffer salt content, sequence length and duplex melt
temperature (T[m]) among other variables. See, Maniatis et al.[62]. With
similar sequence lengths, the buffer salt concentration and temperature
provide
useful variables for assessing sequence identity (homology) by hybridization
techniques. For example, where there is at least 90 percent homology,
hybridization is commonly carried out at 68° C in a buffer salt such as
6XSCC
diluted from 20XSSC. See Sambrook et al. [59]. The buffer salt utilized for
final Southern blot washes can be used at a low concentration, e.g., O.1XSSC
and at a relatively high temperature, e.g., 68° C, and two sequences
will form a
25 hybrid duplex (hybridize). Use of the above hybridization and washing
conditions together are defined as conditions of high stringency or highly
stringent conditions. Moderately stringent conditions can be utilized for
hybridization where two sequences share at least about 80 percent homology.
Here, hybridization is carried out using 6XSSC at a temperature of about
50-55° C. A final wash salt concentration of about 1-3XSSC and at a
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temperature of about 60-68° C are used. These hybridization and washing
conditions define moderately stringent conditions.
In particular, specific hybridization occurs under conditions in which a
high degree of complementarity exists between a nucleic acid comprising the
5 sequence of an isolated sequence and another nucleic acid. With specific
hybridization, complementarity will generally be at least about 70%, 75%, 80%,
85%, preferably about 90-100%, or most preferably about 95-100%.
As used herein, homology or identity is determined by BLAST (Basic
Local Alignment Search Tool) analysis using the algorithm employed by the
10 programs blastp, blasts, blasts, tblastn and tblasta (Karlin et al. Proc.
Natl.
Acad. Sci. USA 87: 2264-2268 (1990) and Altschul, S. F. J. Mol. Evol. 36:
290-300(1993), both of which are herein incorporated by reference) which are
tailored for sequence similarity searching. The approach used by the BLAST
program is to first consider similar segments between a query sequence and a
15 database sequence, then to evaluate the statistical significance of all
matches
that are identified and finally to summarize only those matches which satisfy
a
preselected threshold of significance. For a discussion of basic issues in
similarity searching of sequence databases, see Altschul et al. (Nature
Genetics
6: 119-129 (1994)) which is herein incorporated by reference. The search
20 parameters for histogram, descriptions, alignments, expect (i.e., the
statistical
significance threshold for reporting matches against database sequences),
cutoff, matrix and filter are at the default settings. The default scoring
matrix
used by blastp, blastz, tblastn, and tblastx is the BLOSUM62 matrix
(Henikoff et al. Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992), herein
25 incorporated by reference). For blasts, the scoring matrix is set by the
ratios of
M (i.e., the reward score for a pair of matching residues) to N (i.e., the
penalty
score for mismatching residues), wherein the default values for M and N are 5
and -4, respectively.
The nucleic acids of the present invention can be used in a variety of
30 ways in accordance with the present invention. For example, they can be
used
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as nucleic acid probes to screen other cDNA and genomic DNA libraries so as
to select by hybridization other DNA sequences that encode homologous NaN
sequences. Contemplated nucleic acid probes could be RNA or DNA labeled
with radioactive nucleotides or by non-radioactive methods (for example,
S biotin). Screening may be done at various stringencies (through manipulation
of the hybridization Tm, usually using a combination of ionic strength,
temperature and/or presence of formamide) to isolate close or distantly
related
homologs. The nucleic acids may also be used to generate primers to amplify
cDNA or genomic DNA using polymerase chain reaction (PCR) techniques.
10 The nucleic acid sequences of the present invention can also be used to
identify
adjacent sequences in the genome, for example, flanking sequences and
regulatory elements of NaN. The nucleic acids may also be used to generate
antisense primers or constructs that could be used to modulate the level of
gene
expression of NaN. The amino acid sequence may be used to design and
15 produce antibodies specific to NaN that could be used to localize NaN to
specific cells and to modulate the function of NaN channels expressed on the
surface of cells.
Vectors and Transform~e~~ Host Cells:
The present invention also comprises recombinant vectors containing and
20 capable of replicating and directing the expression of nucleic acids
encoding a
NaN sodium channel in a compatible host cell. For example, the insertion of a
DNA in accordance with the present invention into a vector using enzymes
such as T4 DNA ligase, may be performed by any conventional means. Such an
insertion is easily accomplished when both the DNA and the desired vector
25 have been cut with the same restriction enzyme or enzymes, since
complementary DNA termini are thereby produced. If this cannot be
accomplished, it may be necessary to modify the cut ends that are produced by
digesting back single-stranded DNA to produce blunt ends, or by achieving the
same result by filling in the single-stranded termini with an appropriate DNA
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polymerase. In this way, blunt-end ligation may be carried out. Alternatively,
any site desired may be produced by ligating nucleotide sequences (linkers)
onto the DNA termini. Such linkers may comprise specific oligonucleotide
sequences that encode restriction site recognition sequences.
5 Any available vectors and the appropriate compatible host cells may be
used [59, 60]. Commercially available vectors, for instance, those available
from New England Biolabs Inc., Promega Corp., Stratagene Inc. or other
commercial sources are included.
The transformation of appropriate cell hosts with an rDNA (recombinant
10 DNA) molecule of the present invention is accomplished by well known
methods that typically depend on the type of vector used and host system
employed. Frog oocytes can be injected with RNA and will express channels,
but in general, expression in a mammalian cell line (such as HEK293 or CHO
cells) is preferred. With regard to transformation of prokaryotic host cells,
15 electmporation and salt treatment methods are typically employed, see, for
example, Cohen et al. [6l ]; and [62]. With regard to transformation of
vertebrate cells with vectors containing rDNAs, electroporation, cationic
lipid or
salt treatment methods are typically employed [63, 64].
Successfully transformed cells, i.e., cells that contain an rDNA molecule
20 of the present invention, can be identified by well known techniques. For
example, cells resulting from the introduction of an rDNA of the present
invention can be cloned to produce single colonies. Cells from those colonies
can be harvested, lysed and their DNA content examined for the presence of the
rDNA using conventional methods [65, 66] or the proteins produced from the
25 cell assayed via an immunological method. If tags such as green fluorescent
protein are employed in the construction of the recombinant DNA, the
transfected cells may also be detected in vivo by the fluorescence of such
molecules by cell sorking.
For transient expression of recombinant channels, transformed host cells
30 for the measurement of Na+ current or intracellular Na+ levels are
typically
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prepared by co-transfecting constructs into cells such as HEK293 cells with a
fluorescent reporter plasmid (such as pGreen Lantern-1, Life Technologies,
Inc.) using the calcium-phosphate precipitation technique [27]. HEK293 cells
are typically grown in high glucose DMEM (Life Technologies, Inc)
5 supplemented with 10% fetal calf serum (Life Technologies, Inc). After 48
hrs,
cells with green fluorescence are selected for recording [28].
For preparation of cell lines continuously expressing recombinant
channels, the NaN construct is cloned into other vectors that carry a
selectable
marker in mammalian cells. Transfecdons are carried out using the calcium
phosphate precipitation technique [27J. Human embryonic kidney (HEK-293),
Chinese hamster ovary (CHO) cells, derivatives of either or other suitable
cell
lines are grown under standard tissue culture conditions in Dulbeccos's
modified
Eagle's medium supplemented with 10% fetal bovine serum. The calcium
phosphate-DNA mixture is added to the cell culture medium and left for 15-20
hr, after which time the cells are washed with fresh medium. After 48 hrs,
antibiotic (G418, Geneticin, Life Technologies) is added to select for cells
which have acquired neomycin resistance. After 2-3 weeks in 6418, 10-20
isolated cell colonies are harvested using sterile l4ml pipette tips. Colonies
are
grown for another 4-7 days, split and subsequently tested for channel
expression
using whole-cell patch-clamp recording techniques and RT-PCR.
ethod ; f Measuring Na+ Current Flow:
Na+ currents are measured using patch clamp methods [29], as described
by Rizzo et al. [30] and Dib-Hajj et al. [28J. For these recordings data are
acquired on a Macintosh Quadra 950 or similar computer, using a program such
as Pulse (v 7.52, HEKA, German). Fire polished electrodes typically (0.8-1.5
MVO are fabricated from capillary glass using a Sutter P-87 puller or a
similar
instrument. In the most rigorous analyses, cells are usually only considered
for
analysis if initial seal resistance is <5 Gohm, they have high leakage
currents
(holding current <0.1 nA at -80 m~, membrane blebs, and an access resistance
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<5 Mohm. Access resistance is usually monitored throughout the experiment
and data are not used if resistance changes occur. Voltage errors are
minimized
using series resistance compensation and the capacitance artifact is canceled
using computer controlled amplifier circuitry or other similar methods. For
comparisons of the voltage dependence of activation and inactivation, cells
with
a maximum voltage ermr of tlOmV after compensation are used. Linear leak
subtraction is usually used for voltage clamp recordings. Membrane currents
are typically filtered at 5 KHz and sampled at 20 KHz. The pipette solution
contains a standard solution such as: 140 mM CsF, 2 mM MgCI" 1 mM EGTA,
10 and 10 mM Na-HEPES (pH 7.3). The standard bathing solution is usually 140
nM NaCI, 3 mM KCI, 2 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, and 10 mM
glucose (pH 7.3).
Voltage clamp studies on transformed cells or DRG neurons, using
methods such as intracellular patch-clamp recordings, can provide a
quantitative
measure of the sodium current density (and thus the number of sodium channels
in a cell), and channel physiological properties. These techniques, which
measure the currents that flow through ion channels such as sodium channels,
are described in Rizzo et al. [21J. Alternatively, the blockage or enhancement
of sodium channel function can be measured using optical imaging with
20 sodium-sensitive dyes or with isotopically labeled Na. These methods which
are described in Rose, et al., (J. Neurophysiology, 1997 in press) [67] and by
Kimelberg and Walz [31 ), measure the increase in intracellular concentration
of
sodium ions that occurs when sodium channels are open.
h~easurement oflntracellular Sodium ~~Na*7.l
25 The effects of various agents on cells that express Na'" can be determined
using ratiometric imaging of [Na+]i using SBFI or other sinular ion-sensitive
dyes. In this method, as described by Sontheimer et al. [32], cytosolic-free
Na+
is measured using an indicator for Na+, such as SBFI (sodium-binding
benzofuran isophthalate; [33]) or a similar dye. Cells are first loaded with
the
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membrane-permeable acetoxymethyl ester form of the dye (which is dissolved
in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mTV)7. Recordings
are obtained on the stage of a microscope using a ratiometric imaging setup
(e.g., finm Georgia Instruments). Excitation light is provided at appropriate
5 wavelengths (e.g., 340:385 nm). Excitation light is passed to the cells
through a
dichroic reflector (400 nm) and emitted light above 450 nm is collected.
Fluorescence signals are amplified, e.g., by an image intensifier (GenIISyS)
and
collected with a CCD camera, or similar device, interfaced to a frame grabber.
To account for fluorescence rundown, the fluorescence ratio 340:385 is used to
10 assay cytosolic-free Na*.
For calibration of SBFI's fluorescence, cells are perfused with
calibration solutions containing known Na* concentrations (typically 0 and 30
mM, or 0, 30, and 50 mM [Na*]), and with ionophones such as gramicidin and
monensin (see above) after each experiment. As reported by Rose and Ransom
15 [34], the 345/390 nm fluorescence ratio of intracellular SBFI changes
monotonically with changes in [Na*];. Experiments are typically repeated on
multiple (typically at least 4) different coverslips, providing statistically
significant measurements of intracellular sodium in control cells, and in
cells
exposed to various concentrations of agents that may block, inhibit or enhance
20 Na*.
Method to Measure Na* Influx via Measuring; llNa or 86Rb.
~Na is a gamma emitter and can be used to measure Na* flux [31 ], and ~Rb*
can be used to measure Na*/K*-ATPase activity [32]. g6Rb* ions are taken up by
the Na+/K*-ATPase-like K* ions, but have the advantage of a much longer
25 half life than ''2K* [35]. Thus, measurement of the unidirectional
ouabain-sensitive ~Rb* uptake provides a quantitative method for assaying
Na*/K*-ATPase activity which provides another indicator of the electrical
firing
of nerve cells. Following incubation of cells expressing NaN with the isotope
~Na*, the cellular content of the isotope is measured by liquid scintillation
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counting or a similar method, and cell protein is determined using a method
such as the bicinchoninic acid protein assay [36] following the modifications
described by Goldschmidt and Kimelberg [37] for cultured cells. ~Na and g6Rb*
fluxes are determined in the presence and absence of agents that may block,
S inhibit, or enhance NaN. This permits determination of the actions of these
agents on NaN.
Method to Ident' '~A,gent<s that Modulate NaN Mediated Current:
Several approaches can be used to identify agents that are able to
modulate (i.e., block or augment) the Na* current through the NaN sodium
channel. In general, to identify such agents, a model cultured cell line that
expresses the NaN sodium channel is utilized, and one or more conventional
assays are used to measure Na* current. Such conventional assays include, for
example, patch clamp methods, the ratiometric imaging of [Na*],, and the use
of
~Na and ~Rb as described above.
In one embodiment of the present invention, to evaluate the activity of a
candidate compound to modulate Na* current, an agent is brought into contact
with a suitable transformed host cell that expresses NaN. After mixing or
appropriate incubation time, the Na* current is measured to determine if the
agent inhibited or enhanced Na* current flow.
20 Agents that inhibit or enhance Na* current are thereby identified. A
skilled artisan can readily employ a variety of art-recognized techniques for
determining whether a particular agent modulates the Na* current flow.
Because Na* is preferentially expressed in pain-signaling cells, one can
also design agents that block, inhibit, or enhance Na* channel function by
measuring the response of laboratory animals, treated with these agents, to
acute
or chronic pain. In one embodiment of this aspect of the invention, laboratory
animals such as rats are treated with an agent for instance, an agent that
blocks
or inhibits (or is thought to block or inhibit) NaN. The response to various
painful stimuli are then measured using tests such as the tail-flick test and
limb
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withdrawal reflex, and are compared to untreated controls. These methods are
described in Chapter 1 S of Reference [38]. In another embodiment of this
aspect of the invention, laboratory animals such as rats are subjected to
localized injection of pain-producing inflammatory agents such as formalin
5 [39], Freunds adjuvant [40] or carageenan, or are subjected to nerve
constriction
[41,42] or nerve transection [43] which produce persistent pain. The response
to various normal and painful stimuli are then measured, for example, by
measuring the latency to withdrawal from a warm or hot stimulus [38] so as to
compare control animals and animals treated with agents that are thought to
modify NaN.
The preferred inhibitors and enhancers of NaN preferably will be
selective for the NaN Na+ channel. They may be totally specific (like
tetrodotoxin, TTX, which inhibits sodium channels but does not bind to or
directly effect any other channels or receptors), or relatively specific (such
as
lidocaine which binds to and blocks several types of ion channels, but has a
predilection for sodium channels). Total specificity is not required for an
inhibitor or enhancer to be efficacious. The ratio of its effect on sodium
channels vs. other channels and receptors, may often determine its effect and
effects on several channels, in addition to the targeted one, may be
efficacious
[44].
It is contemplated that modulating agents of the present invention can
be, as examples, peptides, small molecules, naturally occurring and other
toxins
and vitamin derivatives, as well as carbohydrates. A skilled artisan can
readily
recognize that there is no limit as to the structural nature of the modulating
agents of the present invention. Screening of libraries of molecules may
reveal
agents that modulate NaN or current flow through it. Similarly, naturally
occundng toxins (such as those produced by certain fish, amphibians and
invertebrates) can be screened. Such agents can be routinely identified by
exposing a transformed host cell or other cell which expresses a sodium
channel
to these agents and measuring any resultant changes in Na+ current
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Recombinant Protein Expression. Synthesis and Pur; cation:
Recombinant NaN pioteins can be expressed, for example, in E. coli
strains HB 101, DHSa or the protease deficient strain such as CAG-4S6 and
purified by conventional techniques.
S The peptide agents of the invention can be prepared using standard solid
phase (or solution phase) peptide synthesis methods, as is known in the art.
In
addition, the DNA encoding these peptides may be synthesized using
commercially available oligonucleotide synthesis instrumentation and produced
recombinantly using standard recombinant production systems. The production
10 using solid phase peptide synthesis is necessitated if non-gene-encoded
amino
acids are to be included.
Antibodies and Immunodetection:
Another class of agents of the present invention are antibodies
immunoreactive with the Na+ channel. These antibodies may block, inhibit, or
1 S enhance the Na+ current flow through the channel. Antibodies can be
obtained
by immunization of suitable mammalian subjects with peptides, containing as
antigenic regions, those portions of NaN, particularly (but not necessarily)
those
that are exposed extracellularly on the cell surface. Such immunological
agents
also can be used in competitive binding studies to identify second generation
20 inhibitory agents. The antibodies may also be useful in imaging studies,
once
appropriately labeled by conventional techniques.
Production of T~genic Animals:
Transgenic animals containing and mutant, knock-out or modified NaN
genes are also included in the invention. Transgenic animals wherein both NaN
2S and the SNS/PN3 gene are modified, disrupted or in some form modified are
also included in the present invention. Transgenic animals are genetically
modified animals into which recombinant, exogenous or cloned genetic material
has been experimentally transferred. Such genetic material is often referred
to
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as a "bransgene". The nucleic acid sequence of the transgene, in this case a
form
of NaN, may be integrated either at a locus of a genome where that particular
nucleic acid sequence is not otherwise normally found or at the normal locus
for
the transgene. The transgene may consist of nucleic acid sequences derived
5 from the genome of the same species or of a different species than the
species of
the target animal.
The term "germ cell line transgenic animal" refers to a transgenic animal
in which the genetic alteration or genetic information was introduced into a
germ line cell, thereby confernng the ability of the transgenic animal to
transfer
10 the genetic information to offspring. If such offspring in fact possess
some or
all of that alteration or genetic information, then they too are transgenic
animals.
The alteration or genetic information may be foreign to the species of
animal to which the recipient belongs, foreign only to the particular
individual
recipient, or may be genetic information already possessed by the recipient.
In
15 the last case, the altered or introduced gene may be expressed differently
than
the native gene.
Transgenic animals can be produced by a variety of different methods
including tnansfection, electmporation, microinjection, gene targeting in
embryonic stem cells and recombinant viral and retmviral infection (see, e.g.,
20 U.S. Patent No. 4,736,866; U.S. Patent No. 5,602,307; Mullins et al. (1993)
Hypertension 22(4):630-633; Brenin et al. (1997) Surg. Oncol. 6(2)99-110;
Tuan (ed.), Recombinant Gene Expression Protocols, Methods in Molecular
Biology No. 62, Humans Press (1997)).
A number of recombinant or transgenic mice have been produced,
25 including those which express an activated oncogene sequence (U.S. Patent
No.
4,736,866); express simian SV 40 T-antigen (U.S. Patent No. 5,728,915); lack
the expression of interferon regulatory factor 1 (IItF-1) (U.S. Patent No.
5,731,490); exhibit dopaminergic dysfunction (U.S. Patent No. 5,723,719);
express at least one human gene which participates in blood pressure control
30 (U.S. Patent No. 5,731;489); display greater similarity to the conditions
existing
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in naturally occurring Alzheimer's disease (U.S. Patent No. 5,720,936); have a
reduced capacity to mediate cellular adhesion (U.S. Patent No. 5,602,307);
possess a bovine growth hormone gene (Clutter et al. (1996) Genetics
143(4}:1753-1760); or, are capable of generating a fully human antibody
response (McCarthy (1997) The Lancet 349(9049):405).
While mice and rats remain the animals of choice for most transgenic
experimentation, in some instances it is preferable or even necessary to use
alternative animal species. Transgenic procedures have been successfully
utilized in a variety of non-marine animals, including sheep, goats, pigs,
dogs,
cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see,
e.g.,
Kim et al. (1997) Mol. Reprod. Dev. 46(4):515-526; Houdebine (1995) Reprod.
Nutr. Dev. 35(6):609-617; Petters (1994) Reprod. Fertil. Dev. 6(5):643-645;
Schnieke et al. (1997) Science 278(5346):2130-2133; and Ainoah (1997) J.
Animal Science 75(2):578-585}.
The method of introduction of nucleic acid fragments into recombination
competent mammalian cells can be by any method which favors
co-transformation of multiple nucleic acid molecules. Detailed procedures for
producing transgenic animals are readily available to one skilled in the art,
including the disclosures in U.S. Patent No. 5,489,743 and U.S. Patent No.
5,602,307.
The specific examples presented below are illustrative only and are not
intended to limit the scope of the invention.
EXAMPLES
Example 1: Cloning and Characterization of the Rat NaN Codine
a. RNA Preparation
Dorsal root ganglia (DRG) from the lumber region (L4-LS) were
dissected from adult Sprague-Dawley rats and total cellular RNA was isolated
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by the single step guanidinum isothiocyanate-acid phenol procedure [45]. For
analytical applications, DRG tissues were dissected from a few animals at a
time. The quality and relative yield of the RNA was assessed by elechophoresis
in a 1% agarose gel. Because of the limited starting material (4 DRGs weigh on
average 10 mg), quantifying the RNA yield was not attempted. PolyA+ RNA
was purified from about 300 pg of total DRG RNA (28 animals) using the
PolyATract isolation system according to the manufacturers recommendations
(Promega). Half of the purified RNA was used for the preparation of Marathon
cDNA (see below) without further quantification.
b. Reverse Transcr~~tion
For analytical applications, first strand cDNA was synthesized
essentially as previously described [46]. Briefly, total RNA was reverse
transcribed in a 25 ~1 final volume using 1 p,M random hexamer (Boehringer
Mannheim) and 500 units Superscript II reverse transcriptase (Life
Technologies) in the presence of 100 units of RNase Inhibitor (Boehringer
Mannheim). The reaction buffer consisted of 50 mM Tris-HCl (pH 8.3), 75
mM KCI, 3 mM MgCl2, 10 mM DTT and 125 pM dNTP. The reaction was
allowed to proceed at 37°C for 90 min., 42°C for 30 min, then
terminated by
heating to 65°C for 10 min.
c. First-Strand cDNA Synthesis
The Marathon cDNA synthesis protocol was followed according to the
manufacturer's instruction as summarized below (all buffers and enzymes are
purchased from the manufacturer (Clontech):
Combine the following reagents in a sterile 0.5-ml microcentrifuge tube:
1 ~g (1-4 p.l) PolyA+ RNA sample, 1 p.l cDNA Synthesis Primer (10 ~.M) and
sterile H20 to a final volume of 5 p.l. Mix contents and spin the tube briefly
in a
microcentrifuge. Incubate the mixture at 70°C for 2 min., then
immediately
quench the tube on ice for 2 min. Touch-spin the tube briefly to collect the
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condensation. Add the following to each reaction tube: 2 wl SX First-Strand
Buffer, 1 pl dNTP Mix (10 mM), 1 ~1 [a-32PJdCTP (1 p,Ci/~1), 1 ~.l AMV
Reverse Transcriptase (20 units/~1) for a 10 ~1 volume. The radiolabeled dCTP
is optional (used to determine yield of cDNA) and is replaced by sterile HZO
if
5 not used. Mix the contents of the tube by gently pipetting and touch-spin
the
tube to collect the contents at the bottom. Incubate the mixture at
42°C for 1 hr
in an air incubator to reduce condensation and enhance the yield of the first
strand cDNA. Place the tube on ice to terminate first-strand synthesis.
d. Second Strand cDNA S,vnthesis
10 Combine the following components in the reaction tube from above:
48.4 ltl Sterile HZO, 16 ~1 SX Second-Strand Buffer, 1.6 pl dNTP Mix (10
mM), 4 ~.120X Second-Strand Enzyme Cocktail for an 80 ~.l total volume. Mix
the contents thoroughly with gentle pipetting and spin the tube briefly in a
microcentrifuge. Incubate the mixture at 16°C for 1.5 hr. then add 2 ~1
(10
15 units) of T4 DNA Polymerise, mix thoroughly with gentle pipetting and
incubate the mixture at 16°C for 45 min. Add 4 ~1 of the EDTA/Glycogen
mix
to terminate second-strand synthesis. Extract the mixture with an equal volume
of buffer-saturated (pH 7.5) phenol:chloroform:isoamyl alcohol (25:24:1). Mix
the contents thoroughly by vortexing and spin the tube in a microcentrifuge at
20 maximum speed (up to 14,000 rpm or 13000xg), 4°C for 10 min. to
separate
layers. Carefully transfer the top aqueous layer to a clean 0.5-ml tube.
Extract
the aqueous layer with 100 ~,1 of chloroform:isoamyl alcohol (24:1), vortex,
and
spin the tube as before to separate the layers. Collect the top layer into a
clean
0.5-ml microcentrifuge tube. Ethanol precipitate the double-stranded cDNA by
25 adding one-half volume of 4 M Ammonium Acetate and 2.5 volumes of room-
temperature 95% ethanol. Mix thoroughly by vortexing and spin the tube
immediately in a microcentrifuge at top speed, room temperature for 20 min.
Remove the supernatant carefully and wash the pellet with 300 p,l of 80%
ethanol. Spin the tube as before for 10 min. and carefully remove the
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supernatant. Air dry the pellet for up to 10 min. and dissolve the cDNA in 10
~,1
of sterile H20 and store at -20°C. Analyze the yield and size of cDNA
by
running 2 ~.1 of the cDNA solution on a I .2% agarose/EtBr gel with suitable
DNA size markers (for example the 1 Kbp ladder, GibcoBRL). If EtBr staining
5 does not show a signal and [a-'2P)dCTP was included in the reaction, dry the
agarose gel on a vacuum gel drying system and expose an x-ray film to the geI
overnight at -70°C.
e. d for Lig ion
Combine these reagents in a 0.5-ml micmcentrifuge test tube, at room
temperature, and in the following order: 5 ~1 ds cDNA, 2 ~,1 Marathon cDNA
Adaptor (10 ~.M), 2 ~.l SX DNA Ligation Buffer, 1 ~.1 T4 DNA Ligase (1
unit/~,1) for a 10 ~1 final voluu~e. Mix the contents thoroughly with gentle
pipetting and spin the tube briefly in a microceatrifuge. Incubate at either:
lb°C
overnight; or mom temperature (19-23°C) for 3-4 hr. Inactivate the
ligase
15 enzyme by heating the mixture at 70°C for 5 min. Dilute 1 ~1 of this
reaction
mixture with 250 ~.1 of Tricine-EDTA buffer and use for RACE protocols.
Store the undiluted adaptor-ligated cDNA at -20°C for future use.
f. pig
For the initial discovery of NaN, we used generic primers designed
20 against highly conserved sequences in domain 1 (D1) of a-subunits I, II and
III
and later added more primers to accommodate the new a-subunits that were
discovered. Thus, we used generic primers that recognize conserved sequences
in all known Na+ channels. The middle of the amplified region shows
significant sequence and length polymorphism (Fig. 6) and [47,48]. Due to
25 codon degeneracy, 4 forward primers (F1-F4) and 3 reverse primers (Rl-R3)
were designed to ensure efficient priming from all templates that might have
been present in the cDNA pool (Table 1 ); however, any of these primers may
bind to multiple templates depending on the stringency of the reaction.
Forward
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primer F1 matches subunits aI, aIII; aNab; aPNl; ail, arHl and aSNS/PN3.
Sequences of individual subunits show 1 or 2 mismatches to this primer: T to C
at position 16 and A to' G at position 18 (aNa6); C to R at position 6 (a~.l);
A
to G at position 18 (arHl) and T to C at position 3 (aSNS). Forward primer
F2 matches subunit aII. Forward primer F3 perfectly matches aNa6 and also
matches arHl with a single mismatch of C to T at position 16. Reverse primer
Rl matches subunits aI, aII, aIII, aNa6, aPNl, a~,l and arHl. This primer
has mismatches compared to 4 subunits: G to A at position 3, A to G at
position 4 and T to G at position 7 (al); T to C at position 1 and A to G at
10 position 19 (aPNl); G to A at position 3 and A~to G at position 7 (a~.l);
an
extra G after position 3, GC to CT at positions 14-15, and A to T at position
21
(arHl). Reverse primer R2 matches subunit aSNS/PN3.
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Table 1: Generic and NaN specific primers used for the ideatification and
cloning of NaN. All primers except the marathon primers, were synthesized at
the department of Pathology, Frogratn for Critical Technologies in Molecular
Medicine, Yale University.
S Forward Primers Reverse Primers
.__~-.
1. GACCCRTGGAATTGGTTGGA 1. CAAGAAGGCCCAGCTGAAGGTGTC
2. AATCCCTGGAATTGGTTGGA 2. GAGGAATGCCCACGCAAAGGAATC
3. GACCCGTGGAACTGGTTAGA 3.
AAGAAGGGACCAGCCAAAGTTGTC
4. GATCTTTGGAACTGGCTTGA 4. ACYTCCATRCANWCCCACAT
S. AACATAGTGCTGGAGTTCAGG S. AGRAARTCNAGCCARCACCA
6. GTGGCCTTTGGATTCCGGAGG 6. TCTGCTGCCGAGCCAGGTA
7. CTGAGATAACTGAAATCGCC
M~thon AP-1 CCATCCTAATACGACTCACTATAGGGC
Marathon AP-2 ACTCACTATAGGGCTCGAGCGGC
1S We used the respective mouse atypical sodium chamiel mNa,2.3
sequence to design forward primer F4 and reverse primer R3 to amplify the
analogous sequence from aNaG, the prestmzed rat homolog of mNa,2.3 [14].
The amplified sequence was cloned into the Srf I situ of the vector
pCR SCR~T (Stratagene). The nucleotide sequence of this &~agtnent shows
88% ide~ity to the respective sequence of mNa,2.3 (Dib-Ha~j and Waxman,
unpublished [68]). The restriction enzyme Xba I was found to be unique to this
subunit. Recently, the sequence of a full length cDNA clone of putative sodium
channel, NaG-like (SCL-11:Y09164), subunit was published [S]. The published
sequence is 99'/o identical to our sequence and confirms the size and
restriction
2S enzyme polymorphism of the NaG PCR product.
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The predicted lengths of amplified products and subunit-specific
restriction enzyme recognition sites are shown in Fig. 6. All subunit
sequences
are based on Genbank database (accession numbers: aI: X03638; aII: X03639;
aIII: Y00766; aNa6: L39018; ahNE Na: X82835; ap.l M26643; arHl M27902
and aSNS X92184; mNa 2.3 L36719).
Subsequently, amplification of NaN sequences 3' terminal to the
aforementioned fi~agment was achieved using NaN-specific primers and two
generic reverse primers, R4 and R5. The sequence of the R4 primer was based
on the amino acid sequence MWV/DCMEV located just N-terminal to domain
10 II S6 segment (see schematic diagram of Fig. 3 of voltage-gated sodium
channel
oc-subunits for reference). The sequence of the RS primer is based on the
amino
acid sequence AWCWLDFL which forms the N-terminal portion of domain III
S3 segment.
Amplification was typically performed in 60 ~,1 volume using 1 pl of
the first strand cDNA, 0.8mM of each primer and 1.75 units of Expand Long
Template DNA polymerise enzyme mixture (Boehringer Mannheim).
Compared to conventional and thermostable DNA polymerises, Expand Long
Template enzyme mixture increases the yield of the PCR products without an
increase in non-specific amplification [49,50]. The PCR reaction buffer
consisted of 50 mM Tris-HCl (pH 9.2), 16 mM (NH4)ZS04, 2.25 mM MgCl2,
2% (v/v) DMSO and 0.1 % Tween 20. As described previously [46],
amplification was carried out in two stages using a programmable thermal
cycler {PTC-200, MJ Research, Cambridge, MA.). First, a denaturation step at
94°C for 4 min, an annealing step at 60°C for 2 min and an
elongation step at
25 72°C for 90 sec. Second, a denaturation step at 94°C for 1
min, an annealing
step at 60°C for 1 min and an elongation step at 72°C for 90
sec. The second
stage was repeated 33 times for a total of 35 cycles, with the elongation step
in
the last cycle extended to 10 min.
Primary RACE amplification was performed in 50 ~.l final volume using
4 ~,1 diluted DRG marathon cDNA template, 0.2 ~,M marathon AP-l and
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NaN-specific primers, 3.5 U Expand Long Template enzyme mixture.
Extension period was adjusted at 1 min/800 by based on the expected product.
5' and 3' RACE amplification was performed using primer pairs marathon
AP-1/NaN specific R6 and NaN specific FS/marathon AP-1, respectively. The
5 PCR reaction buffer consisted of SO mM Tris-HCl (pH 9.2), 16 mM (NH4),SO"
3.0 mM MgCI" 2% (v/v) DMSO and 0.1 % Tween 20. Amplification in three
stages was performed in a programmable thermal cycler (PTC-200, MJ
Research, Cambridge, MA.). An initial denaturation step at 92~C was carried
out for 2 min. This was followed by 35 cycles consisting of denaturation at
92~C for 20 sec; annealing step at 60~C for 1 min, and an elongation step at
68~C.
Finally, an elongation step at 68~C was carried out for S min. Nested
amplification was performed using 2 p,l of a 1/500 diluted primary RACE
product in a final volume of 50 ~,1 under similar conditions to the primary
RACE reactions. Primer pairs AP-2/NaN-specific R7 and NaN-specific
15 F6/marathon AP-2 were used for nested 5' and 3' RACE, respectively.
Secondary RACE products were band isolated from 1 % agarose gels and
purified using Qiaex gel extraction kit (Qiagen Inc.).
A schematic diagram of the putative structure of NaN is shown in Fig.3.
The length of the intracellular loops is highly variable both in sequence and
20 length among the various subunits. The exception is the loop between
domains
III and IV.
Example 2: petermination of the Putative Rat ~nino Acid Sequence for
the NaN Chi
NaN related clones and secondary RACE fi~agments were sequenced at the
25 W. M. Keck Foundation Biotechnology Resource Lab, DNA sequencing group
at Yale University. Sequence analysis including determination of the predicted
amino acid sequence was performed using commercial softwares, Lasergene
(DNASTAR) and GCG, Inc. The putative amino acid sequence of NaN is
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shown in Fig. 2. Predicted transmembrane segments of domains I - IV are
underlined.
Example 3:
Total RNA extraction from trigeminal ganglia of mice, purification of
polyA+ RNA, and Marathon cDNA construction were done as previously
described for the rat. The initial amplification was performed using rat NaN
primers. The forward primer corresponds to nucleotides 765-787 of the rat
sequence (5' CCCTGCTGCGCTCGGTGAAGAAG 3'), and the reverse primer
corresponds to nucleotides 1156-1137 (negative strand) of the rat sequence (5'
GACAAAGTAGATCCCAGAGG 3'). The amplification produced a fragment
of the expected size. The sequence of this fragment demonstrated high
similarity to rat NaN. Other fragments were amplified using different rat
primers and primers designed based on the new mouse NaN sequence that was
being produced. Finally, longer fragments were amplified using mouse
15 Marathon cDNA template and mouse NaN specific primers in combination with
adaptor primers that were introduced during the Marathon cDNA synthesis.
These fragments were sequenced using primer walking and assembled into
Figure 7A.
Mouse NaN nucleotide sequence, like rat NaN, lacks the out-of frame
ATG at the -8 position relative to the translation initiation colon ATG at
position 41 (Fig.7A). Translation termination colon TGA is at position 5314. A
polyadenylation signal (AATAAA) is present at position 5789 and a putative 23
nucleotide polyA tail is present beginning at position 5800. The sequence
encodes an ORF of 1765 a.a. (Fig. 7B), which is 90% similar to rat NaN. The
gene encoding NaN has been named Scnl la.
Chromosomal localization of mouse Nan
A genetic polymorphism between strains C57BL/6J and SPRETBi was
identified by SSCP analysis of a 274 by fragment from the 3'LTTR of Scnll a.
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Genotyping of 94 animals from the BSS backcross panel (Rowe et al., 1994)
demonstrated linkage of Scnll a with markers on distal chromosome 9 (Fig. 10).
No recombinants were observed between Scnlla and the microsatellite marker
D9Mitl9. Comparison of our data with the MGD consensus map of mouse
5 chromosome 9 revealed close linkage of Scnll a with the two other TTX-R
voltage-gated sodium channels, ScnSa (George et al., 1995; Klocke et al.,
1992)
and ScnlOa (Kozak and Sangameswaran, 1996; Souslova et al., 1997).
Example 4: Determination of a Partial Humor NaN Se~quence\
Human DRG tissue was obtained from a transplant donor. Total RNA
10 extraction and cDNA synthesis were performed as described previously.
Forward primer corresponds to s~uence 310-294 (minus strand) of EST
AA446878. The sequence of the primer is 5' CTCAGTAGTTGGCATGC 3'.
Reverse primer corresponds to sequence 270-247 (minus strand) of EST
AA88521 1. The sequence of the primer is
15 5'GGAAAGAAGCACGACCACACAGTC 3'. Amplification was performed as
previously described. PCR amplification was successful and a 2.1 Kbp fragment
was obtained. This fragment was gel purified and sent for sequencing by primer
walking, similar to what is done for mouse NaN. The sequence of the ESTs is
extended in both directions; the additional sequence shows highest similarity
to
20 rat and mouse NaN, compared to the rest of the subunits.
The sequence of a human 2. 1 kbp fragment was obtained using the PCR
forward and reverse primers for sequencing from both ends of the fragment.
Two additional primers were used to cover the rest of the s~uence. The
sequence was then extended in the 5' direction using forward primer 1 (above)
25 and human NaN reverse primer (5'-GTGCCGTAAACATGAGACTGTCG3')
near the 5' end of the 2.1 kb fragment. The partial amino acid sequence is set
forth in Figure 8B.
The partial ORF of the human NaN consists 1241 amino acids. The
sequence is 64% identical to the corresponding sequence of rat NaN (73%
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similar, allowing for conservative substitutions) using the advanced BLAST
pmgram at NIH. Using the Clustal method of alignment (Lasergene software,
DNAStar, Inc.) the human NaN is 68% and 69% similar to mouse and rat NaN,
respectively. The respective mouse and rat sequences are 88% similar.
Example 5: Isolation of an Alternative S,~,g Variant of Rat NaN
A rat NaN cDNA that encodes a C-terminal truncated version of the full-
length rat NaN in Figures 1 and 2 was isolated by sequencing the insert of a
rat
cDNA clone. The variant NaN cDNA encodes an NaN protein lacking the 387
C-terminal amino acids of the full length NaN and containing a novel 94 amino
acid stretch at the C-terminal end. The new sequence arises from the use of a
cryptic donor splice site in exon 23 and a novel exon 23', which is located in
intron 23. The novel C terminal amino acids are: AAGQAMRKQG
DILGPNIHQF SQSSETPFLG CPQQRTCVSF VRPQRVLRVP
WFPAWRTVTF LSRPRSSESS AWLGLVESSG WSGLPGESGP SSLL. The
N-terminal amino acids of the truncated variant are identical to amino acids 1-
1378 of the full length rat NaN of Figure 2. The alternative exon and the
splicing pattern was confirmed by comparing the cDNA sequence and the
genomic sequence in the respective region.
Example 6: Methods to Isolate Other NaN Seouences
a. Isolation off NaN seauences om genomic DNA
The genomic structure of 3 voltage-gated Na+ channel a-subunits have
akeady been determined [51-54J. These genes bear remarkable similarity in
their organization and provide a predictable map of most of the exon/intron
boundaries. Based on the available rat, mouse and human cDNA sequence of
NaN, disclosed herein, PCR primers are designed to amplify NaN homologous
sequences from other species using standard PCR protocols.
RECTIFfED Si~IEET (RULE 91y
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Alternatively, commercially available genomic DNA libraries are
screened with NaN specific probes (based on the rat, mouse, or more
preferably,
the human sequence) using standard library screening procedures [59, 60]. This
strategy yields genomic DNA isolates that can then be sequenced and the
exon/intron boundaries determined by homology to the rat, mouse or human
cDNA sequence.
b. Col t~of full length NaNse~Quences,from human auto~v or
b.l. Isolation o, human ~an~ia total RNA
A full length NaN human cDNA homologue is isolated from human
dorsal root ganglia or trigeminal ganglia or other cranial ganglia from post-
mortem human material, foetuses or biopsy or surgical tissues. Total
ribonucleic acid (RNA) is isolated from these tissues by extraction in
guanidinium isothiocyanate [69] as described in Example 1.
b.2 Determination o, the 1~~ 1 length transcri~ size ,f the human
homolog~~fthe ra_t NaNsodium channel cDNA.
The method of determining transcript size is as described in Example 9.
Example 7: Production of human DRG cDNA library
A cDNA library from human DRG or trigeminal ganglia polyA+ RNA
was prepared in Example 4 using standard molecular biology techniques [59,
60].
PolyA+ mRNA is hybridized to an oligo(dT) primer and the RNA is
copied by reverse transcriptase into single strand cDNA. Then, the RNA in the
RNA-DNA hybrid is fragmented by RNase H as E. coli DNA polymerase I
synthesizes the second-strand fragment. The ends of the double sanded cDNA
are repaired, linkers carrying specific restriction enzyme site (for example,
Eco
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RI) are ligated to the ends using E. coli DNA ligase. The pool of the cDNA
insert is then ligated into one of a variety of bacteriophage vectors that are
commercially available like Lambda-Zap (Stratagene). The procedures are
summarized in more detail as follows:
a. First strand cDNA ~~vnthesis
Dissolve 10 p,g poly(A) + RNA at a concentration of 1 ~eg/~1 in sterile
water. Heat the RNA for 2-5 min. at 65°C-70°C, then quench
immediately on
ice. In a separate tube add in the following order (180 wl total) : 20 ~1 5 mM
dNTPs (500 uM final each), 40 p.l 5x RT buffer (lx final), 10 p,l 200 mM DTT
IO (10 mM final), 20 pl 0.5 mglml oligo (dT)12-18 (50 ~g/ml final), 60 ~l HZO,
p,l (10 U) RNasin (50 U/ml final). Mix by vortexing, briefly
microcentrifuge, and add the mixture to the tube containing the RNA. Add 20
p,l (200 U) AMV or MMLV reverse transcriptase for a final concentration of
1000 U/ml in 200 p.l . Mix by pipetting up and down several times and remove
10 p.l to a separate tube containing 1 p,l of a'iP dCTP. Typically, incubate
both tubes at room temperature for 5 min., then place both tubes at
42°C for 1.5
hr. This radiolabeled aliquot is removed to determine incorporation and permit
an estimation of recovery; this reaction is stopped by adding 1 p,1 of 0.5 M
EDTA, pH 8.0, and stored frozen at -20°C. The radiolabeled reaction
will be
used later to estimate the yield and average size of the cDNA inserts. The
main
reaction is stopped by adding 4 p, l of 0.5 M EDTA, pH 8.0, and 200 p 1
buffered phenol. The mixture is vortexed well, microcentrifuged at room
temperature for 1 min. to separate phases, and the upper aqueous layer is
transferred to a fi~esh tube. Back extract the phenol layer with 1X TE buffer
(10
mM Tris, 1 mM EDTA, pH 7.5) and pool the aqueous layers from the two
extractions. This back extraction of the phenol layer improves the yield. The
cDNA is ethanol precipitated using 7.5 M ammonium acetate (final
concentration 2.0 to 2.5 M) and 95% ethanol. Place in dry ice/ethanol bath 15
min., warm to 4°C, and microcentrifuge at 10 min. at full speed,
4°C, to pellet
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nucleic acids. The small, yellow-white pellet is then washed with ice-cold 70%
ethanol, and microcentrifuged for 3 min. at full speed, 4°C. Again,
remove the
supernatant, then briefly dry the pellet.
b. second strand s, tvn hesis
5 Typically, the pellet from the first-strand synthesis is resuspended in 284
wl water and these reagents are added in the following order (400 p,l total):
4
p.l 5 mM dNTPs (50 uM final each), 80 p.l Sx second-strand buffer (lx final),
12 p.l 5 mM ~i-NAD (150 uM final), 2 ~,1 10 uCi/~,1 a 32P dCTP (50 uCi/ml
final). Mix by vortexing, briefly microcentrifuge, and add: 4 p.l (4 U) RNase
H
(10 U/ml final), 4 p.l (20 U) E. coli DNA ligase (50 U/ml final), and 10 ~,1
(100
U) E. coli DNA polymerase I (250 U/ml final). Mix by pipetting up and down,
briefly microcentrifuge, and incubate 12 to 16 hr at 14°C. After second-
strand
synthesis, remove 4 pl of the reaction to determine the yield from the
incorporation of the radiolabel into acid-insoluble material. Extract the
second-
15 strand synthesis reaction with 400 p.l buffered phenol and back extract the
phenol phase with 200 p 1 TE buffer, pH 7.5, as described above. The double
stranded cDNA is then ethanol precipitated as described above.
To complete the second-strand synthesis the double-stranded cDNA
ends are rendered blunt using a mixture of enzymes. Resuspend the pellet in 42
p,l water then add these reagents in the following order (80 pl total) : 5 ~1
5
mM dNTPs (310 uM final each), 16 ~,1 Sx TA butler (lx final), 1 pl 5 mM ~i-
NAD (62 uM final): Mix by vortexing, microcentrifuge briefly, and add: 4 It 1
of 2 ~.g/ml RNase A (100 ng/ml final), 4 p.l (4 U) RNase H (50 U/ml final), 4
wl (20 U) E. coli DNA ligase (250 U/ml final), and 4 ~,1 (8 U) T4 DNA
polymerase (100 U/ml final). Mix as above and incubate 45 min at 37°C.
Add
120 p 1 TE buger, pH 7.5, and 1 p.l of 10 mg/ml tRNA. Extract with 200 ~ 1
buffered phenol and back extract the phenol layer with 100 p.l TE buffer as
described above. Pool the two aqueous layers and ethanol precipitate as
described above.
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c. Addition oflinkers to double stranded cDNA
Combine these reagents in a 0.5-ml microcentrifuge test tube, at room
temperature, and in the following order: 100 ng ds cDNA, 2 wl linkers/adaptors
( 10 ~, 2 ~,1 5X DNA Ligation Buyer, 1 ~,1 T4 DNA Ligase ( unit/~,1 ) for a
10 ~,1 final volume. Mix the contents thoroughly with gentle pipetting and
spin
the tube briefly in a microcentrifuge. Incubate at either: 16°C
overnight; or
room temperature (19-23°C) for 3-4 hr. Inactivate the ligase enzyme by
heating
the mixture at 70°C for 5 min. This cDNA is typically digested by Eco
RI to
prepare the cohesive ends of the cDNA for ligation into the vector and to
cleave
10 linker concatemers. Typically this reaction consists of the 10 p 1 of the
cDNA,
2 ~,1 of lOX Eco RI buffer (depending on the company of source), 2 pl of Eco
RI (10 units/p.l) and sterile Hi0 to a final volume of 20 p,l. The mixture is
incubated at 37°C for 2-4 hrs.
d. Size,frac 'ovation o~'cDNA
1 S Size exclusion columns are typically used to remove linker molecules
and short cDNA fragments (350 bp). For example, a 1-ml Sepharose CL-4B
column is prepared in a plastic column plugged with a small piece of
sterilized
glass wool (a 5 ml plastic pipet will work fine). The column is equilibrated
with
0.1 M sodium chloride in lx TE (IOmM Tris, 1 mM EDTA, pH 7.5). The
20 cDNA is then loaded onto the column and 200 ~,1 fractions are collected. 2
pl
aliquots of each fiaction are analyzed by gel electrophoresis and
sutoradiography to determine the peak of cDNA elution. Typically, fractions
containing the first half of the peak are pooled and purified by ethanol
precipitation and resuspending in 10 ~,1 distilled water.
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e. Cloning ofcDNA into bacterio~rha~e vector
Bacteriophage vectors designed for the cloning and propagation of
cDNA are provided ready-digested with Eco RI and with phosphatased ends
from commercial sources (e.g., lambda gtl0 from Stratagene). The prepared
5 cDNA is ligated into lambda vectors following manufacturer's instructions.
Ligated vector/cDNA molecules are packaged into phage particles using
packaging extracts available commercially.
Example 8: Screening of HunQan cDNA Libra~,y
a. Labeling ofcDNA, rf~~"probes), or libra screening
10 An RNA probe is used that recognizes nucleotide sequences that are
specific to NaN, such as 1371-1751 of NaN. Other nucleotide sequences can be
developed on the basis of the NaN sequence (Fig. 2, 7 and 8) such as
nucleotides 765-1160 of the human nucleotide sequence. A Hind IIIlBam HI
fragment of NaN was inserted in pBluescript (SK+) vector (Stratagene). The
15 sequence of the resulting construct was verified by sequencing. The
orientation
of the insert is such that the 5' and 3' ends of the construct delineated by
the
Hind III and Bam HI restriction enzyme sites, respectively, are proximal to T7
and T3 RNA polymerise promoters, respectively. Digoxigenin-labeled Sense
(linearized at the Hind III site and transcribed by T7 RNA polymerise) and
20 antisense (linearized at the Bam HI site and transcribed by T3 RNA
polymerise)
transcripts were prepared in vitro using MEGAscript transcription kit (Ambion)
according to manufacturer specifications. Briefly, 1 pg linearized template
was
transcribed with the respective RNA polymerise in a 20 pl final volume
containing the following reagents: 1X enzyme mixture containing the respective
25 RNA polymerise and RNase inhibitor and reaction buffer (Ambion), 7.5 mM
ATP, GTP and CTP nucleotides, 5.625 mM UTP and 1.725 mM Dig-11LTTP
(Boehringer Mannheim). In vitro transcription was carried out at 37°C
for 3 hrs
in a water bath. DNA template was removed by adding 1 p,l.of RNase-free
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DNase I (2U/wl) to each reaction and incubating fiuther at 37°C for
15 min.
The reaction was then stopped by adding 30 pl nuclease-free HZ) and 25 pl of
LiCl precipitation solution (7.5 M Lithium Chloride, 50 mM EDTA).
The mixture was incubated at -20°C for 30 min. The RNA transcripts
were pelleted in a microfuge at 13000xg, 4°C for 15 min. The
supernatant was
removed and the pellet washed once with 100 pl of 75% ethanol. The mixture
was re-centrifuged at 13000xg;, room temperature for 5 min. The pellet was
then air-dried in a closed chamber and subsequently dissolved in 100 ml of
RNase-free H20. The transcript yield and integrity were determined by
10 comparison to a control DIG-labeled RNA on agarose-formaldehyde gel as
described in the DIG/Genius kit according to manufacturer recommendations
(Boehringer Mannheim). Alternatively, a skilled artisan can design radioactive
probes for autoradiographic analysis.
Other regions of the rat, mouse or human NaN sodium channel cDNA,
1 S like 3' untranslated sequences, can also be used as probes in a similar
fashion for
cDNA library screening or Northern blot analysis. Specifically, a probe is
made
using commercially available kits, such as the Pharmacia oligo labeling kit,
or
Genius kit (Boehringer Mannheim).
b. cDNA library screening
20 Recombinant plaques containing full length human homologues of the
NaN sodium channel are detected using moderate stringency hybridization
washes (50-60°C, 5 x SSC, 30 minutes), using non-radioactive (see
above) or
radiolabeled DNA or cRNA NaN specific probes derived from the 3'
untranslated or other regions as described above. Libraries are screened using
25 standard protocols [59, 60] involving the production of nitrocellulose or
nylon
membrane filters carrying recombinant phages. The recombinant DNA is then
hybridized to NaN specific pmbes (see above). Moderate stringency washes are
carried out.
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Plaques which are positive on duplicate filters (i.e., not artefacts or
background) are selected for further purification. One or more rounds of
screening after dilution to separate the phage are typically performed.
Resulting
plaques are then purified, DNA is extracted and the insert sizes of these
clones
characterized. The clones are cross-hybridized to each other using standard
techniques [59) and distinct positive clones identified.
Typically, overlapping clones that encode the channel are isolated.
Standard cloning techniques are then used to produce a full length cDNA
construct that contains any 5' untranslated sequence, the start codon ATG, the
coding sequence, a stop codon and any 3' untranslated sequence, a poly A
consensus sequence and possibly a poly A run. If overlapping clones do not
produce sufficient fragments to assemble a full length cDNA clone, alternative
methods like RACE (PCR-based) could be used to generate the missing pieces
or a full length clone.
c. Characterization of,~j~ human homol~~e ful~ h clone
A NaN specific cDNA sequence from the full-length clone is used as a
probe in Northern blot analysis to determine the messenger RNA size in human
tissue for comparison with the rat and mouse messenger RNA size.
Confirmation of biological activity of the cloned cDNA is carried out using
methods similar to those described for the rat NaN.
Example 9: Polymerase chain_ re-~~tion ~$~~~ches to clone
Total RNA and poly A+ RNA is isolated from human dorsal root
ganglia or trigeminal ganglia or other cranial ganglia from post-mortem human
material or foetuses or biopsy/surgical tissues as described above.
Preparation
of cDNA and PCR-based methods are then used as described previously in
Example 1.
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Using degenerate PCR primers derived from the rat NaN specific coding
sequence (see Fig. 2), the cDNA is amplified using the polymerise chain
reaction [69]. A skilled artisan could utilize the many variables which can be
manipulated in a PCR reaction to derive the homologous sequences required.
These include, but are not limited to, varying cycle and step temperatures,
cycle
and step times, number of cycles, thermostable polymerise, and Mg2+
concentration. A greater specificity can be achieved using nested primers
derived from further conserved sequences from the NaN sodium channel.
Amplification is typically performed in 60 ~1 volume using 1 pl of the
first strand cDNA, 0.8 mM of each primer and 1.75 units of Expand Long
Template DNA polymerise enzyme mixture (Boehringer Mannheim).
Compared to conventional and thermostable DNA polymerises, Expand Long
Template enzyme mixture increases the yield of the PCR products without an
increase in non-specific amplification [49,50]. The PCR reaction buffer
consists of 50 mM Tris-HCl (pH 9.2), 16 mM (NH4)ZS04, 2.25 mM MgCl2, 2%
(v/v) DMSO and 0.1 % Tween 20. As described previously [46], amplification
is carried out in two stages using a probable thermal cycler (PTC-200, MJ
Research, Cambridge, MA.). First, a denaturation step at 94°C for 4
min, an
annealing step at 60°C for 2 min and an elongation step at 72°C
for 90 sec.
Second, a denaturation step at 94°C for 1 min, an annealing step at
60°C for 1
min and an elongation step at 72°C for 90 sec. The second stage is
repeated 33
times for a total of 35 cycles, with the elongation step in the last cycle
extended
to 10 min. In addition, control reactions are performed alongside the samples.
These should be: 1 ) all components without cDNA, (negative control) and 2)
all
reaction components with primers for constitutively expressed product, e.g,
GAPDH.
The products of the PCR reactions are examined on 1-1.6% (w/v)
agarose gels. Bands on the gel (visualized by staining with ethidium bromide
and viewing under UV light) representing amplification products of the
approximate predicted size are then cut from the gel and the DNA purified.
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The resulting DNA may be sequenced directly or is ligated into suitable
vectors such as, but not limited to, pCR II (Invitrogen) or pGEMT (Promega).
Clones are then sequenced to identify those containing sequence with
similarity
to the rat, mouse or partial human NaN sodium channel sequence.
Example 10: Clone anal3r~is
Candidate clones from Example 9 are fiuther characterized by
conventional techniques. The biological activity of expression products is
also
confirmed using conventional techniques.
Example 11:
tissues
Commercially available human fetal cDNA libraries and/or cDNA pools
are screened with NaN specific primers (by PCR) or probes (library screening)
using PCR standard PCR protocols and standard library screening procedures as
described above.
Example 12: Northern Blot of rat DRG or Trigeminal Neurons with
10-30 p,g total DRG and/or RNA from DRG or trigeminal (for positive
tissues) and cerebral hemisphere, cerebellum and liver (for negative tissues)
is
electrophoresed in denaturing 1 % agarose-formaldehyde gel or agarose-glyoxal
gel, and then is transferred to a nylon membrane as described in achieved in
multiple steps, as detailed in standard molecular biology manuals [59, 60J.
Radiolabeled (specific activity of >10$ dpm/ug) or Digixoginen-labeled RNA
probes are typically used for Northern analysis. An antisense RNA probe (see
Example 20, which describes in situ hybridization with a NaN specific probe)
is
created by in vitro synthesis from a sense DNA fragment. The membrane
carrying the immobilized RNA in wetted with 6x SSC, and the membrane is
placed RNA-side-up in a hybridization tube. One ml formamide
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prehybridization/hybridization solution per 10 cm2 of membrane is added.
Prehybridization and hybridization are usually carried out in glass tubes in a
commercial hybridization oven. The tubes are place in a hybridization oven and
incubated, with rotation, at 60°C for 15 min to 1 hr. The desired
volume of
probe is pipeted into the hybridization tube, and the incubation is continued
with rotation overnight at 60°C. The probe concentration in the
hybridization
solution should be 10 ng/ml if the specific activity is 10$ dpm/ug or 2 ng/ml
if
the specific activity is 109 dpm/ug (use 2-10 ng/ml of Digixogenin labeled
probe).
The hybridization solution is poured off and an equal volume of 2x
SSC/0.1% SDS is added. Incubation with rotation for 5 min at room
temperature is carried out. The wash solution is changed, and this step is
repeated. To reduce background, it may be beneficial to double the volume of
the wash solutions. The wash solution is replaced with an equal volume of 0.2x
SSC/0.1% SDS and the tube is incubated for 5 min with rotation at room
temperature. The wash solution is changed and this step is repeated (this is a
low-stringency wash). For moderate or high stringency conditions, further
washes are done with wash solutions pre-warmed to moderate (42°C) or
high
(68°C) temperatures. The final wash solution is removed and the
membrane
rinsed in 2x SSC at room temperature. Autoradiography is then performed for
up to 1 week. Alternatively, signal is detected using chemiluminescence
technology (Arnersham) if non-radioactive probes are used. The transcript size
is calculated from the signal from the gel in comparison with gel molecular
weight standard markers.
Example 13: tissue ecific distribution of NaN by RT-PCR
NaN specific forward (5' CCCTGCTGCGCTCGGTGAAGAA 3') and
reverse primer (5' GACAAAGTAGATCCCAGAGG 3'), were used in RT-PCR
assays using cDNA template prepared from multiple rat. These primers amplify
NaN sequence between nucleotides 765 and 1156 (392 bp) and are NaN specific
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as judged by lack of similarity to sequences in the database (using programs
like
BLASTN from the National Library of M~icine). Amplification was typically
performed in a 60 lel volume using 1 pl of the first strand of cDNA, 0.8 p,M
of
each primer and 1.75 units of Expand Long Template DNA polymerase enzyme
mixture (Boehringer Mannheim). Compared to conventional and thermostable
DNA polymerases, Expand Long Template enzyme mixture increases the yield
of the.PCR products without an increase in non-specific amplification [49,
50].
The PCR reaction buffer consisted of 50 mM Tris-HCl (pH 9.2), l6mM
(NH4)ZSO,, 2.25 mM MgClz, 2% (v/v) DMSO and 0.1% Tween 20. As
described previously [71 ], amplification was carried out in two stages using
a
programmable thermal cycler (PTC-200, MJ Research, Cambridge, MA.). First,
a denaturation step is performed at 94°C for 4 min., followed by an
annealing
step at 60 ° C for 2 min, and then an elongation step at 72 ° C
for 90 sec. Second,
a denaturation step is performed at 94°C for 1 min, followed by an
annealing
15 step at 60 ° C for 1 min, and then an elongation step at 72 °
C for 90 sec. The
second stage was repeated 33 times for a total of 25-35 cycles, with the
elongation step in the last cycle extended to 10 min.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an
internal control to ensure that a lack of NaN signals in different tissues was
not
20 due to degraded templates or presence of PCR inhibitors. Rat GAPDH
sequences were co-amplified using primers which amplify a 66 by product that
corresponds to nucleotides 328-994 (accession number: M17701). The
amplified product spans multiple exon/intron splice sites, based on the
structure
of the human gene [72]. Dnase I treatment was routinely performed prior to
25 reverse transcription to prevent amplification of GAPDH sequences from
genomic processed pseudogenes that may have contaminated the total RNA
preparation [73].
NaN is primarily and preferentially expressed in DRG and trigeminal
ganglia neurons. Figure 4 shows the results of screening by RT-PCR for the
30 expression of NaN in various neuronal and non-neuronal tissues. Lanes 1, 2,
4,
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9 and 16 show a single amplification product co-migrating with the 400 by
marker, consistent with NaN specific product of 392 bp. Lanes 1 and 16, 2, 4
and 9 contain products using DRG, cerebral hemisphere, retina; and trigeminal
ganglia, respectively. Using this assay, NaN was not detected in cerebellum,
optic nerve, spinal cord, sciatic nerve, superior cervical ganglia, skeletal
muscle,
cardiac muscle, adrenal gland, uterus, liver or kidney (lanes 3, 5-8, and 10-
15,
respectively). The attenuated NaN signal in cerebral hemisphere and retina,
and
the absence of this signal in the remaining tissues is not due to degraded RNA
or the presence of PCR inhibitors in the cDNA templates as comparable
10 GAPDH amplification products were obtained in a parallel set of PCR
reaction
(data not shown).
Example 14: Transformation of a Host Cell with the NaN Co 'ng
Sequence
Transformed host cells for the measurement of Na+ current or
intracellular Na+ levels are usually prepared by co-transfecting constructs
into
cells such as HEK293 cells with a fluorescent reporter plasmid (pGreen
Lantern-I, Life Technologies, Inc.) using the calcium-phosphate precipitation
technique [27]. HEK293 cells are typically grown in high glucose DMEM (Life
Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies,
20 Inc). After 48 hrs, cells with green fluorescence are selected for
recording [28].
For preparation of cell lines continuously expressing recombinant
channels, the NaN construct is cloned into other vectors that carry a
selectable
marker in mammalian cells. Transfections are carried out using the calcium
phosphate precipitation technique [27]. Human embryonic kidney (HEK-293),
Chinese hamster ovary (CHO) cells, or other suitable cell lines are grown
under
standard tissue culture conditions in Dulbeccos's modified Eagle's medium
supplemented with 10% fetal bovine serum. The calcium phosphate-DNA
mixture is added to the cell culture medium and left for 15-20 hr, after which
time the cells are washed with fresh medium. After 48 hrs, antibiotic (G418,
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Geneticin, Life Technologies) is added to select for cells which have acquired
neomycin resistance. After 2-3 weeks in 6418, 10-20 isolated cell colonies are
harvested using sterile l Oml pipette dps. Colonies are grown for another 4-7
days, split and subsequently ested for channel expression using whole-cell
patch-clamp recording techniques and RT-PCR.
Example 15: Production of NaN specific Antibodies
Antibodies specific to the rat, mouse or human NaN are produced with
an immunogenic NaN-specific peptide by raising polyclonal antibodies in
rabbits. In one example, the peptide CGPNPASNKD~FEKEKDSED (rat
amino acids 285-304) was selected because it fits the criteria for
immunogenecity and surface accessibility. This peptide sequence does not
match any peptide in the public databases. The underlined cysteine (C) residue
was changed to Alanine (A) to prevent disulfide bond formation. This amino
acid change is not expected to significantly affect the specificity of the
antibodies.
Peptide synthesis, rabbit immunization, and affnity purification of the
antipeptide antibodies were performed using standard procedures. Purified
antibodies were then tested on DRG neurons in culture. Immunostaining
procedures using these antibodies before and after blocking with excess
peptide
were performed according to standard procedures.
DRG neurons after 16-24 h in culture were processed for
immunocytochemical detection of NaN pmtein as follows. Coverslips were
washed with complete saline solution (137 mM NaCI, 5.3 xnM KCI, 1 ITIM
M902 25 mM sorbitol, 10 mM HEPES, 3 mM CaC 12 pH 7.2), fixed with 4%
paraformaldehyde in 0. 14 M phosphate buffer for 10 min at 4°C, washed
with
three 5-min with phosphate-buffered saline (PBS), and blocked with PBS
containing 20% normal goat serum, 1 % bovine serum albumin and 0. 1
Triton X- 100 for 15 minutes. The coverslips were incubated in anti-NaN
antibody (1:100 dilution) at 4°C overnight. Following overnight
incubation,
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coverslips were washed extensively in PBS and then incubated with goat anti-
rabbit IgG-conjugated to Cy3 (1:3000; Amersham) for 2 h at room temperature.
The coverslips were rinsed with PBS and mounted onto glass slides with Aqua-
poly-mount. The neurons were examined with a Leitz Aristoplan light
5 microscope equipped with epifluorescence and images were captured with a
Dage DC330T color camera and Scion CG-7 color PCI frame grabber (see
Figure 7).
Example 16: N~~ression is altered in a neuro~rain model
The CCI model of neuropathic pain (Bennett and Xie) was used to study
the plasticity of sodium channel expression in DRG neurons. Twenty two adult,
femal Sprague-Dawley rats, weighing 240-260g; were anesthetized with
pentobarbital sodium (50 mg/kg ip) and the right sciatic nerve exposed at the
mid-thigh. Four chromic gut (4-0) ligatures were tied loosely around the nerve
as described by Bennett and Xie (1988) Pain 33, 87-107. The incision site was
15 closed in layers and: a bacteriostatic agent administered intramuscularly.
Previous studies have shown that transection of the sciatic nerve induces
dramatic changes in sodium currents of axotomized DRG neurons, which is
paralleled by significant changes to transcripts of various sodium channels
expressed in these neurons. Sodium currents that are TTX-R and the transcripts
of two TTX-R sodium channels (SNS/PN3 and NaN) are significantly
attenuated while a rapidly repriming silent TTX-S current emerges and the
transcript of a-III sodium channel, which produces a TTX-S current, is up-
regulated. We have discovered that CCI-induced changes in DRG neurons, 14
days post-surgery, mirror those of axotomy. Transcripts of NaN and SNS, the
two sensory neuron-specific TTX-R channels, are significantly down-regulated
as is the TTX-R sodium current, while transcripts of the TTX-S a-III sodium
channel are up-regulated, in small diameter DRG neurons. These changes may
be partly responsible for making DRG neurons hyperexcitable, that contributes
to the hyperalgesia that results from this injury.
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Example 17: Assa3rs for agents which modulate the activity of the NaN
channel using hatch clam~,ethods
Cells lines expressing the cloned Na'" channel are used to assay for
agents which modulate the activity of the NaN channel, e.g., agents which
block
or inhibit the channel or enhance channel opening. Since the channel
activation
is voltage dependent, depolarizing conditions may be used for observation of
baseline activity that is modified by the agent to be tested. Depolarization
may
be achieved by any means available, for example, by raising the extracellular
potassium ion concentration to about 20 to 40 nM, or by repeated electrical
pulses.
The agent to be tested is incubated with HEK 293 or other transformed
cells that express the Na+channel [28]. After incubation for a sufficient
period
of time, the agent induced changes in Na+ channel activity can be measured by
patch-clamp methods [29]. Data for these measurements are acquired on a
Macintosh Quadra 950, or similar computer, using a program such as Pulse (v
7.52, HEKA, German). Fire-polished electrodes (0.8-1.5 MVI~ are fabricated
from capillary glass using a Sutter P-87 pulley or a similar instrument. Cells
are
usually only considered for analysis if initial seal resistance is <5 Gohm,
they
have high leakage currents (holding current <0.1 nA at -80 mV), membrane
blebs, and an access resistance <5 Mohm. Access resistance is monitored and
data is not used if resistance changes occur. Voltage errors are minimized
using
series resistance compensation and the capacitance artifact will be canceled
as
necessary using computer-controlled amplifier circuitry or other similar
methods.
For comparisons of the voltage dependence of activation and
inactivation, cells with a maximum voltage error of <10 mV after
coiripensation
are usually used. Linear leak subtraction is used for voltage clamp
recordings.
Membrane currents are typically filtered at 5 KHz and sampled at 20 KHz. The
pipette solution contains a standard solution such as: 140 mM CsF, 2 mM
MgCl2, 1 mM EGTA, and 10 mM Na-HEPES (pH 7.3). The standard bathing
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solution is a standard solution such as 140 mM NaCI, 3 mM KCI, 2 mM
MgCl2, 1 mM CaCl2, 10 mM HEPES, and 10 mM glucose (pH 7.3).
Tetrodotoxin (TTX)-resistant and TTX-sensitive Na+ currents are
measured by exposure to appropriate concentrations of TTX and/or by pre-pulse
protocols which distinguish between TTX-sensitive and TTX-resistant currents
on the basis of their distinct steady-state inactivation properties [22,55].
Data are collected using standard pulse protocols and are analyzed to
measure sodium current properties that include voltage-dependence, steady-
state
characteristics, kinetics, and re-priming. Measurements of current amplitude
and cell capacitance provides an estimate of Na+ current density, thereby
permitting comparisons of channel density under different conditions [22,30].
Cells are studied in the current clamp mode to study patterns of spontaneous
and
evoked action potential generation, threshold for firing, frequency response
characteristics, and response to de- and hyperpolarization, and other aspects
of
15 electrogenesis [55]. These measurements are carried out both in control
cells
expressing NaN and in cells expressing NaN that also have been exposed to the
agent to be tested.
Example 18: Assays for agents which modulate the activit<r of the NaN
The agent to be tested is incubated with cells exhibiting NaN channel
activity. After incubation for a sufficient period of time, the agent inducod
changes in Na+ channel are measured by ratiometric imaging of [Na+ ], using
SBFI. In this method, cytosolic-free Na+ is measured using an indicator for
Na+,
such as SBFI (sodium-binding benzofuran isophthalate; [33]) or a similar dye.
Cells are first loaded with the membrane-permeable acetoxymethyl ester form
of SBFI (SBFI/AM) or a similar dye (usually dissolved in dimethyl sulfoxide
(DMSO) at a stock concentration of 10 mM). Recordings are obtained on the
stage of a microscope using a commercially available ratiometric imaging setup
(e.g., from Georgia Instruments). Excitation light is provided at appropriate
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wavelengths (e.g., 340:385 nm). Excitation light is passed to the cells thmugh
a
dichroic reflector (400 nm) and emitted light above 450 nm was collected.
Fluorescence signals are amplified, e.g., by an image intensifier (GenIISyS)
and
collected with a CCD camera, or similar device, interfaced to a frame grabber.
5 To account for fluorescence rundown, the fluorescence ratio 340:385 is used
to
assay cytosolic-free Na+.
Far calibration of SBFI's fluorescence, cells are perfused with
calibration solutions containing known Na+ concentrations (typically 0 and 30
mM, or 0, 30, and 50 mM [Na''], and gramicidin and monensin. As reported by
Rose and Ransom [34], the 345/390 nm fluorescence ratio of intracellular SBFI
changes monotonically with changes in [Na+ ];. Experiments are repeated on
multiple (typically at least 4) different coverslips, providing statistically
significant measurements of intracellular sodium in control cells, and in
cells
exposed to various concentrations of agents that may block, inhibit or enhance
15 the activity of the channel. Use of this method is illustrated in
Sontheimer et al.
[32].
Example 19: Assays for agents which modul~.te the activity of the NaN
channel by scin 'gLap~ic im~~g
Cells lines expressing the cloned Na+ channel are used to assay for
agents which modulate the activity of the NaN channel, e.g., agents which
block
the channel or enhance channel opening. For example, the agent to be tested is
incubated with HEK 293 or other transformed cells that express the Na+ channel
[28]. After incubation for a sufficient period of time, the agent induced
changes
in Na''"~ channel activity are detected by the measurement of Na+ influx by
25 isotopic methods. ZfiTa is a gamma emitter and can be used to measure Na+
flux
[31 ) and 86Rb+ can be used to measure Na+/K+ATPase activity which provides a
measure of Na channel activity [32] ~Rb+ ions are taken up by the
Na''!K+ATPase like K+ ions, but have the advantage of a much longer half life
~~ 42K+ [35). Thus, measurement of the unidirectional ouabain-sensitive 86Rb+
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uptake provides a quantitative method for assaying Na+/K+-ATPase activity
which follows action potentials.
Following incubation of cell expressing NaN to the isotope, the cellular
content of the isotope is measured by liquid scintillation counting or a
similar
method, and cell protein is determined using a method such as the
bicinchoninic
acid protein assay [36] following the modifications [37] for cultured cells.
~Na
and 86Rb+ fluxes are determined in the presence and absence of agents that may
block, inhibit, or enhance Na+. This permits deternunation of the actions of
these agents on NaN.
10 Example 20: j~, situ hybridization
a. Probes
Probes are prepared as described above in Example 5.
b. DRG Neuron Culture
Cultures of DRG neurons from adult rats were established as described
previously [70]. Briefly, lumbar ganglia (L4, LS) from adult Sprague Dawley
female rats were freed from their connective sheaths and incubated
sequentially
in enzyme solutions containing collagenase and then pepsin. The tissue was
triturated in culture medium containing 1:1 Dulbecco's modified Eagle's
medium (DMEM) and Hank's F12 medium and 10% fetal calf serum, 1.5
mg/ml trypsin inhibitor, I .5 mg/ml bovine serum albumin, 100 U/ml penicillin
and 0.1 mg/ml streptomycin and plated at a density of 500-1000 cells/mmi on
polyomithine/laminin coati coverslips. The cells were maintained at
37°C in
a humidified 95% air/5% C02 incubator overnight and then processed for in situ
hybridization cytochemistry as described previously [56, 57]. Trigeminal
ganglia can be cultured by a skilled artisan using similar methods.
c. Tissue Pre~ation
Adult female Sprague Dawley rats were deeply anesthetized, e.g., with
chloral hydrate and perfused through the heart, first with a phosphate-
buffered
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saline (PBS) solution and then with a 4% paraformaldehyde in 0.14 M
Sorensen's phosphate buffer, pH 7.4, at 4°C. Following perfusion
fixation,
dorsal root ganglia at levels L4 and LS and trigeminal ganglia were collected
and placed in fresh fixative at 4°C. After 2-4 hours, the tissue was
transferred
to a solution containing 4% paraformaldehyde and 30% sucrose in 0.14 M
phosphate buffer and stored overnight at 4°C. Fifteen ~,m sections were
cut and
placed on poly-L-lysine-coated slides. The slides were processed for in situ
hybridization cytochemistry as previously described [24, 56]. Following in
situ
hybridization cytochemistry, the slides were dehydrated, cleared and mounted
with Permount. The results are shown in Fig. 5.
Sections of DRG hybridized with NaN sense riboprobe showed no
specific labeling (panel C, Fig. 5). In DRG (panel A, Fig. 5) and trigeminal
(panel B) sections hybridized with a NaN antisense riboprobe, with the NaN
signal present in most small (<30 mm diam.) neurons; in contrast, most large
15 (>30 mm diam.) neurons did not exhibit NaN hybridization signal. Sections
of
spinal cord, cerebellum and liver hybridized with an antisense NaN ribopmbe
showed no specific signal (panels D, E and F respectively).
Example 21: bite Seauencg,~
The following are the marine intronic microsatellite sequences. These
microsatellites may be polymorphic in the human SCNl 1 a gene and could be
used as markers to screen for mutant alleles that are associated with a
disease.
Such screening methods, for instance, hybridization or amplification assays,
are
readily available. See Sambrook et al. or Ausubel et al.
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Intron 4; microsatellite is dTdg
AGTTTAATGTTGAGTGAATTGTGGTGGTGATTTCCCACTTGAG
GCCTTTGTGTTAAAGCCCAATGTGTGTGTGTGTGTGTGTGTGTGTGTG
TGTGTGTGTGTGTGTGTGTGTGTGTGGTTGGGGGGTGGTGGCAGAGT
CTGGTATTGGTAAGGTGAGAGCAATCCCAGAACGTCCACCTGCTCTT
CCATTTTATTAATCAGGCAGGCCTCT
Intron 5; microsatellite is dGdTdG (dNdG2)x (XS-30)
GTAAGCCACTGGCTCTTAACTAAAATGCTCGTTGGCATTAGAA
CATTTCTGAGCTGGGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGT
GGTGGTGGTGGTGGTGGTGGTGATGGTGGTGGTGGAGGTGGNGGTG
GAGGTGGTGGCTGTGGTGGTGGNGGTGGTGGTGGTGGTGGANGTGG
ANGTGGTGGCGTGGTGGTGGNGGTGGTGGTGGAGGTGGTGGCTGTG
GTGGTNGTGGTGGC
Intron 6; microsatellite is dCdA
TGTGCATGC>'TGATTCCCAGCTCCTATGGTCTGATTACTCGGT
CCTTAGGAGCAAGGCCAGACTGTCCACCCTGACACACACACACACA
CACACACACACACACACACACACACACACACACAGTGTAGAGAATT
ACCTCATTCTTGGAGTTTCTCTGGAAAAGGAATGTCTCAAAGCCAAG
TTCACAGAGCAACAGCTG
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Intmn 8; 5' microsatellite is dTdC followed by a stretch of dT
TGTTAGAAACTCTAAGACAATGAAGCACCATGCTGGAAATAA
GAGCACAAACTCTTTCTTCATGCATTACCCACTGCTTGTGCTTTCACC
TTAGTGCTCGTGCTCTCTCTTTCTCTCTCTCTCTCTCTCTCTCTCTCTC
TCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTTTTTTTTTTTT
Intron 8; 3' microsatellite is dCdA
CACACACACACACACACACACACACACACACACACACACAG
AGAAACACTGTCGCAGTCATACATATAAAGATAAATACATCTTAAA
AAAAGAACCATGTGATTGAGTTATAAAATATTCCAACTTAT
Intron l OB; microsatellite is dCdA followed, three nucleotides
downstream by dCdA3
AGGTCATTTCCTCTGCAGTGTGCTTGGCAGGAAAAACTTCCTG
GCTATTCAAGTCAGTGCCCTGCTTGATCATCCATGTATCACACACAC
ACAAAACAAACAAACAAACAAACAAAACCCTGGGGAAGAAGGAAG
AGGTTAAGCACATAGGCAGAGAGCAGCCAGGCTGACTCAGAGCAAA
CACCTGATCATTCTTCCAT
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Intron 12; microsatellite is dpydG (dt/dCdG)
GTGCTGGGATCAAAGGCGTGCGCCGCCACCACGCCCGGCCCC
TTTTTATGTTTCAAATTTACTTTTATCATGTGCACGTGTGTGGGTGCG
TGCATGTGTGTGCGTGCGTGTGCGTGTGNGTGTGNGTGTGTGTGTGT
S GTGTGTGTGTGTGTGTGTGTG
Intron 14; microsatellite is dCdA
CACACACACACACACACACACACACACACACACACACACACA
CACACACACACACACTTGCATCTTTGAGTTAATTGGATAGGCTGAGT
CTTACACCGGAATCATACTGTTGC
Intmn 1 SA; microsatellige is dCdA
CCAATGAGAGACTCTTGTCTCA,AAAAA.GCCATGGTGTCCAGA
TCCTGAGGAATAACACCTAAGAATGTGCTCTGGCCTGAAAACACAC
ACACACACACACACACACACACACACACACACAGTTTTATTTATTTA
TTTAAAAAAATATGTCTCTAGGCATTGCTGAAATGTCTCCTACAGGA
1S TTAAGTCAACCAGAGCCA
It should be understood that the foregoing discussion and examples
merely present a detailed description of certain preferred embodiments. It wiU
be apparent to those of ordinary skill in the art that various modification
and
equivalents can be made without departing from the spirit and scope of the
invention.
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