Note: Descriptions are shown in the official language in which they were submitted.
CA 02307062 2000-04-20
WO 99120754 PCT/US98/22321
A PH SENSITIVE POTASSIUM CHANNEL IN SPERMATOCYTES
CROSS-REFERENCES TO RELATED APPLICATIONS
This application claims the benefit of USSN 60/063,138, filed October 22,
1997, and USSN 60/076,172, filed February 27, 1998, both of which are
incorporated by
'reference.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with Government support under Grant No. RO1-
NS24785, awarded by the National Institutes of Health. The Government has
certain rights
in this invention.
FIELD OF THE INVENTION
The invention provides isolated nucleotide and amino acid sequences of
Slo3, a pH sensitive potassium channel expressed in sperm; antibodies to Slo3;
methods of
screening for Slo3 inhibitors and activators; and methods of identifying Slo3
homologs.
BACKGROUND OF THE INVENTION
Potassium channels are found in a wide variety of animal cells such as
nervous, muscular, glandular, immune or epithelial tissue. The channels
regulating these
currents open and allow the escape of potassium under certain conditions. The
outward
flow of potassium ions upon opening of these channels makes the interior of
the cell more
negative, counteracting depolarizing voltages applied to the cell. These
channels are
regulated, e.g., by calcium sensitivity, voltage-gating, and ATP-sensitivity.
The Drosophila Slol gene encodes a calcium-activated potassium channel
present in both neurons and muscle (Elkins et al., Proc. Natl. Acad. Sci.
U.S.A. 83:8415
(1986); Atkinson et al., Science 253:551 (1991); and Adelman et al., Neuron
9:209 (1992)).
Mammalian homologs of dSlol were cloned and found to be "Maxi" or BK (large
conductance) channel types, as the single channel conductance was 272 pS with
symmetrical potassium concentrations. Slol channels cloned from mouse and
human show
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
2
strong conservation of sequence and functional properties (Butler et al.,
Science 261:221-
224 (1993); Dworetzky et al., Brain Res. Mol. Brain Res. 27:189-193 (1994);
Tseng-Crank
et al., Neuron 13:1315-1330 (1994); McCobb et al., Am. J. Physiol. 269:H767-
H777
(1995); and Wallner et al., Rec. Chart. 3:185-199 (1995)). One proposed role
of the Slol
S channel is to provide negative feedback for the entry of calcium into cells
via voltage-
dependent calcium channels. Perhaps because of the versatility of this
mechanism, Slol
channels are expressed in many tissues, including brain, skeletal and smooth
muscle,
auditory hair cells, pancreas, and adrenal gland (Many, Nature 291:497-500
(1981); Pallotta
et al., Nature 293:471-474 (1981); Petersen & Mauryama, Nature 307:693-696
(1984);
Tabcharani & Misler, Biochim. Biophys. Acta. 982:62-72 (1990); Neely & Lingle,
J.
Physiol. 453:97-131 (1992)). In these tissues, Slol channels are involved in
diverse
functions such as regulating arteriolar smooth muscle tone (Brayden & Nelson,
Science
256:532-535 (1992)), tuning of hair cell frequency (Fucks, Curr. Op.
Neurobiol. 2:457-461
(1992); Wu et al., Prog. Biophys. Mol. Bio. 63:131-158 (1996)), and modulation
of
transmitter release at nerve terminals (Robitaille & Charlton, J. Neurosci.
12:297-305
(1995); Knaus et al., J. Neurosci. 16:955-963 (1996)), all situations in which
both
membrane potential and intracellular calcium are critical factors. While
numerous family
members of every type of voltage-gated K+ channel have been found, to date the
Slol
channel has remained the sole functionally characterized representative of the
Slo family
(Wei et al., Neuropharmacology 35:805-829 (1996)).
Spermatocytes require proteins tailored to fulfill roles unique to the process
of germ cell development and fertilization. Cellular signaling in spermatic
cells is tightly
regulated to prevent inappropriate activation of the irreversible steps that
prepare the sperm
to fertilize the oocyte. Many of these steps are triggered and coordinated by
changes in
membrane potential and intracellular Ca2+ concentration and pH. Between mating
and
fertilization, sperm undergo capacitation, a process which later enables them
to fertilize the
oocyte. Capacitation involves an increase in cytosolic pH (pHi), which
promotes metabolic
and swimming activity (Babcock et al., Proc. Natl. Acad. Sci. USA 80:1327-1331
(1983);
Babcock & Pfeiffer, J. Biol. Chem. 262:15041-15047 (1987); Vredenburgh-Wilberg
&
Parrish, Mol. Reprod. Dev. 40:490-502 (1995)). This increase in pHi is
accompanied by
changes in membrane potential and a rise in cytoplasmic Ca2+, which trigger
the acrosome
reaction upon contact with the oocyte (Arnoult et al., J. Cell Biol. 134:637-
645 (1996);
Florman, Dev. Biol. 165:152-164 (1994)). Because of the central importance of
these
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
events in development, many efforts have been made to identify the specific
proteins,
including ion channels, which regulate spermatic function. In particular,
there have been
reports of channels present in spermatocytes and spermatids that have been
proposed to play
central roles in these reactions (Cook & Babcock, J. Biol. Chem. 268:22402-
22407 (1993),
including voltage dependent calcium channels (Florman et al., Dev. Biol.
152:304-214
(1992); Arnoult et al., Proc. Natl. Acad. Sci. USA 93:13004-13009 (1996);
Lievano et al.,
FEBSLett. 388:150-154 (1996); Santi et al., Am. ,I. Physiol. 271:C1583-C1593
(1996)).
Apart from a cyclic nucleotide gated channel, however, few of these channels
have been
directly cloned from testis (Weyand et al., Nature 368:859-863 (1994)).
SUMMARY OF THE INVENTION
Potassium channels have evolved to play specialized roles in many
inexcitable tissues. The present invention provides for the first time
isolated nucleotide and
amino acid sequences of Slo3, a potassium channel with novel functional
properties,
abundantly expressed in spermatocytes. The physiological reactions that sperm
undergo to
achieve fertilization include changes in both pHi and membrane potential.
Although Slo3 is
a member of the Slo family, to which the large-conductance, calcium-activated
Slol
potassium channel belongs, Slo3 channels are not gated by calcium. Slo3
channels,
however, are activated by changes in intracellular pH and membrane potential.
Slo3
channels also exhibit markedly lower selectivity for K+ over Na+ than most
voltage-gated
K+ channels.
In one aspect, the invention provides an isolated nucleic acid encoding a
polypeptide monomer of a pH sensitive potassium channel. The monomer (i) has a
calculated molecular weight of between 120-156 kDa; (ii) has a unit
conductance of
approximately 80-120 pS when the monomer is in a functional tetrameric form of
a
potassium channel and is expressed in a Xenopus oocyte; (iii) has increased
activity above
approximately intracellular pH of 7.1; and (iv) specifically binds to
polyclonal antibodies
generated against SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:16 or SEQ ID N0:18.
In one embodiment, the nucleic acid encodes mSlo3 or hSlo3. In another
embodiment, the nucleic acid encodes SEQ ID NO:1, SEQ ID N0:16, or SEQ ID
N0:18.
In one embodiment, the nucleic acid selectively hybridizes under moderate
stringency
hybridization conditions to SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:17, or SEQ ID
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
4
N0:19. In one embodiment, the nucleic acid has a nucleotide sequence of SEQ ID
N0:2,
SEQ ID N0:4, SEQ ID N0:17, or SEQ ID N0:19.
In one embodiment, the nucleic acid is amplified by primers that selectively
hybridize under stringent hybridization conditions to the same sequence as the
primer sets
selected from the group consisting of
CTCGAACTCCCTAAAATCTTACAGAT (SEQ ID N0:8) and
TTCCGTTGAGCCAGGGGTCACCAGAATT (SEQ ID N0:9);
TCTGCTTTGTGAAGCTAAATCT (SEQ ID NO:10) and
TTTCAAAGCCTCTTTAGCGGTAA (SEQ ID NO:11); and
TTATGCCTGGATCTGCACTCTACATG (SEQ ID N0:12) and
ATAGTTTCCGTCTACTACCGAAA (SEQ ID N0:13).
In another embodiment, the nucleic acid is amplified by primers that
selectively hybridize under stringent hybridization conditions to the same
sequence as the
primer sets selected from the group consisting of:
GGCAGCGCTCATTCTTTCCTCCTT (SEQ ID N0:14) and
TGCCCAAAACCTCAACCCAA.A.ATA (SEQ ID NO:15).
In another aspect, the invention provides an isolated nucleic acid encoding at
least 15 contiguous amino acids from a pH sensitive potassium channel
polypeptide
monomer, said monomer having an amino acid sequence of SEQ ID NO:1, SEQ ID
N0:16,
or SEQ ID N0:18 and conservatively modified variants thereof.
In one embodiment, the nucleic acid encodes a pH sensitive potassium
channel polypeptide monomer having: (i) a unit conductance of 80-120 pS when
the
monomer is in a functional tetrameric form of a potassium channel and is
expressed in a
Xenopus oocyte; (ii) a molecular weight of between 120-156 kDa; and (iii)
increased
activity above an intracellular pH of 7.1; and where the nucleic acid either:
(i) selectively
hybridizes under moderate stringency hybridization conditions to a nucleotide
sequence of
SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:17 or SEQ ID N0:19; or (ii) encodes a
protein
which could be encoded by a nucleic acid that selectively hybridizes under
moderate
stringency hybridization conditions to a nucleotide sequence of SEQ ID N0:2,
SEQ ID
N0:4, SEQ ID N0:17 or SEQ ID N0:19.
In another aspect, the invention provides an isolated nucleic acid encoding a
polypeptide monomer of a pH sensitive potassium channel, the sequence: (i)
encoding a
monomer having a core domain that has greater than 60% amino acid sequence
identity to
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
amino acids 35-641 of an mSlo3 core domain as measured using a sequence
comparison
algorithm; and (ii) specifically binding to polyclonal antibodies raised
against the core
domain of SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:16, or SEQ ID N0:18.
In another aspect, the invention provides an isolated polypeptide monomer of
S a pH sensitive potassium channel, the monomer: (i) having a calculated
molecular weight of
between 120-156 kDa; (ii) having a unit conductance of approximately 80-120 pS
when the
monomer is in a functional tetrameric form of a potassium channel and is
expressed in a
Xenopus oocyte; (iii) having increased activity above approximately
intracellular pH of 7.1;
and (iv) specifically binding to polyclonal antibodies generated against SEQ
ID NO:1, SEQ
ID N0:3, SEQ ID N0:16, or SEQ ID N0:18.
In another aspect, the invention provides an antibody that selectively binds
to
mSlo3 or hSlo3.
In another aspect, the invention provides an expression vector comprising a
nucleic acid encoding a polypeptide monomer of a pH sensitive potassium
channel, the
monomer: (i) having a calculated molecular weight of between 120-156 kDa; (ii)
having a
unit conductance of approximately 80-120 pS when the monomer is in a
functional
tetrameric form of a potassium channel and is expressed in a Xenopus oocyte;
(iii) having
increased activity above approximately intracellular pH of 7.1; and (iv)
specifically binding
to polyclonal antibodies generated against SEQ ID NO:1, SEQ ID N0:3, SEQ ID
N0:16, or
SEQ ID N0:18.
In another aspect, the invention provides a host cell comprising the
expression vector.
In another aspect, the invention provides a method for identifying a
compound that increases or decreases ion flux through a pH sensitive potassium
channel,
the method comprising the steps of: (i) contacting the compound with a
eukaryotic host cell
or cell membrane in which has been expressed a pH sensitive potassium channel
monomer
polypeptide: (a) having a calculated molecular weight of between 120-156 kDa;(
b) having
a unit conductance of approximately 80-120 pS when the monomer is in the
functional
tetrameric form of a potassium channel and is expressed in a Xenopus oocyte;
and (c)
specifically binding to polycional antibodies generated against SEQ ID NO:1,
SEQ ID
N0:3, SEQ ID N0:16, or SEQ ID N0:18; and (ii) determining the functional
effect of the
compound upon the cell or cell membrane expressing the pH sensitive potassium
channel.
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
6
In one embodiment, the increased or decreased flux of ions is determined by
measuring whole cell conductance. In one embodiment, the pH sensitive
potassium channel
monomer polypeptide is recombinant.
In another aspect, the invention provides method of detecting the presence of
Slo3 in mammalian tissue, the method comprising the steps of (i) isolating a
biological
sample from a patient; (ii) contacting the biological sample with a Slo3-
specific reagent that
selectively binds to Slo3; and, (iii) detecting the level of Slo3-specific
reagent that
selectively associates with the sample.
In one embodiment, the Slo3 specific reagent is selected from the group
consisting of Slo3 specific antibodies, Slo3 specific oligonucleotide primers,
and Slo3
nucleic acid probes. In one embodiment, the sample is from a human.
In another aspect, the invention provides in a computer system, a method of
screening for mutations of Slo3 genes, the method comprising the steps of: (i)
receiving
input of a first nucleic acid sequence encoding a pH sensitive potassium
channel protein
1 S having a nucleotide sequence of SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:17,
SEQ ID
N0:19 and conservatively modified versions thereof; (ii) comparing the first
nucleic acid
sequence with a second nucleic acid sequence having substantial identity to
the first nucleic
acid sequence; and (iii) identifying nucleotide differences between the first
and second
nucleic acid sequences.
In one embodiment, the second nucleic acid sequence is associated with a
disease state.
In another aspect, the invention provides in a computer system, a method for
identifying a three-dimensional structure of Slo3 proteins, the method
comprising the steps
of:(i) receiving input of at least about 10 amino acids of an amino acid
sequence of a pH
sensitive potassium channel monomer or at least about 30 nucleotides of a
nucleic acid
encoding the protein, the protein having an amino acid sequence of SEQ ID
NO:1, SEQ ID
N0:3, SEQ ID N0:16, or SEQ ID N0:18, and conservatively modified versions
thereof;
and (ii) generating a three-dimensional structure of the protein encoded by
the amino acid
sequence.
In one embodiment, the amino acid sequence is a primary structure and said
generating step includes the steps of: (i) forming a secondary structure from
said primary
structure using energy terms encoded by the primary structure; and (ii)
forming a tertiary
structure from said secondary structure using energy terms encoded by said
secondary
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
7
structure. In one embodiment, the generating step includes the step of forming
a quaternary
structure from said tertiary structure using anisotropy terms encoded by the
tertiary
structure. In one embodiment, the method further comprises the step of
identifying regions
of the three-dimensional structure of the protein that bind to ligands and
using the regions to
identify ligands that bind to the protein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure lA-B: Primary sequence of mSlo3
Figure lA: Alignment of primary sequence of mSlo3 with BK Ca2+-
activated K+ channels mSlol (mouse) and dSlol (Drosophila). Hydrophobic
segments are
designated SO through S 10. Segments S 1-S6 represent the transmembrane
segments that
surround pore of the channel. The region designated "Calcium Bowl" has been
implicated
in the regulation of mSlo 1 by calcium. The core and tail domain structure of
Slo 1 has been
conserved (Wei et al., Neuron 13:671-681 (1994)). mSlo3 residues 35 through
641
encompass SO through S8, the core domain, and share 56% and 50% identity with
mSlol
and dSlol while interspecies homologs mSlol and dSlol exhibit 62% identity in
this region.
mSlo3 residues 686-1136 encompassing S9 and 510, the tail, share 39% identity
with
mSlol and dSlol while the interspecies homologs mSlol and dSlol share 68%
identity in
this region. A region having no significant homology between mSlol and mSlo3
is found
between SS and S9. An arrowhead indicates a phenylalanine residue (F) in a
region critical
for ion selectivity.
Figure 1B: Kyte-Doolittle hydrophilicity plots of mSlo3 and mSlol. The
mbr5 mSlol (Butler et al., 1992) and splice variant A2C2E2G5I0 dSlol (Atkinson
et al.,
Science 253:551-553 (1991); Adelman et al., Neuron 9:209-216 (1992)) sequences
are
shown. SIo2 is a more distantly related sequence present in the nematode
database (Wei et
al., Neuropharmacology 35:805-829 (I996).
Figure 2A-D: Expression of mSlo3 transcripts is largely restricted to the
testis
Figure 2A: RT-PCR of mouse brain, heart, skeletal muscle, kidney, testis,
lung and liver with mSlo3-specific primers, with (+) and without (-) addition
of reverse
transcriptase. An expected 156 by product is detected only with testis RNA.
Similar results
were obtained with two additional mSlo3 primer pairs specific to S8 to S9 and
S9 to S 10
regions.
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCTNS98/22321
8
Figure 2B: Control RT-PCR assays with a-actin specific primers produce an
expected 537 by product from all tissues. Additional negative controls with
genomic DNA
(10 ng) or primers alone are also shown.
Figure 2C: Northern blot analysis of total RNA (20 mg) from mouse brain,
heart, skeletal muscle, kidney, testis, lung and liver reveals an abundant
mSlo3 transcript
only in testis, with an approximate size of 4 kb.
Figure 2D: Northern blot analysis of polyadenylated RNA (2 mg) from
human tissues (spleen, thymus, prostate, testis, uterus, small intestine,
colon, and
leukocytes) with mSlo3, reveals a cross-hybridizing mRNA species only in
testis, with an
approximate size of 4 kb. For controls, the same blots were hybridized with a
human (3-actin
probe and results are shown below.
Figure 3A-D: mSlo3 channel sensitivity to voltage and pH from individual
inside-out
patches
1 S Figure 3A: Currents at constant voltage (+80 mV). Activity increases at
higher pH. The cytoplasmic surface was exposed to recording solution at
indicated pH;
Npo at pH 7.1, 7.3, 7.6, and 8.0 was 0.000, 0.014, 0.031, and 0.258,
respectively, based on
the presence of a minimum of five channels.
Figure 3B: Reversibility of pH effect. Activity in a single patch is shown
sequentially from top to bottom at indicated pH.
Figure 3C: Currents at constant pH 7.6. Activity increases at positive
potentials.
Figure 3D: Macroscopic current traces and corresponding current-voltage
relations. The diminished current at pH 7.1 was restored when the same patch
was exposed
to pH 8. The slight decrease in current amplitude between the conditions at pH
8 was likely
due to current rundown. Holding potential was -40; on-line leak subtraction
was employed.
Figure 4A-D: mSlo3 whole cell currents from Xenopus oocytes
Figure 4A: Manipulation of intracellular pH alters current amplitude. (Left)
Control currents at start of experiment. (Middle) Diminished current amplitude
after
intracellular acidification (12.5 minute perfusion with NaHC03 replacing NaCI
in nd96).
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
9
(Right) Recovery during alkalinization (10 minute perfusion with nd96
supplemented with
30 mM NH4C1). Voltage families are from -80 to +60 mV in 10 mV increments.
Figure 4B: Representative tail currents in 10 mM K+, 88 mM Na+ at
11°C.
Figure 4C: Tail current amplitude versus voltage for the currents shown in
S Figure 4B. Instantaneous currents at the time of the voltage jump were
calculated from
exponential fits of tail currents extrapolated back to time zero. Currents are
plotted versus
test potential (large filled squares). A Goldman-Hodgkin-Katz (GHK) current
equation was
fitted to the tail current-voltage relation (solid line) with Na+/K+
permeability ratio, P =
0.1 S; calculated underlying K+ (small triangles) and Na+ (small squares)
currents are also
shown.
Figure 4D: mSlo3 exhibits relatively low selectivity for K+ over Na+.
Reversal potential was determined by measuring tail currents in varying
external [K+]; [Na+]
was also varied so that the total monovalent concentration ([K+J + [Na+]) was
98 mM.
Points were fitted with a GHK equation where the Na+/K+ permeability ratio, P,
was
allowed to vary freely. Drosophila Shab reversal potentials were determined as
a control
(Wei et al., 1990). For mSlo3 reversal potential at 2, 5, 10, 50, and 98 [K+],
n = 6, 3, 5, 6, 7,
respectively; for dShab at 2, 10, 50, and 98, n = 3, 1, 3, 3.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
The present invention provides for the first time isolated nucleotide and
amino acid sequences for Slo3 and demonstrates that Slo3 is a pH sensitive
potassium,
voltage-gated channel. Functionally, Slo3 is expressed in spermatocytes, is
voltage-gated,
and is pH sensitive. Furthermore, Slo3 exhibits markedly lower selectivity for
K+ over Na+
than most voltage-gated potassium channels. Among a wide panel of tissues,
both mSlo3
(mouse Slo3) and hSlo3 (human Slo3) mRNA were detected in testis, where the
mRNA was
abundantly expressed in developing spermatocytes. This expression pattern and
sensitivity
to both pH and voltage, indicate that Slo3 is involved in sperm capacitation
and/or the
acrosome reaction, essential steps in fertilization.
When mSlo3 protein (mouse Slo3) is recombinantly expressed in Xenopus
oocytes, the homotetramer channel protein has a unitary conductance of between
80 and
120 pS (as measured with symmetrical potassium concentrations); and for
example, at a
concentration of 160 mM potassium, the channel has a conductance of 106 pS.
Unitary
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
conductance may be conveniently determined using single channel or macroscopic
channel
inside-out or outside-out patch clamp configurations or whole cell recordings
(see Wei et
al., 1994, supra). Patch clamp and whole cell recording methods are well known
in the art
(see, e.g., Franciolini, F.-rperientia, 42:589-594 (1986); and Sakmann et al.,
Annual Review
5 ofPhysiology, 46:455-472 (1984)).
The isolated Slo3 proteins within the scope of the present invention include
those which when expressed in a cell from a quiet line, define a functionality
and
pharmacology indicative of a Slo3 channel. A quiet line is a cell line that in
its native state
(e.g., not expressing Slo3 channels) has low or uninteresting electric
activity, e.g., a CHO
10 cell line. For example, a control cell (without expression of a Slo3
channel of the present
invention) and an experimental cell (expressing a Slo3 channel) are maintained
under
conditions standard for measurement of electrophysiological parameters as
provided in the
working examples disclosed herein.
For example, a cell expressing a SIo3 channel of the present invention can
have a conductance of between 80-120 pS, can comprise an Slo3 channel protein
monomer
of about 120 to 156 kD, can exhibit pH sensitivity (e.g., increased activity
at above
approximately pHi 7.1 ) and voltage-gating, can exhibit amino acid identity of
at Ieast 60%,
and more preferably at least 70%, 80%, 90% or 95% in an alignment with the
core domain
of the exemplary mouse and human Slo3 channel sequences disclosed herein, and
can be
specifically reactive, under immunologically reactive conditions, with an
antibody raised to
an exemplary Slo3 sequence disclosed herein (e.g., SEQ ID NO:1, 3, 16 and 18).
Such
standard methods aid in the identification of Slo3 proteins of the present
invention.
Structurally, the full length nucleotide sequence of mSlo3 (SEQ ID N0:2)
encodes a protein of 1113 amino acids (SEQ TD NO:1 ) with a predicted
molecular mass of
126 kDa. hSlo3 encodes a protein of a similar size and expression pattern (see
Example II).
Slo3 is a member of the Slo of potassium channel protein family as evidenced
by sequence
homology to the BK calcium-activated potassium channel (SIol; see Figure lA).
The
hydrophilicity profiles of Slol and SIo3 sequences indicate 11 hydrophobic
segments, SO
through 510, which can be divided into "core" and "tail" domains (Figure 1B).
Within the
core domain (hydrophobic regions SO-S8 of Slo3 proteins) mSlo3 and the
sequenced region
of hSlo3 share at least 61.5% amino acid identity, while mSlol and mSlo3 share
51%
identity in this region. Homology in the core domain is much higher than in
the tail
domain, which is involved in calcium sensing (Wei et al., Neuron 13:671-681
(1994)).
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
11
Two notable differences suggest possible functional distinctions between
mSlol and mSlo3. First, the "Calcium Bowl," a hyperconserved aspartate-rich
region
involved in calcium sensing, is absent in Slo3 (Schreiber & Salkoff, Biophys.
J. 73:13SS-
1363 (1997)). Second, mSlo3 contains GFG rather than GYG in the conserved pore
S signature sequence involved in K+ ion selectivity (Yool & Schwarz, Nature
349:700-704
( 1991 ); Hartmann et al., Science 2S 1:942-944 ( 1991 ); Heginbotham &
MacKinnon,
Biophys. J. 66:1061-1067 (1994)). This difference shows that ionic selectivity
differs
between the two channels.
Specific regions of the SIo3 nucleotide and amino acid sequence may be
used to identify polymorphic variants, interspecies homologs, and alleles of
Slo3. This
identification can be made in vitro, e.g., under stringent hybridization
conditions and
sequencing, or by using the sequence information in a computer system for
comparison with
other nucleotide sequences. Typically, identification of polymorphic variants
and alleles of
Slo3 is made by comparing the amino acid sequence of the core domain (amino
acids 3S-
1 S 641 of mSlo3, SO through S8 of Slo3}. This domain is also useful for
identifying members
of the Slo family,. For example, potassium channel proteins that share at
least 60% or
greater amino acid identity in the core domain are Slo3 proteins, while those
that share
approximately SO% or less homology are members of the Slo family. Another
useful region
for identifying homologs of SIo3 is the region between S8 and S9. This region
is not highly
conserved between mSlol and mSlo3, and so can be used to identify interspecies
homologs
of Slo3. Antibodies that bind specifically to the core domain of Slo3 can also
be used to
identify alleles, interspecies homologs, and polymorphic variants.
Polymorphic variants, interspecies homologs, and alleles of Slo3 are
confirmed by expressing the putative Slo3 polypeptide monomer and examining
functional
2S characteristics, such as voltage-gating and pH sensitivity. This assay is
used to demonstrate
that a protein having about 60% or greater, preferably 75-80% or greater amino
acid identity
to the core domain of SIo3 shares the same functional characteristics as Slo3
and is
therefore a species of SIo3. Typically, Slo3 having the amino acid sequence of
SEQ ID
NO:1 or 3 is used as a positive control in comparison to the putative Slo3
protein to
demonstrate the identification of a polymorphic variant, allele, or
interspecies homologue of
SIo3.
For example, measurements of the cell expression the putative Slo3 are taken
to detect induction of ion flux (e.g., by radiotracer), or a change in ionic
conductance of the
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
12
cell (e.g., by patch clamp), or a change in voltage (e.g., by fluorescent
dye). If the presence
of an ion channel is indicated by a pH induced or voltage-gated change,
subsequent tests are
used to characterize the channel as a Slo3 channel of the present invention.
For example,
pH sensitivity can be determined as described above, using inside out patches
in saline of
different pH. Whole cell recordings in sodium bicarbonate or ammonium chloride
can also
be used. Compounds such as rotenone and FCCP can be used to acidify the cell,
and
protonofors can also be used to determine pH sensitivity. Preferably, at least
two
characteristics are determined, more preferably at least 3, or 4 are
determined.
Characteristics of Slo3 channels of the present invention are disclosed more
fully herein.
The present invention also provide polymorphic variants of the mSlo3
depicted in SEQ ID NO:1: variant #1, in which a valine residue is substituted
for an
isoleucine residue at amino acid position 21; variant #2, in which an
isoleucine residue is
substituted for a leucine residue at amino acid position 5; and variant #3, in
which a serine
residue is substituted for an alanine residue at amino acid position 25.
The present invention also provide polymorphic variants of the hSlo3
depicted in SEQ ID N0:16: variant #1, in which a valine residue is substituted
for an
isoleucine residue at amino acid position 23; variant #2, in which an
isoleucine residue is
substituted for a leucine residue at amino acid position 6; and variant #3, in
which a serine
residue is substituted for an alanine residue at amino acid position 25.
The isolation of Slo3 protein for the first time provides a means for assaying
for compounds that increase or decrease the ion channel activity of this pH
sensitive
potassium channel, which is involved in sperm physiology. SIo3 nucleic acids
and proteins
are useful for testing inhibitors or activators of Slo3 using in vitro or in
vivo assays, e.g.,
expressing Slo3 in cells or cell membranes and then measuring flux of ions
through the
channel. Such inhibitors or activators identified using Slo3 can be used
therapeutically to
treat infertility conditions related to sperm physiology, or as
contraceptives. SIo3
expression also provides a convenient diagnostic marker for spermatocytes.
Spenmatocytes
lacking Slo3 expression may be indicative of sperm that lack the capability of
undergoing
capacitation or acrosome reactions, which are essential for fertilization.
Antibodies or other
probes for Slo3 can be also used in vitro as diagnostic tools to examine Slo3
expression.
SIo3 can also be used to study sperm physiology in vitro, e.g., the
capacitation and
acrosome reactions that are essential for sperm activity.
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCTNS98/22321
13
Portions of the Slo3 nucleotide sequence may be used to identify homologs
of the channel, as well as variants or mutations of the channel that may be
associated with
disease. This identification can be made in vitro or by using the sequence
information in a
computer system for comparison with other nucleotide sequences. Similarly,
these portions
of Slo3 nucleotide sequence may be used to determine the presence of a Slo3
channel
mRNA or channel protein in a particular tissue of interest. Information
derived from the
SIo3 nucleotide sequence may also be used to identify the chromosomal
localization of the
Slo3 gene or genes using chromosomal panels, radiation hybrid screening,
fluorescent in
situ hybridization methods (FISH), or by comparison of the sequence with
computer nucleic
acid databases. The Slo3 channel or fragments thereof may also be used to
treat diseases
using gene therapy. A SIo3 nucleotide sequence information may also be used to
construct
models of the ion channel protein in a computer system, these models
subsequently being
used to identify compounds that can modulate channel function.
Furthermore, the invention provides assays for SIo3 activity where SIo3 acts
as a direct or indirect reporter molecule, e.g., as part of a chimera with
another channel
protein such as Slol. Such uses of Slo3 as a reporter molecule in assay and
detection
systems have broad applications, e.g., Slo3 can be used as a reporter molecule
to measure
changes in potassium concentration, membrane potential, current flow, ion
flux,
transcription, signal transduction, receptor-ligand interactions, second
messenger
concentrations, in vitro, in vivo, and ex vivo. In one embodiment, Slo3 can be
used as an
indicator of current flow in a particular direction (e.g., outward or inward
potassium flow),
and in another embodiment, Slo3 can be used as an indirect reporter via
attachment to a
second reporter molecule such as green fluorescent protein.
II. Definitions
Unless otherwise indicated, nucleic acids are written left to right in 5' to
3'
orientation; amino acid sequences are written left to right in amino to
carboxy orientation,
respectively. Numeric ranges are inclusive of the numbers defining the range.
The terms
defined below are more fully defined by reference to the specification as a
whole.
By "pH sensitive potassium channel" or "Slo3 channel" is meant a
membrane channel which is voltage-gated, pH sensitive (e.g., with increased
activity as
measured by increased current amplitude above about pHi 7.1), and has a
unitary
conductance of from about 80-120 pS when measured under a symmetrical
potassium
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/Z2321
14
concentration of 160 rnM in a Xenopus oocyte using the conditions specified in
Example
IV. An Slo3 channel comprises multiple Slo3 channel proteins as subunits,
typically four
Slo3 channel proteins (e.g., full length or substantially full length Slo3
channel proteins).
A "Slo3 core domain" refers to the amino acids corresponding to the SO-S8
region of a Slo3 polypeptide, e.g., amino acids 35-641 of mSlo3 (SEQ ID NO:1
).
By "pH sensitive potassium channel subunit" or "Slo3 channel protein" is
meant a polypeptide of a molecular weight of between about 120-156 kDa having
a core
domain with at least about 60% identity to the core domain of SEQ ID NO:I, 3,
18, or 18,
and having the characteristic of pH sensitivity and/or voltage gating. These
proteins serve
as monomers of the Slo3 channel. Thus, a Slo3 channel protein can have the
functional
characteristics to form a heteromeric or homomeric protein with the functional
characteristics of an Slo3 channel, or be a peptide fragment thereof. This
term refers both to
recombinant and naturally occurnng forms of SIo3. Both recombinant and
naturally
occurring Slo3 can be used in the methods of the invention described herein,
e.g., in assays
to identify inhibitors or activators of Slo3.
The term Slo3 therefore refers to polymorphic variants, alleles, mutants, and
interspecies variants of Slo3 that: (1) have greater than 60% amino acid
sequence identity to
a Slo3 core domain; or (2) bind to antibodies raised against an immunogen
comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
N0:3,
SEQ ID N0:16, or SEQ ID N0:18 and conservatively modified variants thereof; or
(3)
specifically hybridize under stringent hybridization conditions to a sequence
selected from
the group consisting of SEQ ID N0:2, SEQ ID N0:17, SEQ ID N0:19 and
conservatively
modified variants thereof; or (4) are amplified by primers that specifically
hybridize under
stringent hybridization conditions to the same sequence as a primer set
consisting of SEQ
ID NOS:9 and 9, 10 and 11, 12 and 13 or 14 and 15.
"pH sensitive" refers to a characteristic of Slo3 channels, where the channels
have increased current amplitude in response to changes in intracellular pH
(pHi).
Typically, pH sensitive channels show increased current amplitude above
approximately
pHi 7.1. For example, mSlo3 at a pHi of 6.8 did not show increased activity,
while activity
was substantially increased at pHi 7.8. pH sensitivity can be measure using a
number of
assays. For example, single channel recordings are made from inside out
patches that have
been perfused with saline of different pHs and the open probability of the
channel vs. the
pH is plotted to determine pH sensitivity. In another example, macroscopic
current is
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
examined with an inside out patch perfused with saline of varying pHs, and the
amplitude of
the current is measured. In another example, a whole cell recording is made
with a two
electrode voltage clamp, where the cell is in sodium bicarbonate solution
(lowers
intracellular pH) or ammonium chloride solution (raises intracellular pH).
The phrase "voltage-gated" activity or "voltage-gating" refers a
characteristic of a potassium channel composed of individual polypeptide
monomers or
subunits. Generally, the probability of a voltage-gated potassium channel
opening increases
as a cell is depolarized. Voltage-gated potassium channels primarily allow
efflux of
potassium because they have greater probabilities of being open at membrane
potentials
10 more positive than the equilibrium potential for potassium (EK) in typical
cells. EK is the
combination of the voltage potential and [K+] potential at which there is no
net flow of
potassium ion. This value, also known as the "reversal potential" or the
"Nernst" potential
for potassium, depends on the relative concentrations of potassium found
inside and outside
the membrane, and is typically between -60 and -100 mV for mammalian cells.
Some
15 voltage-gated potassium channels undergo inactivation, which can reduce
potassium efflux
at higher membrane potentials. These channels can also allow potassium influx
in certain
instances when they remain open at membrane potentials negative to EK (see,
e.g., Adams
& Nonner, in Potassium Channels, pp. 40-60 (Cook, ed., 1990)).
Typically, the channel protein is composed of four or more subunits and the
channel can be heteromeric or homomeric. "Homomeric" refers to a potassium
channel
composed of the same type of subunit (alpha subunits), while "heteromeric"
refers to a
potassium channel composed of two or more different types of subunits, (alpha
and beta
subunits). Beta subunits alone typically do not form a channel. Heteromeric
channels can
have heteromeric pores, or can have homomeric pores composed of four alpha
units, with a
beta subunit associated outside the pore region. Voltage-gated potassium
channels
composed of Slo are typically homomeric, having four Slo subunits. The
characteristic of
voltage gating can be measured by a variety of techniques for measuring
changes in current
flow and ion flux through a channel, e.g., by changing the [K+] of the
external solution and
measuring the activation potential of the channel current (see, e.g., U.S.
Patent No.
5,670,335), by measuring current with patch clamp techniques or voltage clamp
under
different conditions, and by measuring ion flux with radiolabeled tracers or
voltage-
sensitive dyes under different conditions.
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCTNS98/22321
16
The terms "isolated" "purified" or "biologically pure" refer to material that
is
substantially or essentially free from components which normally accompany it
as found in
its native state. Purity and homogeneity are typically determined using
analytical chemistry
techniques such as polyacrylamide gel electrophoresis or high performance
liquid
chromatography. A protein that is the predominant species present in a
preparation is
substantially purified. In particular, an isolated SIo3 nucleic acid is
separated from open
reading frames which flank the Slo3 gene and encode proteins other than Slo3.
The term
"purified" denotes that a nucleic acid or protein gives rise to essentially
one band in an
electrophoretic gel. Particularly, it means that the nucleic acid or protein
is at least 85%
pure, more preferably at least 95% pure, and most preferably at least 99%
pure.
The term "subsequence" in the context of a referenced nucleic acid sequence
includes reference to a contiguous sequence from the nucleic acid having fewer
nucleotides
in length than the referenced nucleic acid. In the context of a referenced
protein,
polypeptide, or peptide sequence, "subsequence" refers to a contiguous
sequence from the
1 S referenced protein having fewer amino acids than the referenced protein.
The term "heterologous" when used with reference to portions of a nucleic
acid indicates that the nucleic acid comprises two or more subsequences which
are not
found in the same relationship to each other in nature. For instance, the
nucleic acid is
typically recombinantly produced, having two or more sequences from unrelated
genes
arranged to make a new functional nucleic acid.
"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and
polymers thereof in either single- or double-stranded form. The term
encompasses nucleic
acids containing known nucleotide analogs or modified backbone residues or
linkages,
which are synthetic, naturally occurring, and non-naturally occurnng, which
have similar
binding properties as the reference nucleic acid, and which are metabolized in
a manner
similar to the reference nucleotides. Examples of such analogs include,
without limitation,
phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl
phosphonates, 2-
O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
Unless otherwise indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g., degenerate codon
substitutions,
as described below) and complementary sequences, as well as the sequence
explicitly
indicated. The term nucleic acid is used interchangeably with gene, cDNA,
mRNA,
oligonucleotide, and polynucleotide.
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCTNS98/22321
17
The terms "polypeptide," "peptide" and "protein" are used interchangeably
herein to refer to a polymer of amino acid residues. The terms apply to amino
acid
polymers in which one or more amino acid residue is an analog or mimetic of a
corresponding naturally occurnng amino acid, as well as to naturally occurnng
amino acid
polymers.
The term "amino acid" refers to naturally occurnng and synthetic amino
acids, as well as amino acid analogs and amino acid mimetics that function in
a manner
similar to the naturally occurring amino acids. Naturally occurring amino
acids are those
encoded by the genetic code, as well as those amino acids that are later
modified, e.g.,
hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs
refers to
compounds that have the same basic chemical structure as a naturally occurring
amino acid,
i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino
group, and an R
group., e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl
sulfonium
Such analogs have modified R groups {e.g., norleucine) or modified peptide
backbones, but
1 S retain the same basic chemical structure as a naturally occurnng amino
acid. Amino acid
mimetics refers to chemical compounds that have a structure that is different
from the
general chemical structure of an amino acid, but that functions in a manner
similar to a
naturally occurring amino acid.
Amino acids may be referred to herein by either their commonly known
three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB
Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to
by their
commonly accepted single-letter codes.
"Conservatively modified variants" applies to both amino acid and nucleic
acid sequences. With respect to particular nucleic acid sequences,
conservatively modified
variants refers to those nucleic acids which encode identical or essentially
identical amino
acid sequences, or where the nucleic acid does not encode an amino acid
sequence, to
essentially identical sequences. Specifically, degenerate codon substitutions
may be
achieved by generating sequences in which the third position of one or more
selected (or all)
codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et
al., Nucleic
Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985);
Rossolini
et al., Mol. Cell. Probes 8:91-98 (1994)). Because of the degeneracy of the
genetic code, a
large number of functionally identical nucleic acids encode any given protein.
For instance,
the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at
every
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
18
position where an alanine is specified by a codon, the codon can be altered to
any of the
corresponding codons described without altering the encoded polypeptide. Such
nucleic
acid variations are "silent variations," which are one species of
conservatively modified
variations. Every nucleic acid sequence herein which encodes a polypeptide
also describes
every possible silent variation of the nucleic acid. One of skill will
recognize that each
codon in a nucleic acid (except AUG, which is ordinarily the only codon for
methionine,
and TGG, which is ordinarily the only codon for tryptophan) can be modified to
yield a
functionally identical molecule. Accordingly, each silent variation of a
nucleic acid which
encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual
substitutions, deletions or additions to a nucleic acid, peptide, polypeptide,
or protein
sequence which alters, adds or deletes a single amino acid or a small
percentage of amino
acids in the encoded sequence is a "conservatively modified variant" where the
alteration
results in the substitution of an amino acid with a chemically similar amino
acid.
Conservative substitution tables providing functionally similar amino acids
are well known
in the art. Such conservatively modified variants are in addition to and do
not exclude
polymorphic variants, interspecies homologs, and alleles of the invention.
The following groups each contain amino acids that are conservative
substitutions for one another:
1 ) Alanine (A), Glycine (G);
2) Serine (S), Threonine (T);
3) Aspartic acid (D), Glutamic acid (E);
4) Asparagine (I~, Glutamine (Q);
5) Cysteine (C), Methionine (M);
6) Arginine (R), Lysine (K), Histidine (H);
7) Isoleucine (I), Leucine (L), Valine (V); and
8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
(see, e.g., Creighton, Proteins (1984)).
A "label" is a composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, or chemical means. For example, useful labels
include 32P,
fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in
an ELISA),
biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal
antibodies are
available (e.g., the peptide of SEQ ID NO:1 can be made detectable, e.g., by
incorporating a
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
19
radio-label into the peptide, and used to detect antibodies specifically
reactive with the
peptide).
As used herein a "probe or primer" is defined as a nucleic acid capable of
binding to a target nucleic acid of complementary sequence through one or more
types of
chemical bonds, usually through complementary base pairing, usually through
hydrogen
bond formation. It will be understood by one of skill in the art that probes
may bind target
sequences lacking complete complementarity with the probe sequence depending
upon the
stringency of the hybridization conditions. The probes are preferably directly
labeled as
with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled
such as with
biotin to which a streptavidin complex may later bind. By assaying for the
presence or
absence of the probe, one can detect the presence or absence of the select
sequence or
subsequence.
A "labeled nucleic acid probe or oligonucleotide" is one that is bound, either
covalently, through a linker, or through ionic, van der Waals or hydrogen
bonds to a label
such that the presence of the probe may be detected by detecting the presence
of the label
bound to the probe.
"Amplification" primers are oligonucleotides comprising either natural or
analog nucleotides that can serve as the basis for the amplification of a
select nucleic acid
sequence. They include, e.g., polymerase chain reaction primers and ligase
chain reaction
oligonucleotides. Amplification primers are used to "amplify" a target nucleic
acid
sequence.
The term "recombinant" when used with reference to a cell, or protein,
nucleic acid, or vector, includes reference to a cell, protein, or nucleic
acid, or vector, that
has been modified by the introduction of a heterologous nucleic acid or the
alteration of a
native nucleic acid to a form not native to that cell, or that the cell is
derived from a cell so
modified. Thus, for example, recombinant cells express genes and proteins that
are not
found within the native (non-recombinant) form of the cell or express native
genes that are
otherwise abnormally expressed, under expressed or not expressed at all.
An "expression vector" is a nucleic acid construct, generated recombinantly
or synthetically, with a series of specified nucleic acid elements which
permit transcription
of a particular nucleic acid in a host cell. The expression vector can be part
of a plasmid,
virus, or nucleic acid fragment. Typically, the expression vector includes a
nucleic acid to
be transcribed operably linked to a promoter.
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
The terms "identical" or percent "identity," in the context of two or more
nucleic acids or polypeptide sequences, refer to two or more sequences or
subsequences that
are the same or have a specified percentage of amino acid residues or
nucleotides that are
the same, when compared and aligned for maximum correspondence over a
comparison
5 window, as measured using one of the following sequence comparison
algorithms or by
manual alignment and visual inspection. This definition also refers to the
complement of a
test sequence. Preferably, the amino acid or nucleotide sequence identity is
at least about
60%, more preferably at least about 75-80%, more preferably about 90-95%. Two
sequences with this level of identiy are "substantially identical."
Preferably, the percent
10 identity exists over a region of the sequence that is at least about 25
amino acids in length,
more preferably over a region that is 50-100 amino acids in length.
When percentage of sequence identity is used in reference to proteins or
peptides, it is recognized that residue positions that are not identical often
differ by
conservative amino acid substitutions, where amino acids residues are
substituted for other
15 amino acid residues with similar chemical properties (e.g., charge or
hydrophobicity) and
therefore do not change the functional properties of the molecule. Where
sequences differ
in conservative substitutions, the percent sequence identity may be adjusted
upwards to
correct for the conservative nature of the substitution. Means for making this
adjustment
are well known to those of skill in the art. Typically this involves scoring a
conservative
20 substitution as a partial rather than a full mismatch, thereby increasing
the percentage
sequence identity. Thus, for example, where an identical amino acid is given a
score of 1
and a non-conservative substitution is given a score of zero, a conservative
substitution is
given a score between zero and 1. The scoring of conservative substitutions is
calculated
according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol.
Sci. 4:11-17
(1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain
View,
California, USA)..
For sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a sequence
comparison
algorithm, test and reference sequences are entered into a computer,
subsequence
coordinates are designated, if necessary, and sequence algorithm program
parameters are
designated. Default program parameters can be used, or alternative parameters
can be
designated. The sequence comparison algorithm then calculates the percent
sequence
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
21
identity for the test sequences) relative to the reference sequence, based on
the designated
or default program parameters.
A "comparison window", as used herein, includes reference to a segment of
any one of the number of contiguous positions selected from the group
consisting of from
25 to 600, usually about 50 to about 200, more usually about 100 to about 150
in which a
sequence may be compared to a reference sequence of the same number of
contiguous
positions after the two sequences are optimally aligned. Methods of alignment
of sequences
for comparison are well-known in the art. Optimal alignment of sequences for
comparison
can be conducted, e.g., by the local homology algorithm of Smith & Waterman,
Adv. Appl.
Math. 2:482 ( 1981 ), by the homology alignment algorithm of Needleman &
Wunsch, J.
Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson &
Lipman, Proc.
Nat'1. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual
alignment and visual inspection (see, e.g., Ausubel et al., supra).
One example of a useful algorithm is PILEUP. PILEUP creates a multiple
sequence alignment from a group of related sequences using progressive,
pairwise
alignments to show relationship and percent sequence identity. It also plots a
tree or
dendogram showing the clustering relationships used to create the alignment.
PILEUP uses
a simplification of the progressive alignment method of Feng & Doolittle, J.
Mol. Evol.
35:351-360 (1987). The method used is similar to the method described by
Higgins &
Sharp, CABIOS 5:151-153 (1989). The program can align up to 300 sequences,
each of a
maximum length of 5,000 nucleotides or amino acids. The multiple alignment
procedure
begins with the pairwise alignment of the two most similar sequences,
producing a cluster
of two aligned sequences. This cluster is then aligned to the next most
related sequence or
cluster of aligned sequences. Two clusters of sequences are aligned by a
simple extension
of the pairwise alignment of two individual sequences. The final alignment is
achieved by a
series of progressive, pairwise alignments. The program is run by designating
specific
sequences and their amino acid or nucleotide coordinates for regions of
sequence
comparison and by designating the program parameters. Using PILEUP, a
reference
sequence is compared to other test sequences to determine the percent sequence
identity
relationship using the following parameters: default gap weight (3.00),
default gap length
weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG
sequence
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCTNS98/22321
22
analysis software package, e.g., version 7.0 {Devereaux et al., Nuc. Acids
Res. 12:387-395
( 1984).
Another example of algorithm that is suitable for determining percent
sequence identity (i.e., substantial similarity or identity) is the BLAST
algorithm, which is
described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for
performing
BLAST analyses is publicly available through the National Center for
Biotechnology
Information (http://www.ncbi.nlm.nih.govn. This algorithm involves first
identifying high
scoring sequence pairs (HSPs) by identifying short words of length W in the
query
sequence, which either match or satisfy some positive-valued threshold score T
when
aligned with a word of the same length in a database sequence. T is referred
to as the
neighborhood word score threshold (Altschul et al., supra). These initial
neighborhood
word hits act as seeds for initiating searches to find longer HSPs containing
them. The
word hits are then extended in both directions along each sequence for as far
as the
cumulative alignment score can be increased. Cumulative scores are calculated
using, for
1 S nucleotide sequences, the parameters M (reward score for a pair of
matching residues;
always > 0) and N (penalty score for mismatching residues, always < 0). For
amino acid
sequences, a scoring matrix is used to calculate the cumulative score.
Extension of the word
hits in each direction are halted when: the cumulative alignment score falls
off by the
quantity X from its maximum achieved value; the cumulative score goes to zero
or below,
due to the accumulation of one or more negative-scoring residue alignments; or
the end of
either sequence is reached. The BLAST algorithm parameters W, T, and X
determine the
sensitivity and speed of the alignment. The BLASTN program (for nucleotide
sequences)
uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=4,
and a
comparison of both strands. For amino acid sequences, the BLASTP program uses
as
default parameters a wordlength (W) of 3, an expectation (E) of 10, and the
BLOSLJM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915
(1989)).
The BLAST algorithm also performs a statistical analysis of the similarity
between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'1. Acad. Sci.
USA 90:5873-
5787 (1993)). One measure of similarity provided by the BLAST algorithm is the
smallest
sum probability (P(N)), which provides an indication of the probability by
which a match
between two nucleotide or amino acid sequences would occur by chance. For
example, a
nucleic acid is considered similar to a reference sequence if the smallest sum
probability in
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PC'T/I1S98/22321
23
a comparison of the test nucleic acid to the reference nucleic acid is less
than about 0.1,
more preferably less than about 0.01, and most preferably less than about
0.001.
An indication that two nucleic acid sequences or polypeptides are
substantially identical, i.e., they have a designated percent identity, is
that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive with the
antibodies
raised against the polypeptide encoded by the second nucleic acid, as
described below.
Thus, a polypeptide is typically substantially identical to a second
polypeptide, for example,
where the two peptides differ only by conservative substitutions. Another
indication that
two nucleic acid sequences are substantially identical is that the two
molecules or their
complements hybridize to each other under stringent conditions, as described
below. A
further indication that two polynucleotides are substantially identical is if
the reference
sequence, amplified by a pair of oligonucleotide primers or a pool of
degenerate primers
that encode a conserved amino acid sequence, can then be used as a probe under
stringent
hybridization conditions to isolate the test sequence from a cDNA or genomic
library, or to
identify the test sequence in, e.g., a northern or Southern blot.
Alternatively, another
indication that the sequences are substantially identical is if the same set
of PCR primers
can be used to amplify both sequences.
The phrase "selectively (or specifically) hybridizes to" refers to the
binding,
duplexing, or hybridizing of a molecule to a particular nucleotide sequence
under stringent
hybridization conditions when that sequence is present in a complex mixture
(e.g., total
cellular or library DNA or RNA), wherein the particular nucleotide sequence is
detected at
least at twice background, preferably 10 times background.
The phrase "stringent hybridization conditions" refers to conditions under
which a probe will hybridize to its target subsequence, typically in a complex
mixture of
nucleic acid, but to no other sequences. Stringent conditions are sequence-
dependent and
will be different in different circumstances. Longer sequences hybridize
specifically at
higher temperatures. An extensive guide to the hybridization of nucleic acids
is found in
Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with
Nucleic
Probes, "Overview of principles of hybridization and the strategy of nucleic
acid assays"
(1993). Generally, stringent conditions are selected to be about 5-10°C
lower than the
thermal melting point (Tm) for the specific sequence at a defined ionic
strength pH. The Tm
is the temperature (under defined ionic strength, pH, and nucleic
concentration) at which
SO% of the probes complementary to the target hybridize to the target sequence
at
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/Z2321
24
equilibrium (as the target sequences are present in excess, at Tm, 50% of the
probes are
occupied at equilibrium). Stringent conditions will be those in which the salt
concentration
is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion
concentration
(or other salts) at pH 7.0 to 8.3 and the temperature is at least about
30°C for short probes
S (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes
(e.g., greater than 50
nucleotides). Stringent conditions may also be achieved with the addition of
destabilizing
agents such as formamide. For selective or specific hybridization, a positive
signal is at
least two times background, preferably 10 time background hybridization.
Exemplary
"highly stringent" hybridization conditions include hybridization in a buffer
comprising
50% formamide, 5x SSC, and 1% SDS at 42°C, or hybridization in a buffer
comprising Sx
SSC and 1% SDS at 65°C, both with a wash of 0.2x SSC and 0.1% SDS
at 65°C.
Exemplary "moderately stringent hybridization conditions" include a
hybridization in a buffer of 40% formamide, 1 M NaCI, and 1 % SDS at
37°C, and a wash in
1X SSC at 45°C. A positive hybridization is at least twice background.
Those ofordinary
skill will readily recognize that alternative hybridization and wash
conditions can be utilized
to provide conditions of similar stringency. Nucleic acids which do not
hybridize to each
other under moderately stringent or stringent hybridization conditions are
still substantially
identical if the polypeptides which they encode are substantially identical.
This may occur,
e.g., when a copy of a nucleic acid is created using the maximum codon
degeneracy
permitted by the genetic code.
The phrase "encodes a protein which could be encoded by a nucleic acid that
selectively hybridizes under moderate stringency hybridization conditions to a
sequence" in
the context of nucleic acids refers to those nucleic acids encoding naturally
occurring
proteins or derivatives of natural proteins, but which are deliberately
modified or engineered
to no longer hybridize to the protein of natural origin under the stated
conditions.
The term "antibody" also includes antigen binding forms of antibodies (e.g.,
Fab, F(ab)2). The term "antibody" refers to a polypeptide substantially
encoded by an
immunoglobulin gene or immunoglobulin genes, or fragments thereof which
specifically
bind and recognize an antigen. The recognized immunoglobulin genes include the
kappa,
lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as
the myriad
immunoglobulin variable region genes. Light chains are classified as either
kappa or
lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon,
which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/C1S98/22321
An exemplary immunoglobulin (antibody) structural unit comprises a
tetramer. Each tetramer is composed of two identical pairs of polypeptide
chains, each pair
having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-
terminus
of each chain defines a variable region of about 100 to 110 or more amino
acids primarily
responsible for antigen recognition. The terms variable light chain (VL) and
variable heavy
chain (VH) refer to these light and heavy chains respectively.
Antibodies exist e.g., as intact immunoglobulins or as a number of well
characterized fragments produced by digestion with various peptidases. Thus,
for example,
pepsin digests an antibody below the disulfide linkages in the hinge region to
produce
10 F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a
disulfide bond.
The F(ab)'2 may be reduced under mild conditions to break the disulfide
linkage in the
hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The
Fab'
monomer is essentially an Fab with part of the hinge region (see, e.g.,
Fundamental
Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are
defined in terms
15 of the digestion of an intact antibody, one of skill will appreciate that
such fragments may
be synthesized de novo either chemically or by utilizing recombinant DNA
methodology.
Thus, the term antibody, as used herein, also includes antibody fragments such
as single
chain Fv, chimeric antibodies (i.e., comprising constant and variable regions
from different
species), humanized antibodies (i.e., comprising a complementarity determining
region
20 (CDR) from a non-human source) and heteroconjugate antibodies (e.g.,
bispecific
antibodies).
An "anti-Slo3" antibody is an antibody or antibody fragment that specifically
binds a polypeptide encoded by a Slo3 gene, cDNA, or a subsequence thereof.
The
antibody can be either a monoclonal or polyclonal antibody.
25 A "chimeric antibody" is an antibody molecule in which (a) the constant
region, or a portion thereof, is altered, replaced or exchanged so that the
antigen binding site
(variable region) is linked to a constant region of a different or altered
class, effector
function and/or species, or an entirely different molecule which confers new
properties to
the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug,
etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or exchanged with
a variable region
having a different or altered antigen specificity.
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
26
The term "immunoassay" is an assay that uses an antibody to specifically
bind an antigen. The immunoassay is characterized by the use of specific
binding properties
of a particular antibody to isolate, target, and/or quantify the antigen.
The phrase "specifically (or selectively) binds to an antibody" or
"specifically (or selectively) immunoreactive with," when refernng to a
protein or peptide,
refers to a binding reaction that is determinative of the presence of the
protein in a
heterogeneous population of proteins and other biologics. Thus, under
designated
immunoassay conditions, the specified antibodies bind to a particular protein
and do not
bind in a significant amount to other proteins present in the sample. Specific
binding to an
antibody under such conditions may require an antibody that is selected for
its specificity
for a particular protein.
For example, antibodies raised to mSlo3 or hSlo3 with the amino acid
sequence encoded in SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:16, and SEQ ID N0:18
respectively, can be selected to obtain polyclonal antibodies specifically
immunoreactive
with that protein and not with other proteins, except for polymorphic variants
and alleles.
This selection may be achieved by subtracting out antibodies that cross-react
with Slo3
molecules from other species. A variety of immunoassay formats may be used to
select
antibodies specifically immunoreactive with a particular protein. For example,
solid-phase
ELISA immunoassays are routinely used to select antibodies specifically
immunoreactive
with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual
(1988), for a
description of immunoassay formats and conditions that can be used to
determine specific
immunoreactivity). Typically a specific or selective reaction will be at least
twice
background signal or noise and more typically more than 10 to 100 times
background.
The phrase "selectively associates with" refers to the ability of a nucleic
acid
to "selectively hybridize" with another as defined above, or the ability of an
antibody to
"selectively (or specifically) bind to a protein, as defined above.
The phrase "functional effects" in the context of assays for testing
compounds affecting the channel includes the determination of any parameter
that is
indirectly or directly under the influence of the channel. It includes changes
in ion flux,
membrane potential, voltage gating, and pH sensitivity and also includes other
physiologic
effects such increases or decreases of transcription or hormone release.
By "determining the functional effect" is meant examining the effect of a
compound that increases or decreases ion flux on a cell or cell membrane in
terms of cell
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
27
and cell membrane function. The ion flux can be any ion that passes through
the channel
and analogs thereof, e.g., potassium, rubidium, sodium, and radioisotopes
thereof.
Preferably, the term refers to the functional effect of the compound on Slo3
channel
activity, e.g., changes in ion flux, current amplitude, voltage gating, pH
sensitivity, and the
like. Such functional effects can be measured by any means known to those
skilled in the
art, e.g., patch clamping, whole cell currents, pH and voltage sensitive dyes,
radioisotope
efflux, inducible markers, and the like.
"Inhibitors," "activators," and "modulators" of Slo3 refer to inhibitory or
activating molecules identified using in vitro assays for Slo3 function.
Inhibitors are
compounds that decrease, block, prevent, delay activation, inactivate,
desensitize, or down
regulate the channel. Activators are compounds that increase, open, activate,
facilitate,
enhance activation, sensitize or up regulate channel activity. Such assays for
inhibitors and
activators include e.g., expressing Slo in cells or cell membranes and then
measuring flux of
ions through the channel and determining changes in polarization (i.e.,
electrical potential).
Methods of measuring changes of cell membrane polarization and ion flux
include voltage-
clamp techniques, determination of whole cell currents, radiolabeled rubidium
flux assays,
and fluorescence assays using voltage-sensitive dyes. Samples or assays that
are treated
with a potential Slo3 activator or inhibitor are compared to control samples
without the
inhibitor, to examine the extent of inhibition. Control samples (untreated
with inhibitors)
are assigned a relative Slo3 activity value of 100. Inhibition of Slo3 is
achieved when the
Slo3 activity value relative to the control is about 90%, preferably 50%, more
preferably
25%. Activation of Slo3 is achieved when the Slo3 activity value relative to
the control is
110%, more preferably 150%, more preferable 200% higher.
By "host cell" is meant a cell which contains an expression vector and
supports the replication or expression of the expression vector. Host cells
may be
prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect,
amphibian, e.g.,
XenopuS, or mammalian cells such as CHO, HeLa and the like. The Slo3 channel
can also
be expressed in a cell membrane derived from such a cell.
"Biological sample" as used herein is a sample of biological tissue or fluid
that contains a Slo3 channel protein or nucleic acid encoding the
corresponding Slo3
channel protein. Such samples include, but are not limited to, seminal fluid
containing
sperm, sperm cells, testis tissue, and brain tissue. Biological samples may
also include
sections of tissues such as frozen sections taken for histological purposes. A
biological
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
28
sample is typically obtained from a eukaryotic organism, preferably a
multicellular
eukaryotes such as insect, protozoa, birds, fish, reptiles, and preferably a
mammal such as
rat, mice, cow, dog, guinea pig, or rabbit, and most preferably a primate such
as macaques,
chimpanzees, or humans.
S By "conductance" is meant electrical conductance. Electrical conductance is
conveniently measured in Siemens (1/ohm = mho). Unitary conductance is
determined by
measuring single channel currents using a patch clamp protocol under
conditions set forth in
Examples III and IV (i.e., in a Xenopus oocyte) using a symmetrical potassium
ion
concentration of 160 mM (see generally, Hille, Ionic Channels of Excitable
Membranes (2d
ed.) In the context of the present invention, "conductance" refers to the
unitary electrical
conductance of a single homomeric protein of the referenced Slo3 channel
protein.
"Functional tetrameric form" refers to expression of a Slo3 protein or
monomer in which a plurality of the Slo3 proteins are assembled to form, by
themselves or
in conjunction with other endogenous Xenopus oocyte molecules, an Slo3
potassium
channel. Expression within a Xenopus oocyte is disclosed in the Examples
provided herein,
e.g., Example III. Typically the Slo3 channel is a homotetramer formed of four
Slo3
monomer proteins.
By "contiguous amino acids from" in the context of a specified number of
amino acid residues from a specified sequence, is meant a sequence of amino
acids of the
specified number from within the specified reference sequence which has the
identical order
of amino acids each of which is directly adjacent to the same amino acids as
in the reference
sequence.
III. Nucleic acids encoding Slo3
The present invention provides isolated nucleic acids of RNA, DNA, or
chimeras thereof, which encode Slo3 channel proteins. Nucleic acids of the
present
invention can be used as probes, for example, in detecting deficiencies in the
level of
mltNA, mutations in the gene (e.g., substitutions, deletions, or additions),
for monitoring up
regulation of Slo3 channels in drug screening assays, or for recombinant
expression of Slo3
channel proteins for use as immunogens in the preparation of antibodies or for
in vitro or in
vivo expression assays.
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
29
A. General Recombinant DNA Methods
This invention relies on routine techniques in the field of recombinant
genetics. Basic texts disclosing the general methods of use in this invention
include
Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989);
Kriegler, Gene
Transfer and Expression: A Laboratory Manual ( 1990); and Current Protocols in
Molecular Biology (Ausubel et al., eds., 1994)).
For nucleic acids, sizes are given in either kilobases (kb) or base pairs
(bp).
These are estimates derived from agarose or acrylamide gel electrophoresis,
from sequenced
nucleic acids, or from published DNA sequences. For proteins, sizes are given
in
kilodaltons (kDa) ar amino acid residue numbers. Proteins sizes are estimated
from gel
electrophoresis, from sequenced proteins, from derived amino acid sequences,
or from
published protein sequences.
Oligonucleotides that are not commercially available can be chemically
synthesized according to the solid phase phosphorarnidite triester method
first described by
Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981}, using an
automated
synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12:6159-
6168 (1984).
Purification of oligonucleotides is by either native acrylamide gel
electrophoresis or by
anion-exchange HPLC as described in Pearson & Reamer, J. Chrom. 255:137-149
(1983).
The sequence of the cloned genes and synthetic oligonucleotides can be
verified after cloning using, e.g., the chain termination method for
sequencing double-
stranded templates of Wallace et al., Gene 16:21-26 (1981).
B. Cloning methods for the isolation of nucleotide sequences
encoding Slo3
In general, the nucleic acid sequences encoding Slo3 and related nucleic acid
sequence homologs are cloned from cDNA and genomic DNA libraries or isolated
using
amplification techniques with oligonucleotide primers. For example, SIo3
sequences are
typically isolated from human nucleic acid (genomic or cDNA) libraries by
hybridizing
with a nucleic acid probe, the sequence of which can be derived from SEQ ID
N0:2, 4, 17,
or 19, preferably from the core domain. A suitable tissue from which Slo3 RNA
and cDNA
can be isolated is testis.
Amplification techniques using primers can also be used to amplify and
isolate Slo3 from DNA or RNA. For example, nucleic acids encoding a mSlo3
channel
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
protein of SEQ ID NO:1 may be obtained by amplification of a mouse testis cDNA
library
or reverse transcribed from mouse testis RNA using isolated nucleic acid
primer pairs
having the sequence:
CTCGAACTCCCTAAAATCTTACAGAT (SEQ ID N0:8) and
5 TTCCGTTGAGCCAGGGGTCACCAGAATT (SEQ ID N0:9);
TCTGCTTTGTGAAGCTAAATCT (SEQ ID NO:10) and
TTTCAAAGCCTCTTTAGCGGTAA (SEQ ID NO:11); or
TTATGCCTGGATCTGCACTCTACATG (SEQ ID N0:12) and
ATAGTTTCCGTCTACTACCGAAA (SEQ ID N0:13).
10 Nucleic acids encoding an hSlo3 channel protein may also be obtained by
amplification of a human testis cDNA library or reverse transcribed human
testis RNA,
using the following primers:
GGCAGCGCTCATTCTTTCCTCCTT (SEQ ID N0:14) and
TGCCCAAAACCTCAACCCAAAATA (SEQ ID NO:15).
15 These primers can be used, e.g., to amplify either the full length sequence
or
a probe of one to several hundred nucleotides, which is then used to screen a
human library
for full-length SIo3. Nucleic acids encoding SIo3 can also be isolated from
expression
libraries using antibodies as probes. Such polyclonal or monoclonal antibodies
can be
raised using the sequence of SEQ ID NO:1, 3, 16, or 18.
20 Slo3 polymorphic variants, alleles, and interspecies homologs that are
substantially identical to the core domain of Slo3 can be isolated using SIo3
nucleic acid
probes and oiigonucleotides under stringent hybridization conditions, by
screening libraries.
Alternatively, expression libraries can be used to clone SIo3 polymorphic
variants, alleles,
and interspecies homologs, by detecting expressed homologs immunologically
with antisera
25 or purified antibodies made against the core domain of Slo3, which also
recognize and
selectively bind to the Slo3 homolog.
To make a cDNA library, one should choose a source that is rich in Slo3
mRNA, e.g., testis tissue. The mRNA is then made into cDNA using reverse
transcriptase,
ligated into a recombinant vector, and transfected into a recombinant host for
propagation,
30 screening and cloning. Methods for making and screening cDNA libraries are
well known
(see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook et al., supra;
Ausubel et
al., supra).
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCTlI1S98/22321
31
For a genomic library, the DNA is extracted from the tissue and either
mechanically sheared or enzymatically digested to yield fragments of about 12-
20 kb. The
fragments are then separated by gradient centrifugation from undesired sizes
and are
constructed in bacteriophage lambda vectors. These vectors and phage are
packaged in
vitro. Recombinant phage are analyzed by plaque hybridization as described in
Benton &
Davis, Science 196:180-182 (1977). Colony hybridization is carried out as
generally
described in Grunstein et al., Proc. Natl. Acad. Sci. USA., 72:3961-3965
(1975).
An alternative method of isolating Slo3 nucleic acid and its homologs
combines the use of synthetic oligonucleotide primers and amplification of an
RNA or DNA
template (see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to
Methods
and Applications (Innis et al., eds, 1990)). Methods such as polymerase chain
reaction
(PCR) and ligase chain reaction (LCR) can be used to amplify nucleic acid
sequences of
Slo3 directly from mRNA, from cDNA, from genomic libraries or cDNA libraries.
Degenerate oligonucleotides can be designed to amplify Slo3 homologs using the
sequences
provided herein. Restriction endonuclease sites can be incorporated into the
primers.
Polymerase chain reaction or other in vitro amplification methods may also be
useful, for
example, to clone nucleic acid sequences that code for proteins to be
expressed, to make
nucleic acids to use as probes for detecting the presence of Slo3 encoding
mRNA in
physiological samples, for nucleic acid sequencing, or for other purposes.
Genes amplified
by the PCR reaction can be purified from agarose gels and cloned into an
appropriate
vector.
Gene expression of Slo3 can also be analyzed by techniques known in the
art, e.g., reverse transcription and amplification of mRNA, isolation of total
RNA or poly
A+ RNA, northern blotting, dot blotting, in situ hybridization, RNase
protection, probing
DNA microchip arrays, and the like.
Synthetic oligonucleotides can be used to construct recombinant Slo3 genes
for use as probes or for expression of protein. This method is performed using
a series of
overlapping oligonucleotides usually 40-120 by in length, representing both
the sense and
non-sense strands of the gene. These DNA fragments are then annealed, ligated
and cloned.
Alternatively, amplification techniques can be used with precise primers to
amplify a
specific subsequence of the Slo3 gene. The specific subsequence is then
ligated into an
expression vector. As described below in Example VI, Slo3 chimeras can be
made, which
combine, e.g., either the core or the tail domain of Slo3 with another domain
of a
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98lZ2321
32
heterologous potassium channel protein to create a chimeric, functional
potassium channel
subunit.
The gene for Slo3 is typically cloned into intermediate vectors before
transformation into prokaryotic or eukaryotic cells for replication and/or
expression. These
intermediate vectors are typically prokaryote vectors, e.g., plasmids, or
shuttle vectors.
Isolated nucleic acids encoding Slo3 channel proteins comprise a nucleic acid
sequence
encoding a Slo3 channel protein selected from the group consisting of SEQ ID
NO:1, 3, 16,
and 18 and subsequences, interspecies homologs, alleles and polymorphic
variants thereof.
In preferred embodiments, the isolated nucleic acid encoding a Slo3 channel
protein is
selected from the group consisting of SEQ ID N0:2, 4, 17, and 19, and
subsequences
thereof.
C. Expression in prokaryotes and eukaryotes
To obtain high level expression of a cloned gene, such as those cDNAs
encoding Slo3, one typically subclones Slo3 into an expression vector that
contains a strong
promoter to direct transcription, a transcription/translation terminator, and
if for a nucleic
acid encoding a protein, a ribosome binding site for translational initiation.
Suitable
bacterial promoters are well known in the art and described, e.g., in Sambrook
et al. and
Ausubel et al. Bacterial expression systems for expressing the Slo3 protein
are available in,
e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235
(1983); Mosbach et
al., Nature 302:543-545 (1983). Kits for such expression systems are
commercially
available. Eukaryotic expression systems for mammalian cells, yeast, and
insect cells are
well known in the art and are also commercially available.
The promoter used to direct expression of a heterologous nucleic acid
depends on the particular application. The promoter is preferably positioned
about the same
distance from the heterologous transcription start site as it is from the
transcription start site
in its natural setting. As is known in the art, however, some variation in
this distance can be
accommodated without loss of promoter function.
In addition to the promoter, the expression vector typically contains a
transcription unit or expression cassette that contains all the additional
elements required for
the expression of the Slo3 encoding nucleic acid in host cells. A typical
expression cassette
thus contains a promoter operably linked to the nucleic acid sequence encoding
Slo3 and
signals required for effiFient polyadenylation of the transcript, ribosome
binding sites, and
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/Z2321
33
translation termination. The nucleic acid sequence encoding Slo3 may typically
be linked
to a cleavable signal peptide sequence to promote secretion of the encoded
protein by the
transformed cell. Such signal peptides would include, among others, the signal
peptides
from tissue plasminogen activator, insulin, and neuron growth factor, and
juvenile hormone
esterase of Heliothis virescens. Additional elements of the cassette may
include enhancers
and, if genomic DNA is used as the structural gene, introns with functional
splice donor and
acceptor sites.
In addition to a promoter sequence, the expression cassette should also
contain a transcription termination region downstream of the structural gene
to provide for
efficient termination. The termination region may be obtained from the same
gene as the
promoter sequence or may be obtained from different genes.
The particular expression vector used to transport the genetic information
into the cell is not particularly critical. Any of the conventional vectors
used for expression
in eukaryotic or prokaryotic cells may be used. Standard bacterial expression
vectors
include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion
expression
systems such as GST and LacZ. Epitope tags can also be added to recombinant
proteins to
provide convenient methods of isolation, e.g., c-myc.
Expression vectors containing regulatory elements from eukaryotic viruses
are typically used in eukaryotic expression vectors, e.g., SV40 vectors,
papilloma virus
vectors, and vectors derived from Epstein-Barr virus. Other exemplary
eukaryotic vectors
include pMSG, pAV009/A+, pMT010/A+, pMAMneo-5, baculovirus pDSVE, and any
other vector allowing expression of proteins under the direction of the SV40
early promoter,
SV40 later promoter, metallothionein promoter, marine mammary tumor virus
promoter,
Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown
effective for
expression in eukaryotic cells.
Some expression systems have markers that provide gene amplification such
as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate
reductase.
Alternatively, high yield expression systems not involving gene amplification
are also
suitable, such as using a baculovirus vector in insect cells, with a Slo3
encoding sequence
under the direction of the polyhedrin promoter or other strong baculovirus
promoters.
The elements that are typically included in expression vectors also include a
replicon that functions in E. coli, a gene encoding antibiotic resistance to
permit selection of
bacteria that harbor recombinant plasmids, and unique restriction sites in
nonessential
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
34
regions of the plasmid to allow insertion of eukaryotic sequences. The
particular antibiotic
resistance gene chosen is not critical, any of the many resistance genes known
in the art are
suitable. The prokaryotic sequences are preferably chosen such that they do
not interfere
with the replication of the DNA in eukaryotic cells, if necessary.
Standard transfection methods are used to produce bacterial, mammalian,
yeast or insect cell lines that express large quantities of Slo3 protein,
which are then purified
using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-
17622 (1989);
Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher,
ed., 1990)).
Transformation of eukaryotic and prokaryotic cells are performed according to
standard
techniques (see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss &
Curtiss,
Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).
Any of the well known procedures for introducing foreign nucleotide
sequences into host cells may be used. These include the use of calcium
phosphate
transfection, polybrene, protoplast fusion, electroporation, liposomes,
microinjection,
plasma vectors, viral vectors and any of the other well known methods for
introducing
cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into
a host
cell (see, e.g., Sambrook et al., supra). It is only necessary that the
particular genetic
engineering procedure used be capable of successfully introducing at least one
gene into the
host cell capable of expressing Slo3.
After the expression vector is introduced into the cells, the transfected
cells
are cultured under conditions favoring expression of Slo3, which is recovered
from the
culture using standard techniques identified below.
IV. Purification of Slo3
Either naturally occurring or recombinant Slo3 can be purified for use in
functional assays. Naturally occurring Slo3 is purified, e.g., from tissue
such as testis
tissue, and any other source of a Slo3 homolog. Recombinant Slo3 is purified
from any
suitable expression system.
Slo3 may be purified to substantial purity by standard techniques, including
selective precipitation with such substances as ammonium sulfate; column
chromatography,
immunopurification methods, and others (see, e.g., Scopes, Protein
Purification: Principles
and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et al., supra; and
Sambrook et al.,
supra).
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
A number of procedures can be employed when recombinant Slo3 is being
purified. For example, proteins having established molecular adhesion
properties can be
reversible fused to Slo3. With the appropriate ligand, Slo3 can be selectively
adsorbed to a
purification column and then freed from the column in a relatively pure form.
The fused
protein is then removed by enzymatic activity. Finally Slo3 could be purified
using
immunoaffinity columns.
A. Purification of Slo3 from recombinant bacteria
Recombinant proteins are expressed by transformed bacteria in large
10 amounts, typically after promoter induction; but expression can be
constitutive. Promotei
induction with IPTG is a one example of an inducible promoter system. Bacteria
are grown
according to standard procedures in the art. Fresh or frozen bacteria cells
are used for
isolation of protein.
Proteins expressed in bacteria may form insoluble aggregates ("inclusion
15 bodies"). Several protocols are suitable for purification of Slo3 inclusion
bodies. For
example, purification of inclusion bodies typically involves the extraction,
separation and/or
purification of inclusion bodies by disruption of bacterial cells, e.g., by
incubation in a
buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCI, 5 mM MgCl2, 1 mM DTT, 0.1 mM
ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3 passages
through a
20 French Press, homogenized using a Polytron (Brinkman Instruments) or
sonicated on ice.
Alternate methods of lysing bacteria are apparent to those of skill in the art
(see, e.g.,
Sambrook et al., supra; Ausubel et al., supra).
If necessary, the inclusion bodies are solubilized, and the lysed cell
suspension is typically centrifuged to remove unwanted insoluble matter.
Proteins that
25 formed the inclusion bodies may be renatured by dilution or dialysis with a
compatible
buffer. Suitable solvents include, but are not limited to urea (from about 4 M
to about 8 M),
formamide (at least about 80%, volume/volume basis), and guanidine
hydrochloride (from
about 4 M to about 8 M). Some solvents which are capable of solubilizing
aggregate-
forming proteins, for example SDS (sodium dodecyl sulfate), 70% formic acid,
are
30 inappropriate for use in this procedure due to the possibility of
irreversible denaturation of
the proteins, accompanied by a lack of immunogenicity and/or activity.
Although guanidine
hydrochloride and similar agents are denaturants, this denaturation is not
irreversible and
renaturation may occur upon removal (by dialysis, for example) or dilution of
the
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
36
denaturant, allowing re-formation of immunologically and/or biologically
active protein.
Other suitable buffers are known to those skilled in the art. Slo3 is
separated from other
bacterial proteins by standard separation techniques, e.g., with Ni-NTA
agarose resin.
Alternatively, it is possible to purify SIo3 from bacteria periplasm. After
lysis of the bacteria, when Slo3 is exported into the periplasm of the
bacteria, the
periplasmic fraction of the bacteria can be isolated by cold osmotic shock in
addition to
other methods known to skill in the art. To isolate recombinant proteins from
the periplasm,
the bacterial cells are centrifuged to fonm a pellet. The pellet is
resuspended in a buffer
containing 20% sucrose. To iyse the cells, the bacteria are centrifuged and
the pellet is
resuspended in ice-cold 5 mM MgS04 and kept in an ice bath for approximately
10 minutes.
The cell suspension is centrifuged and the supernatant decanted and saved. The
recombinant proteins present in the supernatant can be separated from the host
proteins by
standard separation techniques well known to those of skill in the art.
B. Standard Protein Separation Techniques For Purifying SIo3
Solubility Fractionation
Often as an initial step, particularly if the protein mixture is complex, an
initial salt fractionation can separate many of the unwanted host cell
proteins (or proteins
derived from the cell culture media) from the recombinant protein of interest.
The preferred
salt is ammonium sulfate. Ammonium sulfate precipitates proteins by
effectively reducing
the amount of water in the protein mixture. Proteins then precipitate on the
basis of their
solubility. The more hydrophobic a protein is, the more likely it is to
precipitate at lower
ammonium sulfate concentrations. A typical protocol includes adding saturated
ammonium
sulfate to a protein solution so that the resultant ammonium sulfate
concentration is between
20-30%. This concentration will precipitate the most hydrophobic of proteins.
The
precipitate is then discarded (unless the protein of interest is hydrophobic)
and ammonium
sulfate is added to the supernatant to a concentration known to precipitate
the protein of
interest. The precipitate is then solubilized in buffer and the excess salt
removed if
necessary, either through dialysis or diafiltration. Other methods that rely
on solubility of
proteins, such as cold ethanol precipitation, are well known to those of skill
in the art and
can be used to fractionate complex protein mixtures.
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
37
Size Differential Filtration
The molecular weight of Slo3 can be used to isolated it from proteins of
greater and lesser size using ultrafiltration through membranes of different
pore size (for
example, Amicon or Millipore membranes). As a first step, the protein mixture
is
ultrafiltered through a membrane with a pore size that has a lower molecular
weight cut-off
than the molecular weight of the protein of interest. The retentate of the
ultrafiltration is
then ultrafiltered against a membrane with a molecular cut off greater than
the molecular
weight of the protein of interest. The recombinant protein will pass through
the membrane
into the filtrate. The filtrate can then be chromatographed as described
below.
Column Chromatog~raph;~
Slo3 can also be separated from other proteins on the basis of its size, net
surface charge, hydrophobicity, and affinity for ligands. In addition,
antibodies raised
against proteins can be conjugated to column matrices and the proteins
immunopurified.
All of these methods are well known in the art. It will be apparent to one of
skill that
chromatographic techniques can be performed at any scale and using equipment
from many
different manufacturers (e.g., Pharmacia Biotech).
Alternatively, SIo3 protein can be expressed transiently in a cell by
introducing into a cell an RNA encoding the Slo3 protein. The RNA is
transcribed in vitro
according to standard procedures and then introduced into a cell by means such
as injection
or electroporation. The RNA then expresses the Slo3 protein. Such systems are
useful for
measuring single channel and whole cell conductance of a Slo3 channel protein,
e.g., when
the RNA is transiently expressed in cells such as Xenopus oocytes, CHO, and
HeLa cells.
V. Immunological detection of Slo3
In addition to the detection of Slo3 genes and gene expression using nucleic
acid hybridization technology, one can also use immunoassays to detect Slo3.
Immunoassays can be used to qualitatively or quantitatively analyze SIo3. A
general
overview of the applicable technology can be found in Harlow & Lane,
Antibodies: A
Laboratory Manual (1988).
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
38
A. Antibodies to Slo3
Methods of producing polyclonal and monoclonal antibodies that react
specifically with Slo3 are known to those of skill in the art (see, e.g.,
Coligan, Current
Protocols in Immunology (1991); Harlow & Lane, supra; Goding, Monoclonal
Antibodies:
Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature, 256:495-
497 (1975).
Such techniques include antibody preparation by selection of antibodies from
libraries of
recombinant antibodies in phage or similar vectors, as well as preparation of
polyclonal and
monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al.,
Science
246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)).
A number of Slo3 comprising immunogens may be used to produce
antibodies specifically reactive with Slo3., for example, recombinant Slo3 or
a antigenic
fragment thereof such as the core or tail domain, is isolated as described
herein.
Recombinant protein can be expressed in eukaryotic or prokaryotic cells as
described above,
and purified as generally described above. Recombinant protein is the
preferred
immunogen for the production of monoclonal or polyclonal antibodies.
Alternatively, a
synthetic peptide derived from the sequences disclosed herein and conjugated
to a carrier
protein can be used an immunogen. Naturally occurring protein may also be used
either in
pure or impure form. The product is then injected into an animal capable of
producing
antibodies. Either monoclonal or polyclonal antibodies may be generated, for
subsequent
use in immunoassays to measure the protein.
Methods of production of polyclonal antibodies are known to those of skill in
the art. An inbred strain of mice or rabbits is immunized with the protein
using a standard
adjuvant, such as Freund's adjuvant, and a standard immunization protocol. The
animal's
immune response to the immunogen preparation is monitored by taking test
bleeds and
determining the titer of reactivity to Slo3. When appropriately high titers of
antibody to the
immunogen are obtained, blood is collected from the animal and antisera are
prepared.
Further fractionation of the antisera to enrich for antibodies reactive to the
protein can be
done if desired (see Harlow & Lane, supra).
Monoclonal antibodies may be obtained by various techniques familiar to
those skilled in the art. Briefly, spleen cells from an animal immunized with
a desired
antigen are immortalized, commonly by fusion with a myeloma cell (see Kohler &
Milstein,
Eur. J. Immunol. 6:511-519 (1976)). Alternative methods of immortalization
include
transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other
methods well
SUBSTTTUTE SHEET {RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
39
known in the art. Colonies arising from single immortalized cells are screened
for
production of antibodies of the desired specificity and affinity for the
antigen, and yield of
the monoclonal antibodies produced by such cells may be enhanced by various
techniques,
including injection into the peritoneal cavity of a vertebrate host.
Alternatively, one may
isolate DNA sequences which encode a monoclonal antibody or a binding fragment
thereof
by screening a DNA library from human B cells according to the general
protocol outlined
by Huse et al., Science 246:1275-1281 (1989).
Monoclonal antibodies and polyclonal sera are collected and titered against
the immunogen protein in an immunoassay, for example, a solid phase
immunoassay with
the immunogen immobilized on a solid support. Polyclonal antisera with a titer
of 104 or
greater are selected and tested for their cross reactivity against non-Slo3
proteins or even
other homologous proteins from other organisms, using a competitive binding
immunoassay. Specific polyclonal antisera and monoclonal antibodies will
usually bind
with a KD of at least about 0.1 mM, more usually at least about 1 p.M,
preferably at least
about 0.1 p.M or better, and most preferably, 0.01 pM or better.
Once Slo3 specific antibodies are available, Slo3 can be detected by a variety
of immunoassay methods. For a review of immunological and immunoassay
procedures,
see Basic and Clinical Immunology (Stites & Terr eds., 7th ed. 1991 ).
Moreover, the
immunoassays of the present invention can be performed in any of several
configurations,
which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); and
Harlow
& Lane, supra.
B. Immunological binding assays
As explained above, Slo3 expression is associated with sperm physiology,
e.g., capacitation and acrosome reactions. Thus, Slo3 provides a marker with
which to
examine these reactions in sperm. In a preferred embodiment, Slo3 is detected
and/or
quantified using any of a number of well recognized immunological binding
assays (see,
e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a
review of the
general immunoassays, see also Methods in Cell Biology Volume 37: Antibodies
in Cell
Biology (Asai, ed. 1993); Basic and Clinicallmmunology (Stites & Terr, eds.,
7th ed. 1991).
Immunoassays also often utilize a labeling agent to specifically bind to and
label the binding complex formed by the antigen and antibody. The labeling
agent may
itself be one of the moieties comprising the antibody/antigen complex. Thus,
the labeling
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 - PCT/US98/22321
agent may be a labeled Slo3 polypeptide or a labeled anti-Slo3 antibody.
Alternatively, the
labeling agent may be another antibody, which specifically binds to the
antibody/SIo3
complex. In a one embodiment, the labeling agent is a second Slo3 polypeptide
bearing a
label. Alternatively, the second antibody may lack a label, but it may, in
turn, be bound by
5 a labeled third antibody specific to antibodies of the species from which
the second
antibody is derived. The second antibody can be modified with a detectable
moiety, such as
biotin, to which a third labeled molecule can specifically bind, such as
enzyme-labeled
streptavidin. Other proteins capable of specifically binding immunoglobulin
constant
regions, such as protein A or protein G, may also be used.
10 Throughout the assays, incubation and/or washing steps may be required
after each combination of reagents. Incubation steps can vary from about 5
seconds to
several hours, preferably from about S minutes to about 24 hours. However, the
incubation
time will depend upon the assay format, antigen, volume of solution,
concentrations, and the
Like. Usually, the assays will be carned out at ambient temperature, although
they can be
15 conducted over a range of temperatures, such as 10°C to 40°C.
Non-Competitive assay formats
Immunoassays for detecting Slo3 in samples may be either competitive or
noncompetitive. Noncompetitive immunoassays are assays in which the amount of
antigen
20 (in this case the protein) is directly measured. In one preferred
"sandwich" assay, for
example, the anti-Slo3 antibodies can be bound directly to a solid substrate
on which they
are immobilized. These immobilized antibodies then capture Slo3 present in the
test
sample. Slo3 is thus immobilized is then bound by a labeling agent.
25 Competitive assay formats
In competitive assays, the amount of Slo3 present in the sample is measured
indirectly by measuring the amount of an added (exogenous) antigen (i.e., the
Slo3)
displaced (or competed away) from the anti-Slo3 antibody by the antigen
present in the
sample. In one competitive assay, a known amount of, in this case, the Slo3 is
added to the
30 sample and the sample is then contacted with an antibody that specifically
binds to the Slo3.
The amount of SIo3 bound to the antibody is inversely proportional to the
concentration of
Slo3 present in the sample. In a particularly preferred embodiment, the
antibody is
immobilized on a solid substrate. The amount of the SIo3 bound to the antibody
may be
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
41
determined either by measuring the amount of Slo3 present in a Slo3/antibody
complex, or
alternatively by measuring the amount of remaining uncomplexed protein. The
amount of
Slo3 may be detected by providing a labeled Slo3 molecule.
A hapten inhibition assay is another preferred competitive assay. In this
assay Slo3 is immobilized on a solid substrate. A known amount of anti-Slo3
antibody is
added to the sample, and the sample is then contacted with the immobilized
Slo3. The
amount of anti-Slo3 antibody bound to the immobilized Slo3 is inversely
proportional to the
amount of Slo3 present in the sample. Again, the amount of immobilized
antibody may be
detected by detecting either the immobilized fraction of antibody or the
fraction of the
antibody that remains in solution. Detection may be direct where the antibody
is labeled or
indirect by the subsequent addition of a labeled moiety that specifically
binds to the
antibody as described above.
Immunoassays in the competitive binding format can be used for
crossreactivity determinations. For example, a protein partially encoded by
SEQ ID NO:1,
3, 16, or 18 can be immobilized to a solid support. Proteins are added to the
assay that
compete with the binding of the antisera to the immobilized antigen. The
ability of the
above proteins to compete with the binding of the antisera to the immobilized
protein is
compared to Slo3 encoded by SEQ ID NO:1, 3, 16, or 18. The percent
crossreactivity for
the above proteins is calculated, using standard calculations. Those antisera
with less than
10% crossreactivity with each of the proteins listed above are selected and
pooled. The
cross-reacting antibodies are optionally removed from the pooled antisera by
immunoabsorption with the considered proteins, e.g., distantly related
homologs.
The immunoabsorbed and pooled antisera are then used in a competitive
binding immunoassay as described above to compare a second protein, thought to
be
perhaps the protein of this invention, to the immunogen protein (i.e., Slo3
comprising SEQ
ID NO:1, 3, 16, or 18). In order to make this comparison, the two proteins are
each assayed
at a wide range of concentrations and the amount of each protein required to
inhibit 50% of
the binding of the antisera to the immobilized protein is determined. If the
amount of the
second protein required to inhibit SO% of binding is less than 10 times the
amount of the
protein partially encoded by SEQ ID NO:1, 3, 16, or 18 that is required to
inhibit SO% of
binding, then the second protein is said to specifically bind to the
polyclonal antibodies
generated to a Slo3 immunogen.
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/IJS98/22321
42
Other assay formats
Western blot (immunoblot) analysis is used to detect and quantify the
presence of Slo3 in the sample. The technique generally comprises separating
sample
proteins by gel electrophoresis on the basis of molecular weight, transferring
the separated
proteins to a suitable solid support, (such as a nitrocellulose filter, a
nylon filter, or
derivatized nylon filter), and incubating the sample with the antibodies that
specifically bind
the Slo3. The anti-Slo3 antibodies specifically bind to the Slo3 on the solid
support. These
antibodies may be directly labeled or alternatively may be subsequently
detected using
labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that
specifically bind to the
anti-Slo3 antibodies.
Other assay formats include liposome immunoassays (LIA), which use
liposomes designed to bind specific molecules (e.g., antibodies) and release
encapsulated
reagents or markers. The released chemicals are then detected according to
standard
techniques (see Monroe et al., Amer. Clln. Prod. Rev. 5:34-41 (1986)).
Reduction of non-specific binding
One of skill in the art will appreciate that it is often desirable to minimize
non-specific binding in immunoassays. Particularly, where the assay involves
an antigen or
antibody immobilized on a solid substrate it is desirable to minimize the
amount of non-
specific binding to the substrate. Means of reducing such non-specific binding
are well
known to those of skill in the art. Typically, this technique involves coating
the substrate
with a proteinaceous composition. In particular, protein compositions such as
bovine serum
albumin (BSA), nonfat powdered milk, and gelatin are widely used with powdered
milk
being most preferred.
Labels
The particular label or detectable group used in the assay is not a critical
aspect of the invention, as long as it does not significantly interfere with
the specific binding
of the antibody used in the assay. The detectable group can be any material
having a
detectable physical or chemical property. Such detectable labels have been
well-developed
in the field of immunoassays and, in general, most any label useful in such
methods can be
applied to the present invention. Thus, a label is any composition detectable
by
spectroscopic, photochemical, biochemical, immunochemical, electrical, optical
or chemical
SUBSTITUTE SHEET {RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
43
means. Useful labels in the present invention include magnetic beads (e.g.,
DynabeadsT""),
fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and
the like),
radiolabels (e.g., 3H, ~25I, 3sS, ~4C, or32P), enzymes (e.g., horse radish
peroxidase, alkaline
phosphatase and others commonly used in an ELISA), and colorimetric labels
such as
colloidal gold or colored glass or plastic beads (e.g., polystyrene,
polypropylene, latex, etc.).
The label may be coupled directly or indirectly to the desired component of
the assay
according to methods well known in the art. As indicated above, a wide variety
of labels
may be used, with the choice of label depending on sensitivity required, ease
of conjugation
with the compound, stability requirements, available instrumentation, and
disposal
provisions.
Non-radioactive labels are often attached by indirect means. Generally, a
ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand
then binds to
an anti-ligand (e.g., streptavidin) molecule which is either inherently
detectable or
covalently bound to a signal system, such as a detectable enzyme, a
fluorescent compound,
or a chemiluminescent compound. A number of ligands and anti-ligands can be
used in
conjunction with the labeled, naturally occurnng anti-ligands. Alternatively,
any haptenic
or antigenic compound can be used in combination with an antibody.
The molecules can also be conjugated directly to signal generating
compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of
interest as
labels will primarily be hydrolases, particularly phosphatases, esterases and
glycosidases, or
oxidotases, particularly peroxidases. Fluorescent compounds include
fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
Chemiluminescent
compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., lurninol.
For a review
of various labeling or signal producing systems which may be used, see U.S.
Patent No.
4,391,904.
Means of detecting labels are well known to those of skill in the art. Thus,
for example, where the label is a radioactive label, means for detection
include a
scintillation counter or photographic film as in autoradiography. Where the
label is a
fluorescent label, it may be detected by exciting the fluorochrome with the
appropriate
wavelength of light and detecting the resulting fluorescence. The fluorescence
may be
detected visually, by means of photographic film, by the use of electronic
detectors such as
charge coupled devices (CCDs) or photomultipliers and the like. Similarly,
enzymatic
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCTNS98/22321
44
labels may be detected by providing the appropriate substrates for the enzyme
and detecting
the resulting reaction product. Finally simple colorimetric labels may be
detected simply by
observing the color associated with the label. Thus, in various dipstick
assays, conjugated
gold often appears pink, while various conjugated beads appear the color of
the bead.
Some assay formats do not require the use of labeled components. For
instance, agglutination assays can be used to detect the presence of the
target antibodies. In
this case, antigen-coated particles are agglutinated by samples comprising the
target
antibodies. In this format, none of the components need be labeled and the
presence of the
target antibody is detected by simple visual inspection.
VI. Assays for compounds that increase or decrease ion flux
Slo3 and its alleles, polymorphic variants, and interspecies homologs are
subunits of pH sensitive, voltage-gated channels. The activity of such a
channel comprising
a Slo3 subunit can be assess using a variety of in vitro and in vivo assays,
e.g., measuring
current, measuring membrane potential, measuring ion flux, e.g., potassium or
rubidium,
measuring potassium concentration, measuring second messengers and
transcription levels,
and using e.g., voltage-sensitive dyes, radioactive tracers, and patch-clamp
electrophysiology. Furthermore, such assays can be used to test for inhibitors
and activators
of channels comprising Slo3. Such modulators of voltage-gated channel activity
are useful
for investigating and regulating sperm capacitation and the acrosome reaction,
as well as for
treating disorders related to sperm physiology.
Modulators of Slo3 activity are tested using biologically active Slo3, either
recombinant or naturally occurring. The protein can be isolated, expressed in
a cell, or
expressed in a membrane derived from a cell. Modulation is tested using one of
the in vitro
or in vivo assays described herein . Samples or assays that are treated with a
potential Slo3
inhibitor or activator are compared to control samples without the test
compound, to
examine the extent of modulation. Compounds that increase the flux of ions
will cause a
detectable increase in the ion current density by increasing the probability
of a channel
comprising Slo3 being open, by decreasing the probability of it being closed,
increasing
conductance through the channel, and allowing the passage of ions.
Increased or decreased flux of ions may be assessed by determining changes
in polarization (i.e., electrical potential) of the cell or membrane
expressing the Slo3
channel. A preferred means to determine changes in cellular polarization is by
measuring
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
changes in current (thereby measuring changes in polarization) with voltage
clamp and
patch-clamp techniques, e.g., the "cell-attached" mode, the "inside-out" mode,
and the
"whole cell" mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595
(1997)).
Whole cell currents are conveniently determined using the standard methodology
(see, e.g.,
5 Hamil et al., PFlugers. Archiv. 391:85 (1981). Whole cell currents are also
conveniently
determined using the conditions set forth in Example IV. Other known assays
include:
radiolabeled rubidium flux assays and fluorescence assays using voltage-
sensitive dyes (see,
e.g., Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Daniel et
al., J.
Pharmacol. Meth. 25:185-193 (1991); Holevinsky et al., J. Membrane Biology
137:59-70
10 (1994)).
The effects of the test compounds upon the function of the channels can also
be measured by changes in the electrical currents or ionic flux or by the
consequences of
changes in currents and flux. Changes in electrical current or ionic flux are
measured by
either increases or decreases in flux of cations such as potassium or rubidium
ions. The
15 cations can be measured in a variety of standard ways. They can be measured
directly by
concentration changes of the ions or indirectly by membrane potential or by
radiolabeling of
the ions. Consequences of the test compound on ion flux can be quite varied.
Accordingly,
any suitable physiological change can be used to assess the influence of a
test compound on
the channels of this invention. The effects of a test compound can be measured
by a toxin
20 binding assay. When the functional consequences are determined using intact
cells or
animals, one can also measure a variety of effects such as transmitter release
(e.g.,
dopamine), hormone release (e.g., insulin), transcriptional changes to both
known and
uncharacterized genetic markers (e.g., northern blots), cell volume changes
(e.g., in red
blood cells), immuno-responses (e.g., T cell activation), changes in cell
metabolism such as
25 cell growth or pH changes, changes in intracellular second messengers such
as Ca2+ or IP3,
and changes in the sperm capacitation or acrosome reactions.
Assays for compounds capable of inhibiting or increasing potassium flux
through the channel proteins comprising Slo3 can be performed by application
of the
compounds to a bath solution in contact with and comprising cells having an
channel of the
30 present invention (see, e.g., Blatz et al., Nature 323:718-720 (1986);
Park, J. Physiol.
481:555-570 (1994)). Generally, the compounds to be tested are present in the
range from 1
pM to 100 mM.
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
46
Preferably, the Slo3 channel of the assay will be selected from a channel
protein of SEQ ID NO: l, 16, or 18 conservatively modified variant thereof.
Alternatively,
the Slo3 channel of the assay will be derived from a eukaryote and include an
amino acid
subsequence having sequence similarity to the core domain (SO-S8 of Slo3) of
mSlo3 and
hSlo3 channel proteins. Generally, the functional Slo3 channel protein will be
at least 100,
200, 300, or 400-500 amino acids in length. Generally, the sequence similarity
will be at
least 60%, typically at least 70%, generally at least 75%, preferably at least
80%, more
preferably at least 85%, most preferably at least 90%, and often at least 95%.
Thus, Slo3
channel homologs will hybridize, under at least moderate hybridization
conditions, to a
nucleic acid of at least about 100 nucleotides in length from the core domain
of an mSlo3 or
hSlo3 nucleic acid and complementary sequences thereof.
The Slo3 channel homologs will generally have substantially similar
conductance characteristics (e.g., 80-120 pS) and pH sensitivity
characteristics, as described
above. Chimeras formed by expression of at least two of mSlo3 and hSlo3 also
be used, as
well as chimera formed by fusing Slo3 or a Slo3 subsequence to another Slo
molecule such
as Slo 1 or another potassium channel subunit. In a preferred embodiment, the
cell placed in
contact with a compound which is assayed for increasing or decreasing ion flux
is a
eukaryotic cell, e.g., an oocyte ofXenopus (e.g., Xenopus laevis) or a
mammalian cell such
as a CHO or HeLa cell.
As described in Example VI, chimeric Slo3 molecules can be made, which
combine a subsequence such as either the pore or the tail region of Slo3 with
a portion of
another molecule, e.g., the pore or tail of another potassium channel subunit
such as Slol, or
the pore region from a bacterial channel such as KcsA (McKinnon et al.,
Science 280:106-
109 (1998)). Such chimeras provide opportunities to screen and identify
modulators of a
specific Slo3 region, such as the pore or the tail.
Yet another assay for compounds that increase or decrease potassium flux in
pH sensitive potassium channels involves computer assisted drug design, in
which a
computer system is used to generate a three-dimensional structure of Slo3
proteins based on
the structural information encoded by the amino acid sequence. The amino acid
sequence
interacts directly and actively with a preestablished algorithm in a computer
program to
yield secondary, tertiary, and quaternary structural models of the protein.
The models of the
protein structure are then examined to identify regions of the structure that
have the ability
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
47
to bind to ligands. These regions are then used to identify ligands that bind
to the protein or
regions in which SIo3 interacts with other subunits.
The three-dimensional structural model of the protein is generated by
entering channel protein amino acid sequences of at least 10 amino acid
residues or
corresponding nucleic acid sequences encoding a channel protein into the
computer system.
The amino acid sequence of the channel protein is selected from the group
consisting of
SEQ ID NOS:1, 3, 16, and 18 and conservatively modified versions thereof. The
amino
acid sequence represents the primary sequence of the protein, which encodes
the structural
information of the protein. At least 10 residues are entered into the computer
system from
computer keyboards or computer readable substrates that include, but are not
limited to,
electronic storage media (e.g., magnetic diskettes, tapes, cartridges, and
chips), optical
media (e.g., CD ROM), information distributed by Internet sites, and RAM. The
three-
dimensional structural model of the channel protein is then generated by the
interaction of
the amino acid sequence and the computer system. The software is known to
those skilled
1 S in the art.
The amino acid sequence represents a primary structure that encodes the
information necessary to form the secondary, tertiary and quaternary structure
of the
monomer protein and channel. The software looks at certain parameters encoded
by the
primary sequence to generate the structural model. These parameters are
referred to as
"energy terms," and primarily include electrostatic potentials, hydrophobic
potentials,
solvent accessible surfaces, and hydrogen bonding. Secondary energy terms
include van
der Waals potentials. Biological molecules form the structures that minimize
the energy
terms in a cumulative fashion. The computer program is therefore using these
terms
encoded by the primary structure or amino acid sequence to create the
secondary structural
model.
The tertiary structure of the protein encoded by the secondary structure is
then formed on the basis of the energy terms of the secondary structure. The
user at this
point can enter additional variables such as whether the protein is membrane
bound or
soluble, its location in the body, and its cellular location, e.g.,
cytoplasmic, surface, or
nuclear. These variables along with the energy terms of the secondary
structure are used to
form the model of the tertiary structure. In modeling the tertiary structure,
the computer
program matches hydrophobic faces of secondary structure with like, and
hydrophilic faces
of secondary structure with like.
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
48
Finally, quaternary structure of multi-subunit proteins can be modeled in a
similar fashion, using anisotropy terms. These terms interface different
protein subunits to
energetically minimize the interaction of the subunits. In the case of channel
proteins,
typically four identical subunits make up the quaternary structure of the
channel.
S Once the structure has been generated, potential Iigand binding regions are
identified by the computer system. Three-dimensional structures for potential
ligands are
generated by entering amino acid or nucleotide sequences or chemical formulas
of
compounds, as described above. The three-dimensional structure of the
potential ligand is
then compared to that of the channel protein to identify ligands that bind to
the channel
protein. Binding affinity between the protein and ligands is determined using
energy terms
to determine which ligands have an enhanced probability of binding to the
protein.
Computer systems are also used to screen for mutations of Slo3 genes. Such
mutations can be associated with disease states. Once the mutations are
identified,
diagnostic assays can be used to identify patients having such mutated genes
associated with
disease states. Identification of the mutated Slo3 genes involves receiving
input of a first
nucleic acid sequence encoding a pH sensitive potassium channel protein having
an amino
acid sequence selected from the group consisting of SEQ ID NOS:1, 3, 16, 18
and
conservatively modified versions thereof. The sequence is entered into the
computer system
as described above. The first nucleic acid sequence is then compared to a
second nucleic
acid sequence that has substantial identity to the first nucleic acid
sequence. The second
nucleic acid sequence is entered into the computer system in the manner
described above.
Once the first and second sequences are compared, nucleotide differences
between the
sequences are identified. Such sequences can represent allelic differences in
Slo3 genes,
and mutations associated with disease states.
VII. Cellular transfection and gene therapy
The present invention provides packageable Slo3 channel protein nucleic
acids (cDNAs), supra, for the transfection of cells in vitro and in vivo.
These packageable
nucleic acids can be inserted into any of a number of well known vectors for
the
transfection of target cells and organisms as described below. The nucleic
acids are
transfected into cells, ex vivo or in vivo, through the interaction of the
vector and the target
cell. The Slo3 channel protein nucleic acid, under the control of a promoter,
then expresses
the pH sensitive potassium channel protein of the present invention thereby
mitigating the
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US9$l22321
49
effects of absent, partial inactivation, or abnormal expression of the Slo3
channel protein
gene. For example, the Slo3 gene may be used to treat infertility conditions
due to its
involvement in sperm physiology, e.g., capacitation and acrosome reactions.
Such gene therapy procedures have been used to correct acquired and
inherited genetic defects, cancer, and viral infection in a number of
contexts. The ability to
express artificial genes in humans facilitates the prevention and/or cure of
many important
human diseases, including many diseases which are not amenable to treatment by
other
therapies. As an example, in vivo expression of cholesterol-regulating genes,
genes which
selectively block the replication of HIV, and tumor-suppressing genes in human
patients
dramatically improves the treatment of heart disease, AIDS, and cancer,
respectively. For a
review of gene therapy procedures, see Anderson, Science 256:808-813 (1992);
Nabel &
Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166
(1993);
Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van
Brunt,
Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and
Neuroscience
8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44
(1995);
Haddada et al., in Current Topics in Microbiology and Immunology, (Doerfler &
Bohm
eds., 1995); and Yu et al., Gene Therapy, 1:13-26 (1994).
Delivery of the gene or genetic material into the cell is the first critical
step
in gene therapy treatment of disease. A large number of delivery methods are
well known
to those of skill in the art. Such methods include, for example liposome-based
gene
delivery (Mannino & Gould-Fogerite, BioTechniques 6:682-691 (1988); U.S. Pat
No.
5,279,833; WO 91/06309; and Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-
7414
(1987)), and replication-defective retroviral vectors harboring a therapeutic
polynucleotide
sequence as part of the retroviral genome (see, e.g., Miller et al., Mol.
Cell. Biol. 10:4239
( 1990); Kolberg, J. NIH Res. 4:43 ( 1992); and Cornetta et al. Hum. Gene
Ther. 2:215
(1991)). Widely used retroviral vectors include those based upon murine
leukemia virus
(MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV),
human
immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher
et al., J.
Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);
Sommerfelt et al.,
Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller
et al., J. Viroi.
65:2220-2224 (1991); PCT/LJS94/05700.
AAV-based vectors are also used to transduce cells with target nucleic acids,
e.g., in the in vitro production of nucleic acids and peptides, and for in
vivo and ex vivo gene
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S.
Patent No.
4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka,
J.
Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are
described in a
number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al.,
Mol. Cell. Biol.
S 5:3251-3260 (1985); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984);
Hermonat &
Muzyczka, Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); and Samulski et al.,
J. Virol.
63:03822-3828 (1989). Cell lines that can be transfected by rAAV include those
described
in Lebkowski et al., Mol. Cell. Biol. 8:3988-3996 (1988).
10 A. Ex vivo transfection of cells
Ex vivo cell transfection for diagnostics, research, or for gene therapy
(e.g.,
via re-infusion of the transfected cells into the host organism) is well known
to those of skill
in the art. In a preferred embodiment, cells are isolated from the subject
organism,
transfected with an Slo3 channel protein nucleic acid (gene or cDNA), and re-
infused back
15 into the subject organism (e.g., patient). Various cell types suitable for
ex vivo transfection
are well known to those of skill in the art (see, e.g., Freshney et al.,
Culture of Animal Cells,
a Manual of Basic Technigue (3d ed. 1994)).
As indicated above, in a preferred embodiment, the packageabie nucleic acid
which encodes an Slo3 channel protein is under the control of an activated or
constitutive
20 promoter. The transfected cells) express a functional Slo3 channel protein,
which mitigates
the effects of deficient or abnormal Slo3 channel protein gene expression.
B. In vivo transfection
Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing
25 therapeutic nucleic acids can be administered directly to the organism for
transduction of
cells in vivo. Administration is by any of the routes normally used for
introducing a
molecule into ultimate contact with blood or tissue cells. The packaged
nucleic acids are
administered in any suitable manner, preferably with pharmaceutically
acceptable carriers.
Suitable methods of administering such packaged nucleic acids are available
and well
30 known to those of skill in the art, and, although more than one route can
be used to
administer a particular composition, a particular route can often provide a
more immediate
and more effective reaction than another route.
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
51
Pharmaceutically acceptable Garners are determined in part by the particular
composition being administered, as well as by the particular method used to
administer the
composition. Accordingly, there is a wide variety of suitable formulations of
pharmaceutical compositions of the present invention.
The packaged nucleic acids, alone or in combination with other suitable
components, can be made into aerosol formulations (i.e., they can be
"nebulized") to be
administered via inhalation. Aerosol formulations can be placed into
pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Formulations suitable for parenteral administration, such as, for example, by
intravenous, intramuscular, intradermal, and subcutaneous routes, include
aqueous and non-
aqueous, isotonic sterile injection solutions, which can contain antioxidants,
buffers,
bacteriostats, and solutes that render the formulation isotonic with the blood
of the intended
recipient, and aqueous and non-aqueous sterile suspensions that can include
suspending
agents, solubilizers, thickening agents, stabilizers, and preservatives. In
the practice of this
invention, compositions can be administered, for example, by intravenous
infusion, orally,
topically, intraperitoneally, intravesically or intrathecally. The
formulations of packaged
nucleic acid can be presented in unit-dose or mufti-dose sealed containers,
such as ampules
and vials.
Injection solutions and suspensions can be prepared from sterile powders,
granules, and tablets of the kind previously described. Cells transduced by
the packaged
nucleic acid as described above in the context of ex vivo therapy can also be
administered
intravenously or parenterally as described above.
The dose administered to a patient, in the context of the present invention
should be sufficient to effect a beneficial therapeutic response in the
patient over time. The
dose will be determined by the efficacy of the particular vector employed and
the condition
of the patient, as well as the body weight or surface area of the patient to
be treated. The
size of the dose also will be determined by the existence, nature, and extent
of any adverse
side-effects that accompany the administration of a particular vector, or
transduced cell type
in a particular patient.
In determining the effective amount of the vector to be administered in the
treatment or prophylaxis of conditions owing to diminished or aberrant
expression of Slo3
channel protein, the physician evaluates circulating plasma levels of the
vector, vector
toxicities, progression of the disease, and the production of anti-vector
antibodies. In
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCTNS98/22321
52
general, the dose equivalent of a naked nucleic acid from a vector is from
about 1 pg to 100
pg for a typical 70 kilogram patient, and doses of vectors which include a
retroviral particle
are calculated to yield an equivalent amount of therapeutic nucleic acid.
For administration, inhibitors and transduced cells of the present invention
can be administered at a rate determined by the LD-50 of the inhibitor,
vector, or transduced
cell type, and the side-effects of the inhibitor, vector or cell type at
various concentrations,
as applied to the mass and overall health of the patient. Administration can
be
accomplished via single or divided doses.
Transduced cells are prepared for reinfusion according to established
methods (see, e.g., Abrahamsen et al., J. Clin. Apheresis 6:48--53 (1991);
Carter et al., J.
Clin. Apheresis, 4:113-117 (1988); Aebersold et al., J. Immunol. Meth. 112:1-7
(1988);
Muul et al., J. Immunol. Methods 101:171-181 (1987); and Carter et al.,
Transfusion
27:362-365 (1987)). After a period of about 2-4 weeks in culture, the cells
should number
between 1 x 1 Og and 1 x 1012. In this regard, the growth characteristics of
cells vary from
patient to patient and from cell type to cell type. About 72 hours prior to
reinfusion of the
transduced cells, an aliquot is taken for analysis of phenotype, and
percentage of cells
expressing the therapeutic agent.
VIII. Chromosomal assignment of the hSlo3 gene
To identify the chromosomal location of hSlo3, the cDNA sequences
encoding hSlo3 and the 5' and 3' untranslated DNA sequence, as well as DNA
sequence
derived from genomic DNA may be used to map the hSlo3 gene to a site on a
number of
different types of genetic maps. This may be accomplished by mapping methods
which are
well known in molecular genetics, including somatic cell hybrid mapping,
radiation hybrid
(RH) mapping, and chromosome mapping using fluorescent in situ hybridization
(FISH).
An example of one of these methods which is commonly used to map a DNA
sequence is the method of radiation hybrid mapping. This procedure allows one
to establish
with high resolution the position of a DNA sequence within the RH map by
comparison of
the experimental results with those obtained with known DNA markers, and
evaluating the
statistical probability that such a map assignment is non-random (see, e.g.,
Cox et al.,
Science 250:245-250 (1990)).
Typically, a human/hamster somatic cell hybrid panel is used for this
purpose. These panels are commercially available, an example of which is the
commonly
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
53
used Stanford G3 panel, available from Research Genetics Inc. The panel is
composed of
genomic DNA from each of 83 different clonal human/hamster cell hybrid cell
lines. Each
cell line contains fragments of human genomic DNA in addition to the genomic
host DNA
of the hamster cell line from which they were derived. Since the human genomic
DNA is
distributed unevenly among the 83 clonal lines, PCR amplification of a
specific human
DNA fragment using genomic DNA from each of the clonal lines results in an
amplified
product in only those clonal lines containing the fragment of the
corresponding human
genomic DNA. Identification of the lines which produce a positive signal and
which do not
give a pattern that may be deconvoluted to a map position is determined by
comparison of
the pattern with patterns derived from other markers in a database, for
example the RH
server database at the Stanford Human Genome Center. Localization of the RH
mapped
DNA sequence to a site on a human chromosome may then be established using
physical
map information derived from nearby known RH markers that have already been
assigned a
locus on the physical map. This assignment may be accomplished using publicly
available
databases such as the Genome Database.
The chromosomal localization of Slo3 may be used to determine whether a
disease or genetic defect is attributable to changes in the genomic DNA
containing the Slo3
gene. Examples of such changes are well known in the literature and include
point
mutations, insertions, and deletions. Examples of human diseases attributable
to changes in
genomic DNA sequence include cystic fibrosis and long Q-T syndrome.
Association of a
disease with changes in the gene coding for a Slo3 may be accomplished by
examination of
the genetics literature to find diseases for which the chromosomal assignment
is already .
known but for which a specific mutation has not been determined. This can also
be
accomplished by examining genomic DNA sequence of an individual or group of
individuals directly to determine if a mutation has occurred using established
methods or a
combination of both. Examples of such methods include but are not limited to
PCR, single
strand conformational polymorphism (SSCP} analysis, and direct sequencing of
genomic
DNA.
Alternatively, a disease may be mapped to a chromosomal location or a
specific gene without prior knowledge of its identity by positional cloning or
other methods
know to those of skill. The identification of the gene may then be established
by comparing
the chromosomal location or actual DNA sequence with those derived from the
literature or
from databases containing known sequence data such as Genbank
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
54
IX. Kits
Slo3 and its homologs are a useful tool for examining expression and
regulation of pH sensitive, voltage-gated potassium channels, particularly in
developing
sperm. Slo3 specific reagents that specifically hybridize to Slo3 nucleic
acid, such as Slo3
probes and primers, and Slo3 specific reagents that specifically bind to the
Slo3 protein,
e.g., Slo3 antibodies are used to examine expression and regulation.
Nucleic acid assays for the presence of Slo3 DNA and RNA in a sample
include numerous techniques are known to those skilled in the art, such as
Southern
analysis, northern analysis, dot blots, RNase protection, S 1 analysis,
amplification
techniques such as PCR and LCR, and in situ hybridization. In in situ
hybridization, for
example, the target nucleic acid is liberated from its cellular surroundings
in such as to be
available for hybridization within the cell while preserving the cellular
morphology for
subsequent interpretation and analysis. The following articles provide an
overview of the
art of in situ hybridization: Singer et al., Biotechnigues 4:230-250 (1986);
Haase et al.,
Methods in Virology, vol. VII, pp. I89-226 (1984); and Nucleic Acid
Hybridization: A
Practical Approach (Hames et al., eds. 1987). 'In addition, Slo3 protein can
be detected
with the various immunoassay techniques described above, e.g., ELISA, western
blots, etc..
The test sample is typically compared to both a positive control (e.g., a
sample expressing
recombinant Slo3) and a negative control.
The present invention also provides for kits for screening for modulators of
Slo3. Such kits can be prepared from readily available materials and reagents.
For
example, such kits can comprise any one or more of the following materials:
biologically
active Slo3, reaction tubes, and instructions for testing Slo3 activity.
Preferably, the kit
contains biologically active Slo3. A wide variety of kits and components can
be prepared
according to the present invention, depending upon the intended user of the
kit and the
particular needs of the user. For example, the kit can be tailored for in
vitro or in vivo
assays for measuring the activity of a homomeric voltage-gated potassium
channel
comprising an Slo3 subunit.
All publications and patent applications cited in this specification are
herein
incorporated by reference as if each individual publication or patent
application were
specifically and individually indicated to be incorporated by reference.
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity of understanding, it will
be readily
apparent to one of ordinary skill in the art in light of the teachings of this
invention that
certain changes and modifications may be made thereto without departing from
the spirit or
S scope of the appended claims.
EXAMPLES
The following examples are provided by way of illustration only and not by
way of limitation. Those of skill in the art will readily recognize a variety
of noncritical
10 parameters that could be changed or modified to yield essentially similar
results.
Example I: Cloning mSlo3, a pH sensitive potassium channel
The nucleotide sequence for mSlo3 was cloned by searching a database for
EST sequences with homology to mSlol, generating a probe based on the EST
sequence
15 using PCR, and screening a library with the EST probe.
A. Methods
By tBlastn (NCBI), an EST (GenBank accession No. AA072586) was
identified by homology to the C-terminal "tail" of mSlol. The EST derived from
a mouse
20 promyelocytic WEHI-3 cell line cDNA library. A 32P-labeled 1254 by PCR
product
generated from the EST-pBluescript plasmid (Genome Systems) was employed to
isolate
cDNA clones from a WEHI-3 library (Stratagene) by hybridization. The
oligonucleotides
used to generate the probe were 5' GTGGA-TGATACC-GACATGC-TGGAC 3' (sense)
and 5' GAGACCACCTCTC TCCCGTGTCGT 3' (antisense). mSlo3 expression in the
25 WEHI-3 cell line is apparently anomalous; all isolated cDNAs were
inappropriately spliced
or truncated, and PCR analysis of the WEHI-3 cDNA bank using combinations of
primers
homologous to mSlo3 and vector sequence indicated that complete cDNAs were not
represented.
Subsequent screening of a mouse testis cDNA library (Dr. Graeme Mardon)
30 yielded cDNAs that extended to the putative initiator methionine, as shown
in Figure 1.
The reading frame was closed upstream from the indicated methionine. A full
length cDNA
was constructed from two overlapping cDNAs and the presence of full length
transcripts in
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCTNS98/22321
56
testis corresponding to this cDNA was verified by RT-PCR from total testis
RNA. The
entire cDNA was sequenced in both directions.
For expression in Xenopus oocytes, a Kozak initiator sequence was
introduced by PCR and the entire open reading frame was subcloned into the
pOocyte-
S Xpress vector, containing Xenopus 5' and 3' ~3-globin UTRs. The 5' end
generated by PCR
was checked for errors by sequencing in both directions.
B. Results
mSlo3 cDNA was isolated from a testis cDNA library based on its homology
to the large-conductance calcium-activated (BK) potassium channel, mSlo 1
(Butler et al.,
Science 261:221-224 (1993)). The probe was generated from an expressed
sequence tag
identified in the GenBank database. The new channel was termed mSlo3 ("m"
denoting
derivation from mouse). Figure lA illustrates that the 1113 amino acid mSlo3
is similar to
the 1196 amino acid mSlol protein (Butler et al., 1993, supra) as well as
Drosophila Slol
(Atkinson et al., Science 253:551-553 (1991); Adelman et al., 1992, supra).
The
hydrophilicity profiles of both sequences indicate 11 hydrophobic segments, SO
through
S10 (Figure 1B). As with mSlol, these can be divided into "core" and "tail"
domains.
Homology of mSlo3 to mSlol in the core domain (SO-S8) (51%), which is
generally
conserved in the voltage-gated superfamily of K+ channels, is much higher than
in the tail
domain, which is involved in calcium sensing (Wei et al., Neuron 13:671-681
(1994)).
Two notable differences between mSlo3 and mSlol demonstrate their
functional distinctions. First, the "Calcium Bowl," a hyperconserved aspartate-
rich region
involved in calcium sensing, is absent in mSlo3 (Schreiber & Salkoff, Biophys.
J. 73:1355-
1363 ( 1997)). This absence showed that mSlo3 is gated by factors other than
calcium.
Second, mSlo3 contained GFG rather than GYG in the conserved pore signature
sequence
involved in K+ ion selectivity (Yool & Schwarz, Nature 349:700-704 ( 1991 );
Hartmann et
al., Science 251:942-944 (1991); Heginbotham & MacKinnon, Biophys. J. 66:1061-
1067
(1994)). Most K+ selective channels contain GYG. This difference demonstrated
that ionic
selectivity differs between the two channels: mSlo3 has greater permeability
to Na+ than
Slo 1 channels. A block of conservation containing arginine at regular
intervals in the
beginning of the S4 region may underlie the fact that, in addition to other
factors, both
mSlo3 and Slol channels are gated by voltage. mSlo3 core residues 35 through
641 share
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/LJS98/22321
57
51% identity with mSlol. Overall, mSlo3 shares less than 30% amino acid
identity with
mSlol.
Example II: mRNA expression of mSlo3
In situ hybridization, northern analysis, RT-PCR, and cDNA cloning were
used to investigate mSlo3 expression in a variety of tissues. These
experiments
demonstrated that mSIo3 is abundant in spermatocytes.
A. Methods
For each tissue tested with RT-PCR, MMLV reverse transcriptase (GIBCO)
was used on S mg total RNA primed with 25 mM random hexanucleotides
(Boehringer
Mannheim) and 200 mM dNTPs at 42°C, for 1 hr. 0.1 % of each first
strand synthesis was
assayed by PCR, using 1.0 mM oligonucleotide primers, 200 mM dNTPs and 0.0075
units
KlenTaq, cycling 30 times. Reaction products were electrophoresed on 1.5% and
3.0%
agarose gels, using standard Tris-borate (TBE) buffer, and visualized by
staining with
ethidium bromide. PCR primer pairs used were:
mSlo3 (S4 to SS). 5' CTCGAACTCCCTAAAATCTTACAGAT 3' (sense)
and 5' TTCCGTTGAGCCAGGGGTCACCAGAATT 3' (antisense) to generate a 156 by
product; mSlo3 (S8 to S9). 5' TCTGCTTTGTGAAGCTAAATCT 3' (sense) and 5'
TTTCAAAGCCTCTTTAGCGGTAA 3' (antisense) to generate a 690 by product; mSlo3
(S9 to S10); S' TTATGCCTGGATCTGCACTCTACATG 3' (sense) and S'
ATAGTTTCCGTCTACTACCGAAA 3' (antisense) to generate a 221 by product. As a
control, Human (i-actin. 5' GATGATATCGCCGCGCTCGTCGTCGAC 3' (sense) and S'
TCGGTCCAGGTCTGCGTCCTACCGTAC 3' (antisense) to generate a 535 by product.
For northern blot analysis, total RNA or poly A+ RNA was isolated from
freshly dissected mouse and human tissue using Trizol (GIBCO). The mouse
tissues were
brain, heart, skeletal muscle, kidney, lung, liver and testis, the human
tissues were spleen,
thymus, prostate, testis, uterus, small intestine, colon, and leukocytes. 20
mg total RNA or
2 mg poly A+ RNA from each tissue was electrophoresed on a 1 % agarose
denaturing gel,
using MOPS-formaldehyde buffer, then transferred to nitrocellulose. The human
tissue blot
was obtained commercially (Clontech). A PCR product generated using primers 5'
CGGAAACGTCATGTACAATCGAAATCCA 3' (sense) and 5' TTCC-GTTG-AGCC-
AGGG-GTCACCAGAATT 3' (antisense) was labeled using random hexanucleotides
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
58
(Boehringer-Mannheim). Blots were hybridized and washed under standard high
stringency
conditions and exposed to X-ray film for 16 hours. After hybridization with
mSlo3 probes,
blots were rehybridized with a human ~3-actin probe to verify RNA loading. For
examination of human tissues, 2 mg of poly A+ RNA from the tissues described
above were
electrophoresed as described. The probe was the mSlo3 probe described above.
For in situ hybridization, testes from white mice >30 days old were
dissected, frozen, sectioned immediately with a cryostat, collected on slides,
and stored at -
20°C. A partial mSlo3 cDNA (approximately 1 kb, corresponding to coding
sequence for
residues 170-510) was subcloned into pBluescript II KS+ (Stratagene). T3 and
T7 RNA
polymerase (Stratagene) were used to synthesize 32P-UTP-labeled antisense and
sense
probes, respectively, from linearized plasmid. Slides were hybridized
overnight at 55°C.
After washing, slides were dipped in NTB-2 liquid emulsion (Kodak), air dried,
and placed
in light protected boxes at 4°C for 10 days.
B. Results
For RT-PCR and northern analysis, mouse brain, skeletal muscle, lung, liver,
kidney, and heart tissue were examined; only testis produced a positive signal
(Figure 3).
Total RNA blots from the same tissues showed a transcript size of
approximately 4 kb, also
restricted to testis. Using the mouse Slo3 probe, in human tissues, poly A+
RNA on a
northern blot again showed a transcript size of approximately 4 kb, again only
in testis.
For in situ hybridization experiments, testes from white mice >30 days old
were dissected, frozen, and sectioned immediately with a cryostat, collected
on slides, and
stored at -20°C. A partial cDNA (approximately 1 kb) for the potassium
channel mSlo3
was cloned into the Bluescript expression vector (Stratagene). Both sense and
antisense
RNA probes were labeled with uridine 5'-[a-32P]-triphosphate ( 10 pCi/p.l).
Slides were
coverslipped and allowed to hybridized overnight at 55°C. Following
washing, slides were
dehydrated, air dried and dipped in Kodak NTB-2 liquid emulsion, then placed
in air tight
light boxes at 4°C for 10 days.
Labeling was observed in annular rings corresponding to the positions of
spermatocytes in seminiferous tubules. Positive hybridization signals appeared
as white
dots on darkfield micrographs. Dense circular patterns of hybridization
signals
corresponded to the annular clusters of spenmatocytes. The annular structure
shows the
cross-section of a single seminiferous tubule, composed largely of developing
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
59
spermatocytes. Stem cells and primary spermatocytes were at the outer edges,
while more
mature spermatocytes were found near the lumen. Supporting Sertoli cells and
Leydig cells,
difficult to distinguish at this resolution, were present in lower numbers.
Hybridization
signals at the inner margins of the circular patterns of seminiferous tubules
corresponded to
the positions of secondary spermatocytes and possibly even spermatids. Dark-
field
microscopy of the same view shows intense hybridization of antisense probe
with mSlo3
mRNA in developing spermatocytes. The outer edges and interstices between
tubules are
unstained, suggesting that primary and secondary spermatocytes are the
predominant cell
type expressing the message, rather than spermatogonia (spermatic stem cells).
Staining of
the innermost regions of the tubule suggests that even early spermatids may be
expressing
the message. The high density of labeling demonstrates a high degree of mSlo3
expression
in spermatocytes.
In addition to the dense patterns of staining in testis in situ, two
additional
experiments indicated that mSlo3 is abundantly expressed in testis. Northern
analysis of
total RNA isolated from testis gives a positive signal after only 10 hrs. of
exposure. In
addition, cDNAs encoding mSlo3 were abundant in a cDNA library constructed
from adult
testis derived RNA (unique cDNAs encoding mSlo3 represented approximately
0.01% of
total cDNAs in the library). In contrast to its abundance in testes, mSlo3 was
absent or
expressed at much lower levels in other tissues, such as brain, skeletal
muscle, and heart.
Example III: Xenopus oocyte expression of mSlo3
mSlo3 clones were expressed in Xenopus oocytes, in order to analyze
potassium channel function.
A. Methods
As described above in Example I, the entire open reading frame of mSlo3
was assembled in a modified Bluescript II KS+ vector (Stratagene) containing
Xenopus 5'
and 3' ~-globin UTRs. For expression, cRNA was generated from template
linearized at a
unique vector Notl site and transcribed using the mMessage mMachine T3 kit
(Arnbion).
B. Results
A full-length mSlo3 cDNA was cloned into pBSC-MXT, a Bluescript-
derived plasmid (Stratagene) containing Xenopus (3-globin 5' and 3'
untranslated sequences.
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
cDNA expression constructs were linearized at a unique Not-I site, and capped
cRNA was
synthesized using the mMessage mMachine (Ambion). Reactions were precipitated
with
LiCI to remove the DNA template and resuspended in nuclease-free water at a
final
concentration of approximately 1 mg/ml. Oocytes were prepared for injection as
previously
5 described (Wei et al., Science 248:599-603 (1990)}, except for the use of a
Drummond
nanojector. Approximately 50 nl of RNA in nuclease-free water was injected
into each
oocyte. Oocytes were incubated in ND96 medium (Wei et al., 1994, supra) and
analyzed 1-
8 days after injection.
10 Example IV: Electrophysiology
mSlo3 clones expressed in Xenopus oocytes, as described above in Example
III, were analyzed for electrophysiological characteristics.
A. Methods
1 S cRNA (40 nl at approximately 1 mg/p,l) was inj ected into mature Xenopus
oocytes; recordings were made 1-8 days later. For whole cell-recording, medium
nd96
(concentrations in mM; 96 NaCI, 2 KCI, 1.8 CaCl2, 1 MgCl2, S HEPES, pH 7.5)
supplemented with 1 mM DIDS (to block endogenous chloride currents) employed
as bath
solution, or modified as noted in figure legends. Patches were perfused with
either 0 Ca2+
20 EGTA solutions (160 K gluconate, 34 KOH, 10 HEPES, and 10 EGTA) or Ca2+-
containing
( 184 K gluconate, 10 KOH, 10 HEPES, 200 mM hemicalcium gluconate) solutions.
HCl
was used to adjust pH. Pipet solution contained 0.5 K gluconate, 0.5 KCI, 1.1
KOH, 10
HEPES, 159 Na gluconate, and 2 hemiMg giuconate, pH 7.1. Recording. Two-
electrode
voltage clamp was carned out with a TEV-200 amplifier (Dagan). Patch currents
were
25 recorded on either an Axopatch 1B or 200A amplifier (Axon), and digitized
at either 3.4, 5
or 10 kHz. Data acquisition and analysis programs were CCURRENT and CQUANT
(Dr.
Keith Baker) or pClamp6 (Axon). Recordings were made at room temperature
except tail
currents which were recorded at i 1°C using a Pettier device (Cambion).
30 B. Results
When expressed in the Xenopus oocyte expression system, mSlo3 cRNA
produced currents that were sensitive to pH and voltage. Since pHi regulation
is critical in
spermatic function, mSlo3 activity was tested over a range of pHi in detached,
perfused
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
61
patches (Figure 3). mSlo3 currents were small or absent at a pHi of 7.1 or
lower, while
raising pHi resulted in sharp increases in channel activity. Whole-cell mSlo3
currents were
also sensitive to manipulations of intracellular pH (Figure 4). Acidification
of the oocyte
using bicarbonate-based bath solution strongly attenuated currents, while
alkalinization by
ammonium chloride reversed this decrease (Fakler et al., EMBO J. 15:4093-4099
{1996);
Sasaki et al., Biochim. et Biophys. Acta 1137:45-S 1 ( 1992)). These results
are consistent
with those obtained with perfused patches. Despite channel rundown, which
occurred
rapidly over the course of minutes, macroscopic currents could be elicited
from large-
diameter patch electrodes (Figure 3D). These currents revealed that although
mSlo3 is
similar in primary structure to the high conductance Ca2+-activated potassium
channel
mSlol, mSlo3 activation is not sensitive to Ca2+ (Figure 3D). Channel activity
was robust
even in the presence of 10 mM EGTA (0 M Ca2+ conditions in Figure 3D, and all
experiments in Figure 3A-C).
Several other mSlo3 properties were distinct from its closest homolog,
mSlol. mSlo3 single-channel conductance was lower than mSlol (approximately
100 pS
vs. 270 pS in symmetric K+ as derived from the slope of the unit current
amplitude versus
voltage relation; Butler et al., 1992, supra). Unlike Slol, Slo3 was
relatively insensitive to
TEA (EC50 approximately 100 mM versus less than 0.2 mM for mSlo 1 ),
consistent with a
Y to V change at residue 283 near the K+ selective pore (Butler et al., 1992,
supra;
Kavanaugh et al., J. Biol. Chem. 266:7583-7587 (1991)). The highly selective
mSlol
channel toxin blockers charybdotoxin (50 nM) and iberiotoxin (20 nM) did not
affect mSlo3
currents (Miller et al., Nature, 313:316-318 (1985); Galvez et al., J. Biol.
Chem. 265: i 1083-
11090 (1990)). Of particular note, tail current analysis revealed that mSlo3
is less selective
for K+ over Na+ than Slol, having a PK/PNa of approximately S versus >50 for
mSlol
(Figure 4C, D; Tseng-Crank et al., 1994, supra).
These experiments demonstrated that mSlo3 has the following
electrophysiological characteristics when expressed in Xenopus oocytes:
1. mSlo3 is sensitive to pH, being very active above pH 7.1. mSlo3 is
outwardly rectifying.
2. Although mSlo3 is structurally related to the Maxi K calcium-
activated channel, mSlol, mSlo3 is insensitive to calcium.
3. In Xenopus oocytes with the symmetric K+ concentrations used above
(160 mM KCL), mSlo3 has a single channel conductance of approximately 100 pS.
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PC'f/US98/22321
62
4. mSlo3 has a relativity high conductivity to Na+ for a K+ channel; the
PNaIPK ratio is about 0.25.
Example V: Cloning hSlo3, a human homolog of the mSlo3 channel
Sequence of hSlo3 was obtained by PCR and human testis cDNA library
screening, using the mSlo3 sequence as a probe.
A. Methods
The nucleotide sequence of mSlo3 was examined in the core domain, and
PCR primers were synthesized to amplify the corresponding segment from a human
testis
cDNA library (Clontech). The primers were 5' GGCAGCGCTCATTCTTTCCTCCTT - 3'
and 5' TGCCCAAAACCTCAACCCAA.AATA - 3'. PCR was performed at an annealing
temperature of 50°C for 30 seconds and an extension time of 30 seconds
at 72°C for 40
cycles. PCR fragments were subcloned into pCR II (Invitrogen) and sequenced.
The cDNA library (2 x 106 pfu) was subsequently screened with a mSlo3
probe. A 2.6 kb BamHI fragment of mSlo3 was labeled with 32P by random primer
extension, and bacteriophage lambda plaque filter lifts were hybridized in 30%
formamide,
1X Denhardt's, Tris-HCI 20 mM (pH 7.4), 4X SSC, 0.1% SDS, denatured sheared
salmon
sperm DNA 20 ~tg/ml, yeast tRNA 20 ~g/ml at 42°C overnight. Filters
were washed in 2X
SSC/0.1 % SDS at 42°C for 15 minutes x 2, then in 0.2X SSC/0.1 % SDS at
42°C for 30
minutes. Filters were exposed to x-ray film overnight at -80°C and
developed.
Bacteriophage plaques that showed corresponding labeling on the autoradiograms
were
purified to homogeneity, the phage DNA prepared and cDNA inserts excised with
EcoRI
and subcloned into pBluescript II SK (Stratagene) for sequencing. Sequencing
was
performed on an ABI automated sequences.
B. Results
DNA fragments generated by PCR of a human testis cDNA library were
sequenced, and the nucleotide sequence obtained (SEQ ID N0:4) was highly
homologous
with mSlo3, showing approximately 70% sequence identity on the nucleotide
level. The
translated amino acid sequence (SEQ ID N0:3) was 61.5% identical to mSlo3 on
the amino
acid level over the sequence region, with 94% identity in the S i region. PCR
of human
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
63
cDNA libraries derived from brain, pancreas, heart, retina, and leukemia cell
line (Jurkat)
failed to produce the expected band.
Screening of a human testis cDNA library at moderate stringency produced 6
clones, all of which were identified as being hSlo3 by PCR using specific
internal primers.
One clone was partially sequenced, producing a nucleotide sequence which was
identical to
the DNA fragments derived from the PCR of the library. Although the clones
have not been
sequenced in their entirety, the high sequence identity in conjunction with
the localization
of the channel mRNA to human testis by northern blot (Example II) and PCR
establishes
the identity of the human Slo3 channel.
Example VI: Slo3/Slol Chimeras
In this experiment, a Slol core-Slo3 tail chimera has been made, to
demonstrate that two distinct regions of the Slo 1 tail are involved in
calcium sensing. These
regions were demonstrated by re-introducing small regions of the Slol tail
into a calcium-
insensitive BK channel constructed from two Slo family members, Slol and Slo3
(Schreiber
et al, J. Biol. Chem. 273:3509-3516 (1998)). Although these regions are
adjacent, it is
possible that they contribute independently to calcium sensitivity. This
independence is
supported by the fact that one of the regions that we describe, the Calcium
Bowl (Schreiber
et al., Biophys. J. 73:1355-1363 (1997)) has been modified in another family
member,
mSlo2, to be a site of chloride regulation, while the channel retains
regulation by calcium at
a second site.
A. Methods
Wild-type channel constructs. mSlol constructs were based on the mbr5
cDNA construct described previously Butler et al., Science 261:221-224
(1993)); the mSlol
and tail were as described in Wei et al., Neuron 13:671-681 (1994). Briefly,
the mSlol core
begins at the initiator methionine and terminates after S8 in the unconserved
linker region.
The mSlol tail construct begins at a native internal methionine before the S9
hydrophobic
domain. The mSlo3 tail construct is derived form the mSlo3 cDNA (GenBank
accession
number AF039213; Schreiber. et al., J. Biol. Chem. 273:3509-3516 (1998)).
Chimeric mSlol-mSlo3 tail constructs: mSlo3 and chimeric tail constructs
were cloned into pOocyte-Xpress, a Bluescript-derived plasmid (Stratagene)
containing
Xenopus ~3-globin S' and 3' untranslated sequences (Melton et al., Nucl. Acids
Res.
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/2232i
64
12:7035-7056 (1984)). Chimeric constructs were generated by standard overlap
PCR
techniques (Horton et al., Gene 77:61-68 (1989)). Oligonucleotides were
synthesized at the
Washington University Protein and Nucleic Acid Laboratory. The mSlo3 tail
construct
consisted of the C-terminal region of mSlo3 corresponding to that included in
the mSlol
tail, starting with methionine number 687 in mSlo3 (so that N-terminal
residues were
MLDS). The initiator methionine was placed into a Kozak consensus sequence
(Kozak,
Mol. Biol. 196:947-950 (1987)). To generate chimeras, the tail domain was
divided into
segments A through D. First and last residues of each segment are as follows:
Region A:
replaces mSlo3 792 IAVN ... LTEL 870 with mSlol 793 RAVN ... ITEL 885. This
region
begins after S9 and runs to a point just N-terminal to the Calcium Bowl.
Region B: replaces
mSlo3 871 KNPS ... GAVF 906 with mSlol 886 VNDT ... GTAF 918, except that the
C-
terminal end of this fragment is a hybrid of the mSlol and mSlo3 sequences
reading GAAF.
Chimera B tail includes the entire Calcium Bowl region. Region C: replaces
mSlo3 899
STSF ... SEME 941 with mSlo 1 909 TQPF ... PELE 963. This region includes S 10
and 20
residues following S10. Region D: replace mSlo3 939 EMEH ... HLLP 1034 with
mSlol
951 ELEA ... ELVP 1048. Region D is a large segment near the C-terminal of the
protein.
Larger pieces were also generated and denoted by their composition, e.g. BC
was a chimeric
beginning at the N-terminal end of B through the C-terminal end of C.
Reciprocal
experiments, the coexpression of mSlol tail with mSlo3 core, failed to produce
functional
channels.
Xenopus oocyte expression: mSlo3 and chimeric tail constructs were
linearized at a unique Notl site, and capped cRNA was synthesized using the T3
mMessage
mMachine kit (Ambion). Reactions were precipitated with LiCI and resuspended
in
nuclease-free distilled water at a final concentration of approximately 1.0
mg/ml. Oocytes
were prepared for injection as previously described (Wei et al., Science
248:599-603
(1990)) except for the use of a Drummond nanojector. Approximately 50 nl was
injected
into each oocyte. Oocytes were incubated in ND96 medium (Wei et al., Neuron
13:671-681
(1994)) and analyzed 1-8 days after injection.
Electrophysiology: Before patch recording, vitelline membranes were
removed from oocytes in hypertonic stripping solution (200 mM potassium
aspartate, 20
mM KCI, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES). Inside-out patch recordings were
made using buffered calcium perfusion solutions as described previously (Wei.
et al.,
Neuron 13:671-681 (1994)). Methanesulfonate-based perfusion and pipet
(extracellular)
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
solutions contained symmetric K+ (160mM), pH 7Ø mSlol core + mSlo3 channels
were
not sensitive to pH in terms of either changes in VSO or Gmax when tested with
solutions at
pH 7 or 8 containing 184 K gluconate, 10 KOH, 10 HEPES, and 200 ~M hemiCa
gluconate,
pH adjusted with HCI; the pH 7 solution was used in the pipet to provide
symmetric [K+].
5 From pH 7 to pH 8, ~V50 = 8.1 t 2.1 mV; n = 6 patches. The ratio of maximal
conductances for at pH 8 to that at pH 7 was 0.89 t .I2; n=6 patches. Similar
results (no
effect of pH) were obtained with pH 7 and pH 8 solutions containing nominally
zero
calcium (10 mM EGTA). Macroscopic currents were measured with an Axopatch 1B
amplifier (Axon Instruments), digitized at 10 kHz, and filtered at 5 kHz.
Analysis was
10 carried out using pClamp (Axon Instruments ). For characterization, each
patch was
subjected to a family of voltage clamp step pulses in 10 mV increments.
Conductance-
voltage relations were plotted and fitted with Boltzmann functions.
Conductance was then
normalized to the maximal conductance of the Boltzmann fit.
1 S B. Results
Considering the role of calcium in Slo 1 channel activation, it was surprising
to find a closely related channel, SIo3, which was lacking in calcium
sensitivity (Schreiber
et al., J. Biol. Chem. 273:3509-3516 (1998)). Like mSlol, mSlo3 is a large-
conductance,
voltage-gated potassium channel with corresponding core and tail domains.
However,
20 despite their overall homology, mSlo3 is insensitive to calcium. This lack
of calcium
sensitivity has allowed production of a chimeric channel with properties
similar to the Slol
core, but lacking calcium sensitivity. This was accomplished by fusing the
mSlo3 tail with
the mSlol core. The Slol core construct included the N-terminal two-thirds of
mouse Slol,
ending downstream of the S8 hydrophobic segment, in the "linker" region; the
Slo3 tail
25 began at a native internal methionine upstream from S9. Remarkably, the
Slo3 tail + Slol
core hybrid channels produced robust expression. The ability of the Slo3 tail
to form
functional channels with the Slol core indicates that the Slo3 tail can supply
a function
necessary for channel assembly, similar to that of a chaperone, in lieu of the
Slol tail. This
function is likely to be encoded by regions that are conserved between the
mSlol an mSlo3
30 tails.
Hybrid channels consisting of Slo3 tail and Siol core exhibit voltage
sensitivity (as reflected in the slope of the Boltzmann fit to the G-V curve)
characteristic of
the Slol core. This is to be expected if the core determines voltage
sensitivity. Most
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
66
importantly, the chimeric channels were calcium insensitive, which is a
characteristic of
Slo3 channels. Thus, changing [Ca2+J did not alter the voltage range of
activation or the
activation kinetics of the chirneric channels, two gating parameters strongly
altered by Ca2+
in wild-type Slol channels (DiChiara et al., J. Physiol. 489:403-418 (1995);
Cui et al., J.
Gen. Physiol. 109:647-673 (1997); Cox et al., J. Gen. Physiol. 109:633-646
(1997)). These
experiments are further evidence for the importance of the Slol tail in
calcium sensing. This
chimeric calcium-insensitive BK channel has been used to identify specific
regions of the
Slol tail involved in calcium sensing.
Chimera ABCD tail produces wild-type Slol calcium sensitivity: To map the
regions involved in calcium sensitivity, the Slol tail was divided into four
segments, A
through D. To verify that all calcium sensing regions were included, regions
A, B, C, and
D, when transplanted as a unit (chimera ABCD) were shown to completely restore
wild-
type calcium sensing to the mSlo3 tail. This indicates that the structures
essential to Slol
calcium sensing are contained within ABCD, a region comprising approximately
56% of the
total length of the tail domain. These four regions are contiguous or slightly
overlapping
and encompass 269 amino acid residues out of a total of 480 Slo tail residues.
The
hydrophobic region S9 is not included in ABCD. In addition, this result
suggests that the
remaining N- and C-terminal-most regions of the Slol tail that were not
transplanted,
including the S9 hydrophobic segment and the C-terminal 120 amino acids, are
unlikely to
make any direct contribution to calcium sensing. However, the regions outside
of ABCD
may be involved in other roles of the tail that are conserved between Slol and
Slo3. As
mentioned above, one possible function of these regions might be to promote
proper
channel assembly or targeting to the plasma membrane. Each region (A, B. C, or
D) was
then tested for its ability to restore calcium sensitivity by adding it back
to the SIo3 tail, and
testing its calcium sensitivity in SIo3 tail chimera +Slol core channels.
The Calcium Bowl (Region B) restores a portion of wild-type calcium
sensitivity: In a previous paper a series of mutations in the Calcium Bowl
region of the tail
was created to show its involvement in the calcium-sensing process (Schreiber
et al.,
Biophys. J. 73:1355-1363 (1997)). Further evidence showing the involvement of
this region
in Ca2+ sensing would be provided by adding this region back to the Ca2+ -
insensitive
channel and restoring Ca2+ sensitivity. Thus, this small region was added
(Region B) into
the calcium-insensitive Slo3 tail; and then assayed for the calcium-
sensitivity of the
channels formed from this SIo3 modified tail coexpressed with Slol core
(chimera B tail +
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
67
Slol core). The results of this experiment show that the Calcium Bowl does, in
fact, restore
significant calcium sensitivity to the channel. In this experiment, 34
residues encompassing
the Calcium Bowl from mSlol replaced 37 residues of the mSlo3 tail. Within
this region,
12 amino acids were identical; of the 22 additional Slo 1 residues added back,
8 represented
conservative changes. Overall similarity between the sequences included
conservative
changes was 59%. The 12 amino acids in conserved positions allowed an accurate
alignment through this region. Of the 22 amino acids that differ between the
two constructs,
seven are negatively charged amino acids present in Slol but absent in Slo3;
negatively
charged amino acids are favored to coordinate calcium (Marsden, Biochem. Cell.
Biol.
68:587-601 (1990)).
The qualitative effect on Ca2+ on channels formed from chimera B tail + Slol
core was similar to that observed for wild-type mSlol (or mSlol tail + mSlol
core)
channels; as [Ca2+] was raised, the G-V curve shifted to more negative
voltages. Thus,
higher [Ca2+] permits the channel to activate at more negative voltages. As
with wild-type
Slo 1, [Ca2+] had little or no effect on the voltage sensitivity of the
current, as reflected in the
slope of the G-V curve. Although the response to Ca2+ was significant in
channels formed
from chimera B tail + Slol core, the magnitude of the Ca2+ -induced voltage
shifts were not
equal to that of wild-type channels; The OV50 from 4 to 300 ~.M Caz+ was
approximately -
58 p,V for chimera B tail + Slol core, compared with -84 mV for wild-type Slol
tail + Slol
core. The fact that the magnitude of change in the position of the G-V curve,
OVSO/0[Ca2+],
is less than that with the complete Slol tail could be due to several factors,
but it is also
consistent with previous observations that there is likely to be more than one
calcium
sensing site per subunit in mSlol (Schreiber et al., Biophys. J. 73:1355-1363
(1997)). One
possibility is that the Calcium Bowl is an autonomous region that is
sufficient to confer Ca2+
sensing, and an additional region also contributes to the calcium-sensing
function.
A region downstream from the Calcium Bowl also contributes to calcium
sensing: The region downstream from the Calcium Bowl, which includes the S10
hydrophobic segment (region C), also produced calcium-sensitive channels when
incorporated into the Slo3 tail and coexpressed with the Slol core (chimera C
tail + Slol
core channels. However, increasing Ca2+ had a proportionally smaller effect on
chimera C
tail + Slol core channels than on chimera B tail + Slol core channels, as
reflected in the
slope of the V50 versus [Ca2+] relation. As with chimera B tail + Slol core
channels, the
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
68
magnitude of the ~VSO/t1[Ca2+] was less than that for mSlol tail, suggesting
neither region
B nor C can separately account for Slol calcium sensitivity.
B and C make additive contributions to calcium sensitivity: To assay the
effect of combining regions B and C, the chimeric tail construct BC was
created. Regions B
and C together produce channels with higher calcium than either B or C alone.
This shows
simple additivity. Over the range of calcium from 0 to 300 p,M the difference
between the
VSO for the control mSlo3 tail alone and each chimera was -77 mV for chimera B
tail and -
27 mV for chimera C tail. For chimera BC tail, the difference was -100 mV.
This is
remarkably close to the -104 mV obtained by adding the values for chimera B
and C tails.
Thus, these regions appear to make independent contributions to the channel's
calcium
sensitivity.
Other regions of the tail do not confer calcium sensitivity: In addition to
testing regions B and C, the remaining segments of the Slol tail were tested.
Neither region
A nor D produced calcium sensitive channels when incorporated in to the Slo3
tail. Thus,
1 S only two limited regions of the tail, B and C, confer calcium sensing
properties. Both
chimera A tail and D tail shift the starting position of the G-V curve along
the voltage axis,
but calcium ion has very little effect on the magnitude of the shift. this
indicates that in
addition to confernng calcium sensitivity, distinct regions of the tail play a
role in
determining the voltage range of activation of the channel, perhaps by
transducing an
allosteric interaction of tail and core.
Chimera A, a 93 as region of mSlol tail between S9 and S10 N-terminal to
the Calcium Bowl, was also considerably larger than either of the regions that
conferred
calcium sensitivity. Channels with this tail construct showed no significant
response to
calcium. However, the voltage range of activation of channels with the chimera
tail regions,
2S with a VSO in zero calcium 40 mV more positive than that obtained with the
Slo3 tail. In
addition chimera A tail showed a slightly reduced G-V slope, quite similar to
the decrease
in slope seen in 0 p,M calcium with wild-type mSlol tail (Stefani et al.,
Proc. Natl. Acad.Sci
(USA) 94:5427-5431 (1997); Cui et al., J.Gen.Physiol. 109:647-673 (1997)). A
similar
effect of region A in shifting the voltage operating range can be seen in
comparing channels
formed with chimera ABCD tail versus chimera BCD tail: the VSO of chimera ABCD
tail +
Slol core is shifted approximately +60 to +80 mV versus chimera BCD tail at
all [Ca2+].
Viewed simply as a voltage-dependent channel in the absence of calcium ion,
these results
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PC'T/US98/22321
69
suggest region A could be an inhibitory domain responsible for setting the
voltage range of
activation to very positive values in Wild-type Slol channels.
Chimera D, a 98 residue region C-terminal to S 10 was the largest single
domain transplanted. Like chimera A, channels formed with chimera D tail +
Slol core
also showed no sensitivity to calcium ion. However, the voltage range of
activation was
repositioned approximately -40 mV relative to that of the Slo3 tail - Slol
core channels.
Although this shi8 is in the opposite direction of that conferred by chimera A
tail, the net
effect of having both regions, A and B, is apparently inhibitory, because
including both
regions (in chimera ABCD tail) produced channels with a very positively
shifted
current/voltage relation. It is inferred than both region A and B may include,
or
allosterically influence, the transduction interface between core and tail. In
contrast, regions
B and C may influence the transduction interface only in a calcium-dependent
allosteric
manner.
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
SEQUENCE LISTING
mSlo3 amino acid sequence (SEQ ID NO:1):
MSQTLLDSLNQKELTETSCTIEIQAAFILSSLATFFGGLIILFLFRIALKSSRSWKYVKGPRGLLELFSSRRIE
S ANPLRKIrYFHGVFRQRIEMLLSAQTWGQVLVILVFVLSIGSLVIYFINSMDPVRRCSSYEDKIVHGDLSFNAF
FSFYFGLRFWAAEDKIKFWLEMNSIVDIFTIPPTFISYYLKSNWLGLRFLRALRLLELPKILQILQVIKTSNSV
KLSKLLSIVISTWFTAAGFLHLVENSGDPWLNGRNSQTMSYFESIYLVTATMSTVGFGDWAKTSLGRIFIVFF
TLGSLILFANYIPEMVELFSTRKKYTKPYEAVKGKKFIWCGNITVDSVTAFLRNFLHWKSGEINIEIVFLGET
LPCLELETLLKCHTSCTNFVCGTALKFEDLKRVAVENSEACLILANHFCSDLHDEDNSNIMRVLSIKNYYPQTR
IO VIIQILQSQNKVFLSKIPNWDWSAGDNILCFAELKLGFIAQGCLVPGLCTFLTTLFIEQNQKVFPKHPWQKHFL
NGLKNKILTQRLSNDFVGMTFPQVSRLCFVKLNLMLIAIQHKPFFHSCCTLILNPSSQVRLNKDTLGFFIADSS
KAVKRAFFYCSNCHSDVCNPELIGKCNCKIKSRQQLIAPTIMVMKSSLTDFTTSSHIHASMSTEIHTCFSREQP
SLITITTNRPTTNDTVDDTDMLDSSGMFHWCRAMPLDKWLKRSEKAKHEFQNHIWCVFGDAQCTLVGLRNFV
MPLRASNYTRQELKDIVFIGSLEYFQREWRFLRNFPKIHIMPGSALYMGDLIAVNVEQCSMCVILATPYKALSS
IS QILVDTEAIMATLNIQSLRITSPTPGSSKSEVKPSSAFDSKERKQRYKQIPILTELKNPSNIHFIEQMGGLDGM
LKGTSLHLSTSFSTGAVFSDTFLDSLLATSFYNYHWELLQMLVTGGISSEMEHYLVKEKPYKTTDDYEAIKSG
RTRCKLGLLSLDQTVLSGINPRKTFGQLFCGSLDNFGILCVGLYRMIDEEEPSQEHKRFVITRPSNECHLLPSD
LVFCAIPFNTTCGKSDSSPFNFRLKTTLQTRRRHWPRGRISSIRTMPTSPTIFTQSTTRERGGLSTTTPESILW
TR
mSlo3 nucleotide sequence (SEQ ID N0:2):
ATGTCTCAAACATTGCTAGACAGTTTAAATCAGAAGGAGTTGACGGAAACGTCATGTACAATCGAAATCCAGGC
AGCGTTCATTCTTTCCTCCTTGGCGACTTTCTTCGGGGGACTCATCATCTTATTCCTTTTCAGAATAGCCTTGA
AAAGCTCAAGAAGTTGGAAATACGTCAAGGGGCCAAGAGGACTCTTGGAACTATTCTCATCACGTAGAATCGAG
2S GCTAATCCTTTGAGGAAACTTTACTTTCATGGAGTATTTCGTCAGCGCATCGAAATGCTGCTTTCTGCACAGAC
CGTCGTGGGGCAAGTGTTGGTGATCCTTGTCTTTGTACTAAGCATCGGGTCTCTTGTGATCTATTTCATCAATT
CAATGGATCCTGTTCGAAGGTGTTCTTCATATGAAGACAAAATTGTCCATGGGGATTTGAGTTTCAACGCTTTC
TTTAGCTTCTATTTTGGGTTGAGGTTTTGGGCAGCTGAAGACAAGATCAAGTTCTGGTTGGAGATGAATTCAAT
TGTAGACATTTTTACCATCCCGCCAACCTTTATTTCTTATTATTTGAAGAGTAATTGGCTAGGTTTGAGATTTC
3O TAAGAGCTCTGCGGTTGCTCGAACTCCCTAAAATCTTACAGATCCTACAAGTCATCAAGACCAGCAATTCAGTG
AAGCTTTCCAAACTGTTGTCAATAGTTATCAGTACCTGGTTCACGGCAGCAGGATTCCTTCACCTGGTGGAAAA
TTCTGGTGACCCCTGGCTCAACGGAAGAAACTCACAGACTATGTCATACTTTGAGTCTATTTATCTGGTGACAG
CAACAATGTCAACTGTTGGCTTTGGGGACGTGGTGGCCAAGACATCCCTAGGACGGATTTTCATTGTTTTCTTC
ACCCTTGGGAGTTTGATACTATTTGCAAACTACATTCCAGAAATGGTGGAGCTCTTTTCTACCAGGAAGAAATA
3S CACCAAGCCCTACGAAGCAGTCAAAGGAAAAAAGTTCATCGTGGTCTGTGGAAACATCACAGTTGACAGTGTTA
CTGCTTTCCTGAGGAATTTTCTCCACTGGAAGTCCGGGGAAATCAATATTGAGATCGTATTCCTTGGAGAGACT
CTCCCTTGCTTGGAACTGGAGACCTTACTGAAGTGCCACACATCCTGTACCAACTTCGTATGCGGCACCGCACT
GAAGTTCGAGGATCTGAAGCGAGTTGCAGTGGAGAACTCGGAGGCGTGCCTGATTCTAGCCAACCATTTCTGTA
GTGACTTACATGACGAAGACAACTCAAACATTATGAGGGTGCTCTCGATCAAGAACTATTATCCACAGACCAGA
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
'a~ 'll
GTCATCATTCAGATACTTCAGTCTCAAAACAAGGTTTTCCTGTCAAAAATCCCCAACTGGGACTGGAGTGCTGG
AGACAATATCCTCTGCTTTGCAGAGCTAAAGCTCGGATTTATCGCCCAAGGCTGCTTGGTGCCAGGGCTGTGCA
CCTTTCTCACGACTCTGTTCATTGAACAAAACCAAAAGGTTTTTCCTAAACATCCCTGGCAAAAACATTTCTTG
AATGGCTTGAAGAACAAGATTCTGACACAGCGCCTCTCTAACGACTTCGTGGGGATGACATTTCCCCAGGTCTC
S CCGGCTCTGCTTTGTGAAGCTAAATCTCATGCTGATCGCCATCCAACACAAGCCCTTCTTTCACAGTTGTTGCA
CTCTGATACTAAACCCATCATCCCAAGTGAGGCTGAATAAGGACACCTTAGGGTTCTTCATTGCGGACTCCTCC
AAAGCCGTCAAAAGGGCTTTCTTTTACTGTTCCAACTGTCACAGCGATGTGTGCAATCCTGAGCTAATTGGAAA
GTGTAACTGTAAAATCAAGAGCCGACAACAACTCATAGCACCGACCATCATGGTGATGAAAAGCAGCTTGACCG
ATTTCACCACTTCTTCACACATCCACGCTTCTATGTCAACAGAAATTCACACTTGTTTTTCAAGAGAACAGCCT
IO AGTTTGATCACCATTACAACCAACAGACCAACGACAAACGACACAGTGGATGATACCGACATGCTGGACAGCAG
TGGCATGTTTCACTGGTGCAGAGCAATGCCCTTGGACAAGGTGGTTCTGAAACGAAGTGAGAAGGCAAAACACG
AGTTTCAGAACCACATTGTAGTATGCGTGTTTGGAGATGCCCAATGTACCCTGGTGGGGCTTCGGAATTTCGTG
ATGCCCCTGAGAGCCAGCAACTACACCCGGCAGGAGCTGAAGGACATTGTTTTTATTGGGTCTCTGGAGTACTT
CCAGAGAGAATGGCGATTTCTCCGAAACTTTCCCAAGATACACATTATGCCTGGATCTGCACTCTACATGGGAG
IS ATCTGATTGCAGTCAATGTAGAGCAGTGCTCTATGTGCGTCATCTTAGCCACACCCTACAAGGCACTGAGCAGC
CAGATTCTGGTGGACACAGAGGCCATCATGGCCACCCTCAACATCCAGTCCCTGCGGATCACCAGTCCTACTCC
AGGGTCTTCAAAGTCAGAAGTAAAGCCATCATCTGCCTTTGATAGTAAAGAAAGGAAGCAAAGATACAAACAGA
TCCCCATTCTCACTGAACTGAAGAATCCCTCCAACATCCACTTTATTGAGCAGATGGGCGGACTGGATGGAATG
CTCAAAGGGACTAGCTTGCATCTCAGCACTTCTTTCTCCACCGGTGCTGTCTTTTCAGACACCTTCTTGGATTC
ZO TCTCCTGGCCACGTCCTTCTACAATTACCATGTCGTGGAATTACTTCAGATGCTAGTGACTGGAGGCATAAGCT
CTGAGATGGAACACTATTTGGTTAAGGAGAAGCCCTATAAGACAACTGACGACTATGAGGCAATCAAGTCTGGG
AGGACGCGGTGTAAGCTGGGACTCCTCTCTTTAGACCAAACCGTTCTATCAGGCATTAATCCAAGAAAAACCTT
TGGACAGCTGTTCTGTGGCTCATTGGATAATTTCGGGATCCTATGTGTCGGCTTATACCGTATGATTGATGAAG
AGGAACCCAGCCAAGAACACAAAAGGTTTGTGATCACCAGGCCATCCAATGAGTGCCACCTGCTGCCCTCAGAT
ZS CTCGTGTTTTGTGCCATCCCTTTCAACACCACCTGTGGCAAATCAGACAGCAGTCCTTTCAATTTCAGGCTCAA
AACAACTCTACAAACGCGACGACGCCATTGGCCCAGGGGTCGAATTTCTTCGATTCGCACCATGCCGACGAGTC
CCACGATCTTTACCCAGTCGACGACACGGGAGAGAGGTGGTCTCAGCACCACCACTCCCGAGTCTATCCTTTGG
ACACGTTAG
30 hSlo3 amino acid sequence (SEQ ID N0:3):
GLAALILSSFVTLFSGLISLLIFRLIWRXVKKWQIIKGTGIILELFTSGTIARSHVRSLHFQGQFRDHIEMLLS
AQTFVGQVLVILVFVLSIGSLIIYFINSADPVGTLFII
hSIo3 nucleotide sequence (SEQ ID N0:4):
3S GGCTTGGCAGCGCTCATTCTTTCCTCCTTTGTGACCCTCTTCAGTGGACTCATCAGCCTGTTGATCTTCAGGCT
GATCTGGAGAYCTGTTAAAAAATGGCAAATCATCAAGGGAACAGGAATTATCTTGGAACTGTTCACATCAGGTA
CCATCGCTAGGAGCCATGTAAGAAGCCTCCACTTCCAGGGACAATTTCGTGATCATATAGAAATGTTGCTTTCA
GCCCAGACCTTTGTGGGGCAAGTGTTGGTGATCCTTGTCTTTGTACTAAGCATTGGGTCTCTTATAATCTATTT
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCTNS98/22321
CATCAATTCWGCTGACCCTGTTGGAACGCTGTTCATCATATGAAGACAAAACCATTCCTATTGATTTGGTTTTC
AATGCTTTCTTTAGTTTCTATTTTGGGTTGAGGTTTTGGCAAAGCC
hSlo3-a amino acid sequence (SEQ ID N0:5)
GLAAFILSSFVTLFSGLISLLIFRLIWRXVKKWQIIKGTGIILELFTSGTIARSHVRSLHFQGQFRDHIEMLLS
AQTFVGQVLVILVFVLSIGSLIIYFINSADPVGTLFII
hSlo3-b amino acid sequence (SEQ ID N0:6)
GLAALILSSFVTLFTGLISLLIFRLIWRXVKKWQIIKGTGIILELFTSGTIARSHVRSLHFQGQFRDHIEMLLS
lO AQTFVGQVLVILVFVLSIGSLIIYFINSADPVGTLFII
hSlo3-c amino acid sequence (SEQ ID N0:7)
GLAALILSSFVTLFSGLISLLIFRLIWRXVKKWQIIKGTGIILELFTSGTIARSHVRSLHFQGQFRDHIEMLLS
AQTFVGQVLVILVFVLSIGSLIIYFINSMDPVGTLFII
1S
hSlo3-1 amino acid sequence (SEQ ID N0:16)
MFQTKLRNETWEDLPKMSCTTEIQAAFILSSFVTFFSGLIILLIFRLIWRSVKKWQIIKGTGIILELFTSGTIA
RSHVRSLHFQGQFRDHIEMLLSAQTFVGQVLVILVFVLSIGSLIIYFINSADPVGSCSSYEDKTIPIDLVFNAF
FSFYFGLRFMAADDKIKFWLEMNSIVDIFTIPPTFISYYLKSNWLGLRFLRALRLLELPQILQILRAIKTSNSV
ZO KFSKLLSIILSTWFTAAGFIHLVENSGDPWLKGRNSQNISYFESIYLVMATTSTVGFGDWAKTSLGRTFIMFF
TLGSLILFANYIPEMVELFANKRKYTSSYEALKGKKFIWCGNITVDSVTAFLRNFLRDKSGEINTEIVFLGET
PPSLELETIFKCYLAYTTFISGSAMKWEDLRRVAVESAEACLIIANPLCSDSHAEDISNIMRVLSIKNYDSTTR
IIIQILQSHNKVYLPKIPSWNWDTGDNIICFAELKLGFIAQGCLVPGLCTFLTSLFVEQNKKVMPKQTWKKHFL
NSMKNKILTQRLSDDFAGMSFPEVARLCFLKMYLLLIAIEYKSLFTDGFCGLILNPPPQVRIRKNTLGFFIAET
ZS PKDVRRALFYCSVCHDDVFIPELITNCGCKSRSRQHITVPSVKRMKKCLKGISSRISGQDSPPRVSASTSSISN
FTTRTLQHDVEQDSDQLDSSGMFHWCKPTSLDKVTLKRTGKSKYKFRNHIVACVFGDAHSAPMGLRNFVMPLRA
SNYTRKELKDIVFIGSLDYLQREWRFLRNFPQIYILPGCALYSGDLHAANIEQCSMCAVLSPPPQPSSNQTLVD
TEAIMATLTIGSLQIDSSSDPSPSVSEETPGYTNGHNEKSNCRKVPILTELKNPSNIHFIEQLGGLEGSLQETN
LHLSTAFSTGTVFSSSFLDSLLATAFYNYHVLELLQMLVTGGVSSQLEQHLDKDKVYGVADSCTSLLSGRNRCK
3O LGLLSLHETILSDVNPRNTFGQLFCGSLDLFGILCVGLYRIIDEEELNPENKRFVITRPANEFKLLPSDLVFCA
IPFSTACYKRNEEFSLQKSYEIVNKASQTTEDTFRHKLSSHPLIQLLRHCIHQSILTSRELTPSLFLSK
hSlo3-1 nucleotide sequence (SEQ ID N0:17)
ATGTTTCAGACTAAGCTACGAAATGAAACTTGGGAAGACTTGCCAAAAATGTCCTGCACAACTGAGATCCAAGC
3S AGCATTCATTCTCTCTTCCTTTGTGACCTTCTTCAGTGGACTCATCATCCTGTTGATCTTCAGGCTGATCTGGA
GATCTGTTAAAAAATGGCAAATCATCAAGGGAACAGGAATTATCTTGGAACTGTTCACATCAGGTACCATCGCT
AGGAGCCATGTAAGAAGCCTCCACTTCCAGGGACAATTTCGTGATCATATAGAAATGTTGCTTTCAGCCCAGAC
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
ss ~l3
CTTTGTGGGGCAAGTGTTGGTGATCCTTGTCTTTGTACTAAGCATTGGGTCTCTTATAATCTATTTCATCAATT
CTGCTGACCCTGTTGGAAGCTGTTCATCATATGAAGACAAAACCATTCCTATTGATTTGGTTTTCAATGCTTTC
TTTAGTTTCTATTTTGGATTGAGGTTTATGGCAGCTGATGACAAGATCAAGTTCTGGCTGGAGATGAATTCAAT
CGTAGACATCTTTACCATCCCACCAACCTTTATTTCTTATTATTTGAAGAGCAATTGGCTAGGTTTAAGGTTCC
S TAAGAGCCTTGCGCCTGCTAGAACTCCCTCAAATCTTGCAAATTCTACGAGCCATCAAGACCAGTAACTCAGTG
AAGTTTTCCAAACTGCTGTCAATAATTCTCAGTACCTGGTTCACAGCTGCGGGATTCATTCACCTGGTGGAAAA
TTCTGGTGATCCCTGGCTCAAAGGTAGAAATTCACAGAATATATCATATTTTGAGTCAATTTACCTGGTCATGG
CAACAACGTCAACCGTTGGATTTGGAGATGTGGTAGCCAAGACATCCTTAGGACGGACCTTCATCATGTTCTTC
ACACTGGGGAGTTTGATATTATTTGCGAACTATATACCTGAAATGGTGGAACTGTTTGCTAACAAGAGGAAATA
IO CACCAGTTCMTATGAAGCACTCAAAGGAAAGAAGTTTATTGTGGTCTGTGGAAACATCACTGTGGACAGTGTGA
CCGCTTTCCTGAGGAATTTCCTCCGCGACAAGTCAGGAGAGATCAACACTGAAATTGTTTTCCTGGGAGAAACC
CCTCCTTCTTTGGAACTTGAAACCATATTTAAATGCTACTTGGCCTACACAACGTTCATTTCTGGATCTGCAAT
GAAGTGGGAGGATCTGAGGCGAGTTGCGGTGGAATCTGCAGAGGCATGCCTGATTATAGCCAATCCTTTGTGCA
GTGATTCCCATGCTGAAGATATTTCCAACATTATGAGGGTGCTCTCTATCAAGAACTATGATTCTACCACCAGA
IS ATCATCATACAGATACTGCAATCCCATAACAAGGTTTATCTGCCAAAGATTCCCAGCTGGAACTGGGACACCGG
AGACAACATCATCTGCTTTGCTGAATTAAAACTTGGATTTATCGCCCAAGGCTGTTTGGTGCCAGGCTTGTGTA
CCTTCCTAACATCTCTATTTGTGGAGCAAAACAAAAAGGTTATGCCTAAACAGACCTGGAAGAAACACTTCTTG
AATAGCATGAAAAACAAAATTCTGACCCAACGTCTCTCTGATGACTTTGCTGGAATGAGCTTTCCTGAAGTTGC
CCGGCTCTGCTTTCTGAAGATGTACCTCCTGTTGATAGCCATCGAATACAAGTCCCTCTTTACGGATGGTTTCT
ZO GTGGTCTGATACTAAATCCACCTCCACAAGTGAGGATACGTAAGAACACATTAGGGTTCTTTATTGCTGAAACT
CCAAAGGACGTCAGAAGAGCCTTGTTTTACTGTTCAGTCTGTCATGATGATGTGTTCATTCCTGAGCTAATTAC
AAACTGTGGCTGCAAAAGCAGAAGCCGGCAGCACATCACAGTGCCATCGGTAAAGAGAATGAAAAAATGTCTGA
AGGGAATCTCCTCTCGTATATCAGGGCAGGATTCTCCGCCAAGGGTATCTGCAAGCACTTCGAGCATATCAAAC
TTCACCACCAGGACTCTTCAACATGATGTAGAACAAGATTCTGACCAGCTTGATAGCAGTGGGATGTTTCACTG
ZS GTGCAAACCAACCTCTTTGGACAAGGTGACTCTGAAACGAACTGGCAAGTCAAAGTATAAGTTTCGGAACCATA
TTGTAGCATGTGTATTTGGAGATGCCCACTCAGCCCCGATGGGGCTTCGGAACTTTGTAATGCCCTTGAGAGCC
AGCAACTATACCAGGAAGGAGCTGAAGGACATAGTGTTCATTGGGTCTCTGGACTATCTACAGAGAGAATGGCG
ATTTCTCCGGAATTTTCCCCAGATATACATTCTGCCTGGATGTGCACTTTATTCTGGAGACCTCCATGCGGCCA
ACATAGAGCAATGCTCCATGTGTGCTGTCTTGTCCCCCCCACCCCAGCCATCAAGCAACCAGACTTTGGTAGAC
3O ACAGAAGCCATCATGGCAACCCTCACCATCGGATCCTTGCAAATTGACTCCTCCTCTGACCCGTCACCCTCAGT
GTCAGAGGAGACTCCAGGTTACACAAATGGACATAATGAGAAATCAAACTGCCGAAAAGTCCCTATCCTTACTG
AACTGAAAAATCCTTCCAACATTCACTTTATTGAACAGCTTGGTGGACTGGAAGGGTCCCTCCAAGAAACAAAT
CTGCATCTCAGCACTGCCTTTTCTACGGGCACTGTTTTTTCCAGCAGCTTCTTGGATTCTCTGCTGGCCACGGC
CTTCTACAATTATCATGTCCTGGAATTGCTTCAGATGCTGGTGACAGGAGGAGTAAGTTCTCAGCTGGAACAAC
3S ATTTAGATAAGGATAAAGTCTATGGTGTGGCAGATAGCTGCACGTCGCTCTTGTCTGGAAGAAACCGGTGTAAG
CTGGGGCTTCTGTCCTTACACGAAACCATTTTATCAGACGTTAATCCAAGAAACACCTTTGGACAACTGTTCTG
TGGCTCATTAGATCTTTTTGGAATCCTGTGTGTTGGCTTATACCGAATAATTGATGAAGAGGAGCTCAACCCAG
AAAACAAAAGGTTTGTGATCACCCGGCCAGCCAATGAGTTCAAGCTGCTGCCTTCAGATCTTGTGTTTTGTGCC
ATACCCTTCAGCACTGCTTGTTATAAAAGGAATGAAGAGTTCTCATTGCAAAAGTCATATGAAATTGTAAATAA
SUBSTITUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
~ ~j3
AGCATCACAGACAACAGAGGACACATTCAGACACAAATTGTCCTCCCACCCATTGATTCAGTTACTGAGACATT
GTATTCACCAGTCTATTCTTACCAGCCGAGAACTAACTCCCTCTCTTTTCCTAAGCAAATAGG
hSlo3-2 amino acid sequence (SEQ ID N0:18)
S MFQTKLRNETWEDLPKMSCTTEIQAAFILSSFVTFFSGLIILLIFRLIWRSVKKWQIIKGTGIILELFTSGTIA
RSHVRSLHFQGQFRDHIEMLLSAQTFVGQVLVILVFVLSIGSLIIYFINSADPVGSCSSYEDKTIPIDLVFNAF
FSFYFGLRFMAADDKIKFWLEMNSIVDIFTIPPTFISYYLKSNWLGLRFLRALRLLELPQILQILRAIKTSNSV
KFSKLLSIILSTWFTAAGFIHLVENSGDPWLKGRNSQNISYFESIYLVMATTSTVGFGDWAKTSLGRTFIMFF
TLGSLILFANYIPEMVELFANKRKYTSSYEALKGKKFIWCGNITVDSVTAFLRNFLRDKSGEINTEIVFLGET
IO PPSLELETIFKCYLAYTTFISGSAMKWEDLRRVAVESAEACLIIANPLCSDSHAEDISNIMRVLSIKNYDSTTR
IIIQILQSHNKVYLPKIPSWNWDTGDNIICFAELKLGFIAQGCLVPGLCTFLTSLFVEQNKKVMPKQTWKKHFL
NSMKNKILTQRLSDDFAGMSFPEVARGLILNPPPQVRIRKNTLGFFIAETPKDVRRALFYCSVCHDDVFIPELI
TNCGCKSRSRQHITVPSVKRMKKCLKGISSRISGQDSPPRVSASTSSISNFTTRTLQHDVEQDSDQLDSSGMFH
WCKPTSLDKVTLKRTGKSKYKFRNHIVACVFGDAHSAPMGLRNFVMPLRASNYTRKELKDIVFIGSLDYLQREW
~S RFLRNFPQIYILPGCALYSGDLHAANIEQCSMCAVLSPPPQPSSNQTLVDTEAIMATLTIGSLQIDSSSDPSPS
VSEETPGYTNGHNEKSNCRKVPILTELKNPSNIHFIEQLGGLEGSLQETNLHLSTAFSTGTVFSSSFLDSLLAT
AFYNYHVLELLQMLVTGGVSSQLEQHLDKDKVYGVADSCTSLLSGRNRCKLGLLSLHETILSDVNPRNTFGQLF
CGSLDLFGILCVGLYRIIDEEELNPENKRFVITRPANEFKLLPSDLVFCAIPFSTACYKRNEEFSLQKSYEIVN
KASQTTEDTFRHKLSSHPLIQLLRHCIHQSILTSRELTPSLFLSK
hSlo-3-2 nucleotide sequence (SEQ ID N0:19)
ATGTTTCAGACTAAGCTACGAAATGAAACTTGGGAAGACTTGCCAAAAATGTCCTGCACAACTGAGATCCAAGC
AGCATTCATTCTCTCTTCCTTTGTGACCTTCTTCAGTGGACTCATCATCCTGTTGATCTTCAGGCTGATCTGGA
GATCTGTTAP.AAAATGGCAAATCATCAAGGGAACAGGAATTATCTTGGAACTGTTCACATCAGGTACCATCGCT
2S AGGAGCCATGTAAGAAGCCTCCACTTCCAGGGACAATTTCGTGATCATATAGAAATGTTGCTTTCAGCCCAGAC
CTTTGTGGGGCAAGTGTTGGTGATCCTTGTCTTTGTACTAAGCATTGGGTCTCTTATAATCTATTTCATCAATT
CTGCTGACCCTGTTGGAAGCTGTTCATCATATGAAGACAAAACCATTCCTATTGATTTGGTTTTCAATGCTTTC
TTTAGTTTCTATTTTGGATTGAGGTTTATGGCAGCTGATGACAAGATCAAGTTCTGGCTGGAGATGAATTCAAT
CGTAGACATCTTTACCATCCCACCAACCTTTATTTCTTATTATTTGAAGAGCAATTGGCTAGGTTTAAGGTTCC
3O TAAGAGCCTTGCGCCTGCTAgAACTCCCTCAAATCTTGCAAATTCTACGAGCCATCAAGACCAGTAACTCAGTG
AAGTTTTCCAAACTGCTGTCAATAATTCTCAGTACCTGGTTCACAGCTGCGGGATTCATTCACCTGGTGGAAAA
TTCTGGTGATCCCTGGCTCAAAGGTAGAAATTCACAGAATATATCATATTTTGAGTCAATTTACCTGGTCATGG
CAACAACGTCAACCGTTGGATTTGGAGATGTGGTAGCCAAGACATCCTTAGGACGGACCTTCATCATGTTCTTC
ACACTGGGGAGTTTGATATTATTTGCGAACTATATACCTGAAATGGTGGAACTGTTTGCTAACAAGAgGAAATA
3S CACCAGTTCMTATGAAGCACTCAAAGGAAAGAAGTTTATTGTGGTCTGTGGAAACATCACTGTGGACAGTGTGA
CCGCTTTCCTGAGGAATTTCCTCCGCGACAAGTCAGGAGAGATCAACACTGAAATTGTTTTCCTGGGAGAAACC
CCTCCTTCTTTGGAACTTGAAACCATATTTAAATGCTACTTGGCCTACACAACGTTCATTTCTGGATCTGCAAT
GAAGTGGGAGGATCTGAGGCGAGTTGCGGTGGAATCTGCAGAGGCATGCCTGATTATAGCCAATCCTTTGTGCA
GTGATTCCCATGCTGAAGATATTTCCAACATTATGAGGGTGCTCTCTATCAAGAACTATGATTCTACCACCAGA
SUBSTTTUTE SHEET (RULE 26)
CA 02307062 2000-04-20
WO 99/20754 PCT/US98/22321
ATCATCATACAGATACTGCAATCCCATAACAAGGTTTATCTGCCAAAGATTCCCAGCTGGAACTGGGACACCGG
AGACAACATCATCTGCTTTGCTGAATTAAAACTTGGATTTATCGCCCAAGGCTGTTTGGTGCCAGGCTTGTGTA
CCTTCCTAACATCTCTATTTGTGGAGCAAAACAAAAAGGTTATGCCTAAACAGACCTGGAAGAAACACTTCTTG
AATAGCATGAAAAACAAAATTCTGACCCAACGTCTCTCTGATGACTTTGCTGGAATGAGCTTTCCTGAAGTTGC
CCGTGGTCTGATACTAAATCCACCTCCACAAGTGAGGATACGTAAGAACACATTAGGGTTCTTTATTGCTGAAA
CTCCAAAGGACGTCAGAAGAGCCTTGTTTTACTGTTCAGTCTGTCATGATGATGTGTTCATTCCTGAGCTAATT
ACAAACTGTGGCTGCAAAAGCAGAAGCCGGCAGCACATCACAGTGCCATCGGTAAAGAGAATGAAAAAATGTCT
GAAGGGAATCTCCTCTCGTATATCAGGGCAGGATTCTCCGCCAAGGGTATCTGCAAGCACTTCGAGCATATCAA
ACTTCACCACCAGGACTCTTCAACATGATGTAGAACAAGATTCTGACCAGCTTGATAGCAGTGGGATGTTTCAC
IO TGGTGCAAACCAACCTCTTTGGACAAGGTGACTCTGAAACGAACTGGCAAGTCAAAGTATAAGTTTCGGAACCA
TATTGTAgCATGTGTATTTGGAGATGCCCACTCAGCCCCGATGGGGCTTCGGAACTTTGTAATGCCCTTGAGAG
CCAGCAACTATACCAGGAAGGAGCTGAAGGACATAGTGTTCATTGGGTCTCTGGACTATCTACAGAGAGAATGG
CGATTTCTCCGGAATTTTCCCCAGATATACATTCTGCCTGGATGTGCACTTTATTCTGGAGACCTCCATGCGGC
CAACATAGAGCAATGCTCCATGTGTGCTGTCTTGTCCCCCCCACCCCAGCCATCAAGCAACCAGACTTTGGTAG
IS ACACAGAAGCCATCATGGCAACCCTCACCATCGGATCCTTGCAAATTGACTCCTCCTCTGACCCGTCACCCTCA
GTGTCAGAGGAGACTCCAGGTTACACAAATGGACATAATGAGAAATCAAACTGCCGAAAAGTCCCTATCCTTAC
TGAACTGAAAAATCCTTCCAACATTCACTTTATTGAACAGCTTGGTGGACTGGAAGGGTCCCTCCAAGAAACAA
ATCTGCATCTCAGCACTGCCTTTTCTACGGGCACTGTTTTTTCCAGCAGCTTCTTGGATTCTCTGCTGGCCACG
GCCTTCTACAATTATCATGTCCTGGAATTGCTTCAGATGCTGGTGACAGGAGGAGTAAGTTCTCAGCTGGAACA
ZO ACATTTAGATAAGGATAAAGTCTATGGTGTGGCAGATAGCTGCACGTCGCTCTTGTCTGGAAGAAACCGGTGTA
AGCTGGGGCTTCTGTCCTTACACGAAACCATTTTATCAGACGTTAATCCAAGAAACACCTTTGGACAACTGTTC
TGTGGCTCATTAGATCTTTTTGGAATCCTGTGTGTTGGCTTATACCGAATAATTGATGAAGAGGAGCTCAACCC
AGAAAACAAAAGGTTTGTGATCACCCGGCCAGCCAATGAGTTCAAGCTGCTGCCTTCAGATCTTGTGTTTTGTG
CCATACCCTTCAGCACTGCTTGTTATAAAAGGAATGAAGAGTTCTCATTGCAAAAGTCATATGAAATTGTAAAT
ZS AAAGCATCACAGACAACAGAGGACACATTCAGACACAAATTGTCCTCCCACCCATTGATTCAGTTACTGAGACA
TTGTATTCACCAGTCTATTCTTACCAGCCGAGAACTAACTCCCTCTCTTTTCCTAAGCAAATAGT
SUBSTITUTE SHEET (RULE 26)
