Nucleic acid sequence encoding apamin receptor protein

The present invention relates to an isolated nucleic acid fragment comprising a nucleic acid sequence encoding an apamin receptor protein, or biologically active fragment thereof.

Potassium (K) channels are integral membrane proteins of great molecular 
and functional diversity, present in practically all mammalian cells. 
These channels are primarily responsible for maintaining a resting 
membrane potential and are rapidly activated in response to an external 
depolarizing stimulus, binding of certain ligands, or changes in the 
intracellular concentration of calcium or ATP. In the excitable cells such 
as neurons or cardiac myocytes, K-channels determine the duration of the 
action potential thus performing a vital function in the CNS and the 
cardiac functions (reviewed in 1-2) The calcium-activated K-channel 
sub-family consists of at least three discernible ionic currents; a large 
("BK"), an intermediate ("IK") and a small conductance ("SK") channels 
(Reviewed in 3-5). These K-channels are activated in response to a rise in 
the intracellular concentration of calcium [Ca.sup.2+ ]i. In addition to 
[Ca.sup.2+ ]i, the "BK" and "IK" channels are also sensitive to the 
changes in the membrane potential, whereas "SK" channel has no significant 
voltage sensitivity. 
Functionally, the SK-channel is involved in the afterhyperpolarization that 
follows action potentials in many neurons. These include the sympathetic 
ganglionic neurons, hippocampal neurons, neurosecretory neurons and spinal 
moroneurons, as well as the skeletal muscle cells (1, 5-9). Furthermore, 
the SK-channel has been suggested to play a major role in the spontaneous 
transient outward currents in the tracheal smooth muscle cells (10), the 
inhibitory action of the .alpha..sub.1 -adrenoceptors, neurotensin 
receptor and the P.sub.2 -subtype of the ATP receptor (4, 9). 
The neuronal and the skeletal muscle SK-channel is specifically and avidly 
blocked by a bee venom-derived peptide toxin, apamin (5, 11-14). By all 
indications, the apamin receptor complex is either identical to, or 
closely associated with the SK-channel. Apamin is an 18 amino acid 
neurotoxic peptide which has a single class of binding sites in the rat 
brain synaptosomes and brain slices with an apparent dissociation constant 
(K.sub.d) of 10-25 pM (15, 16). Apamin is also capable of a temperature 
dependent and high affinity (K.sub.d =30-150 pM) binding to the detergent 
solubilized brain receptor sites (17-20). The reported B.sub.max value for 
the rat brain synaptosomes and brain slices is 10-30 fmol/mg protein (16, 
17, 20), while that for the detergent solubilized receptor ranges from 
0.45 to 17 fmol/mg protein (18, 19). 
The polypeptide components of the apamin receptor have been studied by 
several groups. Cross-linking experiments using [.sup.125 I]apamin, 
followed by SDS-PAGE and autoradiography have indicated that the apamin 
binding proteins of the rat brain synaptosomal membrane consist of two 
protein species, a major 80-86 KDa protein and, in most reported 
preparations, a minor 50-59 KDa band (17, 21, 22). Partial peptide mapping 
of the two protein bands, using an anti-apamin anti-serum, has shown that 
the smaller polypeptide is likely to be a proteolytic fragment of the 
larger protein and not an additional subunit of the apamin binding protein 
in the brain. Furthermore, in the plasma membrane of the cultured neurons 
or astrocytes, there are additional components with the ability to 
cross-link to [.sup.125 I]apamin. Cross-linking of [.sup.125 I]apamin to 
the membranes from the rat heart, liver and smooth muscle has also 
indicated that a 85-87 KDa polypeptide is the major labeled component of 
the apamin binding complex (23). A second 59 KDa protein was identified in 
the liver membrane only (23). 
The blocking of the small conductance calcium 1 U activated potassium 
channel (sKca) results in prolongation of the action potential, while its 
activation by an increase in the intracellular calcium concentration 
accelerates the rate of hyperpolarization, thus shortening the duration of 
the action potential. In vascular smooth muscle cells (such as those in 
veins and arteries), activation of sKca results in the hyperpolarization 
of the smooth muscle membrane, which in turn results in the inhibition of 
the voltage-gated calcium channels. The inhibition of the latter may then 
lead to the relaxation of the blood vessels and lowering of the blood 
pressure. In the heart, modulation of sKca can be a potentially useful 
means to regulate an arrhythmic heart. In the nervous system, the 
hippocampus of Alzheimer's patients shows a drastic reduction in apamin 
denisty (30). Further apamin receptor in neurons has been reported to be 
involved in the process of learning and memory (42). Thus, manipulation of 
this receptor may also result in improving cognition. Notwithstanding the 
significant therapeutic potential manipulation of sKca may have, 
relatively little is known about the identity of the proteins involved in 
this channel. The present invention now provides a key element in the 
study of the potassium channel function. 
SUMMARY OF THE INVENTION 
The present invention relates to a nucleic acid fragment comprising a 
sequence encoding an apamin receptor, as well as the recombinantly 
produced apamin receptor per se. Such receptors are associated with 
calcium activated potassium channels in a variety of animal tissues, such 
as brain, skeletal, cardiac, vascular smooth muscle, pancreas, kidney and 
liver tissue. An exemplary sequence of Kcal 1.8, a porcine receptor, is 
provided in FIG. 3A; however, the invention also encompasses any 
nucleotide sequence which hybridizes, under medium or high stringency 
conditions (as defined in the Examples below), with a nucleotide sequence 
encoding the amino acid sequence of FIG. 3A, as well as the biologically 
active proteins and fragments encoded by such sequences. By "biologically 
active" is meant proteins or fragments which are capable of eliciting 
production of antibodies capable of binding to the receptor, as well as 
proteins or fragments which are associated with calcium activated K+ 
channels (such as "BK" or "IK") but do not necessarily bind apamin. 
The invention also relates to host cells and recombinant vectors useful in 
expressing the apamin receptor gene and protein. Such hosts will provide a 
convenient basis for development of screens designed to identify compounds 
which are capable of modulating activity of the receptor and thus, 
modulate the activity of the potassium channel. In the heart, modulation 
of this channel provides a means for regulating an arrhythmic heart; thus, 
any drug that can open or close this potassium channel is considered a 
potential antiarrhythmic agent. Similarly, in vascular smooth muscle 
cells, such as those in veins and arteries, activation of the potassium 
channel results in hyperpolarization of the smooth muscle membrane, which 
in turn results in the inhibition of the voltage-gated calcium channels. 
The inhibition of the latter will then lead to relaxation of the blood 
vessels and lowering of blood pressure. The receptor is also associated 
with cognition functions. As noted above, receptor density decreases in 
Alzheimer's patients, and is involved in the process of learning and 
memory. Thus, compounds which activate the receptor may be useful in 
improving impaired cognitive function in Alzheimer's patients, or to 
enhance memory and learning capacity. Therefore, a convenient system 
enabling the detection of compounds that modulate potassium channel 
activity has the potential for identifying drugs with tremendous 
therapeutic utility. Also, the isolated nucleic acid sequence detectably 
labelled can be used as a diagnostic probe for Alzheimer's disease, by 
determining the level of expression of such receptors in peripheral 
neurons of individuals suspected of being affected. Copending and cofiled 
applications which have as common inventors Mohammad Reza Ziai and 
Patricia Tyson Sokol, relating to purified apamin binding proteins Ser. 
No. 07/922,307, and affinity matrix for binding protein purification Ser. 
No. 07/922,604 abandoned in favor of Ser. No. 08/144,210 , are each 
incorporated herein by reference in their entirety.

DETAILED DESCRIPTION OF THE VECTOR 
A full-length apamin binding protein nucleic acid sequence, presumed to be 
associated with a calcium activated K.sup.+ channel, is first isolated 
from a porcine vascular smooth muscle (aorta) expression cDNA library in a 
.lambda.-ZAP vector. The library is screened with polyclonal sera raised 
against a bovine brain apamin receptor. Screening of about 2 million 
plaque forming units yields four positive plaques which are rescreened and 
plaque purified. 
The .lambda.-ZAP is transformed into "pBluescript" plasmid by standard 
techniques, the DNA is digested with the restriction endonucleases EcoRI 
and XhoI to release the cDNA inserts and analyzed by agarose gel 
electrophoresis. One 1.6 Kb cDNA clone (designated Kcal 1.6) is selected 
for Northern hybridization, genomic Southern blotting and DNA sequencing. 
As shown in FIG. 1A, the cDNA Kcal 1.6 detects a single mRNA band of 
approximately 2.1 Kb in the adult rat brain mRNA (lane 1), bovine brain 
mRNA (lane 2) and porcine brain mRNA (lane 3). The probe, however, reveals 
two mRNA bands of 2.1 and 3.0 Kb in size in the Northern blot of mRNA from 
neonatal rat brain (FIG. 1B). These results suggest that in the neonatal 
rat brain, there are two distinct mRNA species which hybridize to Kcal 
1.6, possibly arising by the alternate splicing of mRNA. 
Next, an EcoRI cut-genomic Southern blot is probed with Kcal 1.6 cDNA. As 
shown in FIG. 2, after repeated washing of the blot at high stringency, 
the Kcal 1.6 probe detects a single 14 Kb band in human (lane 1) and in 
monkey (lane 2). However, there are variable patterns of hybridization in 
the rat (lane 3), mouse (lane 4), canine (lane 5), bovine (lane 6), rabbit 
(lane 7) and chicken (lane 8). There is no detectable hybridization with 
the yeast DNA (lane 9). This experiment indicates that there are 
significant sequence homologies among the genes encoding p80 in various 
species. Furthermore, the gene(s) encoding p80 in human and monkey are 
probably more similar than those in other species. 
Kcal 1.6 cDNA is then sequenced. The nucleotide sequence obtained indicates 
that the clone is not of full length and lacks the initiation methionine 
residue. To obtain a full-length clone, Kcal 1.6 is used as a probe and 
the original porcine aorta cDNA library is screened, and positive clones 
analyzed by restriction mapping and electrophoresis for relatedness and 
insert size. One cDNA clone (designated Kcal 1.8), which appears to be 
slightly longer than Kcal 1.6, is selected and sequenced by a Taq 
polymerase sequencing technique. When the nucleotide sequence is 
translated in frame, the cDNA Kcal 1.8 encodes a protein of 437 amino 
acids (FIGS. 3A-E), with an initiation methionine and a stop site. 
Hydrophobicity analysis (FIG. 3F) of the sequence indicates the presence 
of four strongly hydrophobic putative transmembrane domains (TMD1-4), a 
short amino terminus and a long carboxyl terminus. The sequence has some 
interesting features. It contains a strong "EF-Hand" consensus sequence 
(in FIG. 3E, indicated by a *). The EF-Hand consensus sequence is present 
in virtually all calcium binding protein members of calmodulin and 
troponin C families. In fact, the EF-Hand motif in Kcal 1.8 almost 
perfectly matches that of calmodulin, as well as a recently cloned 
component of Drosophila calcium activated K-channel, "Slo" (28). In 
addition, the sequence flanking the putative "EF-Hand" motif of Kcal 1.8 
has significant homology with a number of known calcium binding proteins 
including troponin C, myosin, calreticulin, PEP-19, and several others. 
Since the small conductance calcium-activated potassium channel (skca) 
must have a calcium binding site, it gives further support to the belief 
that Kcal 1.8 indeed encodes skca. If the "EF-Hand" motif is in fact a 
calcium binding site of Kcal 1.8 protein, it places the "EF-Hand" motif on 
the cytoplasmic side of the membrane. The amino acid sequence of Kcal 1.8 
also contains one protein kinase C site, and one tyrosine kinase 
phosphorylation site (not shown). In addition, a "leucine zipper" motif 
can be identified in the C-terminal portion of the protein (FIG. 3E, boxed 
"L"). At present, the significance, if any, of this motif in Kcal 1.8 is 
unclear. However, the presence of these putative phosphorylation sites, 
together with the "EF-Hand" motif are likely to place both N- and 
C-termini of the protein in the cytoplasmic side of the plasma membrane. 
To further confirm Kcal 1.8's identity as an apamin receptor, Kcal cDNA is 
introduced into a stable mammalian expression vector, pRC/CMV, which is 
used to transfect CV-1 cells (African green monkey kidney). Cells stably 
expressing the Kcal 1.8 gene product are selected and are contacted with 
radiolabelled apamin, in the presence or absence of unlabelled apamin. A 
15 number of transfectants show enhanced binding of radio-labelled apamin, 
thereby adding further confirmation of Kcal 1.8's identity. 
The foregoing discussion, and the sequences provided in FIGS. 3A-E, relate 
to a porcine smooth muscle apamin receptor. However, it will be understood 
that the invention encompasses more than the specific exemplary sequences. 
Modifications to the sequence, such as deletions, insertions, or 
substitutions in the sequence which produce silent changes in the 
resulting protein molecule are also contemplated. For example, alteration 
in the gene sequence which reflect the degeneracy of the genetic code, or 
which result in the production of a chemically equivalent amino acid at a 
given site, are contemplated; thus, a codon for the amino acid alanine, a 
hydrophobic amino acid, may be substituted by a codon encoding another 
less hydrophobic residue, such as glycine, or a more hydrophobic residue, 
such as valine, leucine, or isoleucine. Similarly, changes which result in 
substitution of one negatively charged residue for another, such as 
aspartic acid for glutamic acid, or one positively charged residue for 
another, such as lysine for arginine, can also be expected to produce a 
biologically equivalent product. Nucleotide changes which result in 
alteration of the N-terminal and C-terminal portions of the protein 
molecule would also not be expected to alter the activity of the protein. 
It may also be desirable to eliminate one or more of the cysteines present 
in the sequence, as the presence of cysteines may result in the 
undesirable formation of multimers when the protein is produced 
recombinantly, thereby complicating the purification and crystallization 
processes. In some cases, it may in fact be desirable to make mutants of 
the sequence in order to study the effect of alteration on the biological 
activity of the protein. Each of the proposed modifications is well within 
the routine skill in the art, as is determination of retention of 
biological activity of the encoded products. 
The invention also encompasses homologous sequences obtained from other 
species and other tissues. As has already been demonstrated above, the 
nucleic acid sequence depicted in FIGS. 3A-E hybridizes, under relatively 
stringent conditions, with nucleic acid fragments present in a number of 
other species, including human, thus demonstrating the ability to isolate 
other non-porcine sequences. Moreover, apamin receptors from tissue types 
other than vascular smooth muscle are also known to exist. Brain, skeletal 
muscle, and liver, in addition to vascular smooth muscle, have been 
repeatedly demonstrated to express a single class of binding site (4, 
15-20). On the other hand, cardiac tissue seems to exhibit a heterogeneous 
population of target sites. The sequence disclosed in FIGS. 3A-E can thus 
be used as a probe to isolate the corresponding receptors from other 
species and tissues. Alternate receptor types are isolatable as follows. 
cDNA libraries prepared from mRNA from the specific tissue type of 
interest are probed with radiolabelled Kcal 1.8 cDNA and washed under 
medium stringency (e.g., 1.times.SSC, 0.1% SDS, 55.degree. C). Plaques 
which appear positive are rescreened to verify authenticity. The positive 
plaques are then used in plasmid rescue according to techniques known in 
the art. Rescued plasmids are purified, cut with appropriate restriction 
enzymes, and analyzed in an agarose gel stained with ethidium bromide. The 
second gel is transferred to an nitrocellulose filter, probed with 
labelled Kcal 1.8, washed sequentially under a medium, then high 
stringency (0.1.times.SSC, 0.1% SDS, at 65.degree. C.) wash and exposed to 
X-ray film. Those inserts which strongly hybridize to Kcal 1.8 under high 
stringency conditions represent likely receptor cDNA candidates. Further 
confirmation of the identity of these putative receptors can be 
accomplished according to the protocols described in the following 
examples, or in accordance with routine techniques known in the art. Thus, 
the invention encompasses not only the nucleotide and amino acid sequences 
depicted in FIGS. 3A-E, but also nucleotide sequences which hybridize, 
under medium or high stringency conditions, with nucleotide sequence 
encoding the amino acid sequence of FIGS. 3A-E, as well as the 
biologically active proteins or fragments encoded thereby. 
The nucleic acid sequence can be used to express the receptor protein in a 
variety of host cells, both prokaryotic and eukaryotic for the chosen cell 
line. Examples of suitable eukaryotic cells include mammalian cells, plant 
cells, yeast cells, and insect cells. Suitable prokaryotic hosts include 
Escherichia coli and Bacillus subtills. 
Suitable expression vectors are selected based upon the choice of host 
cell. Numerous vectors suitable for use in transforming bacterial cells 
are well known. For example, plasmids and bacteriophages, such as .lambda. 
phage, are the most commonly used vectors for bacterial hosts, and for E. 
coli in particular. In both mammalian and insect cells, virus vectors are 
frequently used to obtain expression of exogenous DNA. In particular, 
mammalian cells are commonly transformed with SV40, polyoma virus, or 
transfected with plasmids such as pRC/CMV; and insect cells in culture may 
be transformed with baculovirus expression vectors. Yeast vector systems 
include yeast centromere plasmids, yeast episomal plasmids and yeast 
integrating plasmids. The invention encompasses any and all host cells 
transformed or transfected by the claimed nucleic acid fragments, as well 
as expression vectors used to achieve this. In particular, the host cells 
chosen for transfection are cells which exhibit only low (i.e., 
background) levels of receptor expression (e.g., see FIG. 4) before 
transcription. 
In a preferred embodiment, nucleic acid sequences encoding an apamin 
receptor are used to transfect eukaryotic cells, preferably mammalian 
cells. For an initial determination of the ability of a given sequence to 
produce an apamin binding protein, transient expression, using plasmids 
such as pcDNAI or PSG5 into which the putative receptor DNA sequence has 
been ligated, and CMT-1 or COS-1 or -7 cells, can be employed. CMT-1 cells 
are transfected using the calcium phosphate precipitation method, and 
within 24 hours of transfection, the SV40 large T antigert is induced with 
addition of zinc to the medium. Seventy-two hours after transfection, 
cells are harvested for either RNA isolation or apamin binding assays. 
Expression is compared between cDNA and mock-transfected cells to 
determine if receptor activity is achieved by transfected cells. A 
positive host cell is preferably one which exhibits about twice the 
background level of apamin binding observed in non-transfected host cells 
of the same type. 
For use of the sequences in screen development, stable expression of the 
DNA may be desirable. In this case, the DNA encoding the receptor is 
ligated into a stable vector containing a selectable marker, such as 
pRC/CMV, pcDNAI Neo, pXTI, or pMAM Neo. The plasmid DNA is linearized and 
introduced into an appropriate cell line for such vectors, e.g., CV-1, 
CHO, HEepG-2 or NIH3T3 cells, by electroporation. Successfully transfected 
cells are identified by selection and isolated clones are picked and 
amplified. To determine transcription of Kcal message, cellular RNA is 
isolated and separated electrophoretically on agarose gel. Detection of 
endogenous and exogenous mRNA is accomplished using Kcal 1.8 as a probe. 
Identification of exogenous (transfected) mRNA is accomplished by probing 
with a 400 bp fragment from the 5' untranslated region of cDNA, since this 
region is most divergent among species, diminishing the incidence of 
cross-hybridization. 
The ability of any given isolated DNA sequence to yield a functional apamin 
receptor is determinable by a simple apamin binding assay. Transfected 
cells are prepared as previously described (41). Binding assays are 
performed by a standard procedure (16), and values for maximum binding of 
ligand to receptor (Bmax) and dissociation constant (Kd) for each cell 
line is calculated. 
Further evaluation of the measurement of potassium channel activity in 
cultured transfectant cells is accomplished by .sup.86 Rb efflux assay 
(30, incorporated herein by reference). Briefly, stably transfected cells 
are loaded overnight with .sup.86 Rb in microtiter plates; the medium is 
then discarded and adherent cells washed three times to remove isotope. 
Cells are then incubated for 30 minutes at 37.degree. C. with an isotonic 
buffer containing 20 mM CaCl.sub.2 and 100 .mu.M calcium ionophore A23187. 
The supernatants from wells are recovered and counted. The cell layer is 
solubilized in Triton X-100 and also counted, and the percent efflux of 
.sup.86 Rb calculated as described. The experiment is carried out in the 
presence or absence of 1 mM apamin (an sKca blocker) or 1 .mu.M 
charybdotoxin (a BKca blocker), and control experiments carried out in 
parallel with cells being incubated with buffer, but without added 
ionophore. The percent efflux in transfectants harboring cloned DNA mock 
transfectants, and wild-type CV-1 cells (to measure endogenous efflux) are 
compared. Such assays are also useful in determining the effect of 
structural change in the channel in its function, and also to evaluate 
functional differences between different receptor subtypes. This assay is 
useful both in confirming activity of a putative receptor/channel as well 
as confirming the effects 
DEPOSIT OF BIOLOGICAL MATERIALS 
The following biological materials were deposited with the American Type 
Culture Collection, 12301 Parklawn Drive, Rockville, Md., on Jun. 18, 
1992, and given the Accession Numbers indicated: 
______________________________________ 
Material Accession No. 
______________________________________ 
E. coli containing ATCC 69017 
pBluescript plasmid 
containing Kcal 1.8 
______________________________________ 
The present invention is further illustrated by the following non-limiting 
examples. 
EXAMPLES 
1. Screening Expression Library 
A porcine aorta expression cDNA library in .lambda.-Uni ZAP .lambda.R 
(Stratagene, La Jolla, Calif.) is probed with a 1:1000 dilution of a 
murine anti-apamin binding protein polyclonal antiserum (M2) using the 
Vectastain ABC kit (Vector Laboratories Inc., Burlingame, Calif.) as the 
secondary antibody and detection system. Approximately 2.times.10.sup.6 
plaque forming units are screened in this manner. 
Four positive plaques are selected from the first round of screening. These 
are subjected to a re-screen and plasmids (pBluescript) containing the 
cDNA inserts are rescued using a helper phage. The parent plasmid DNA is 
digested with the restriction endonucleases EcoRI and XhoI to release the 
cDNA inserts and analyzed by agarose gel electrophoresis. One 1.6 Kb cDNA 
clone (designated Kcal 1.6) is selected for Northern hybridization, 
genomic Southern blotting and DNA sequencing. For Northern hybridization, 
polyA mRNA is isolated from frozen rat tissues using "Fast Track" mRNA 
isolation kit (Invitrogen, San Diego, Calif.) or purchased from Clontech 
Labs (Palo Alto, Calif.). Genomic Southern blot, "Zoo-blot" is purchased 
from Clontech Labs and processed as described by the manufacturer. As 
shown in FIG. 1A, the cDNA Kcal 1.6 detects a single mRNA band of 
approximately 2.1 Kb in the adult rat brain mRNA (lane 1) bovine brain 
mRNA (lane 2) and porcine brain mRNA (lane 3). The probe, however, reveals 
two mRNA bands of 2.1 and 3.0 Kb in size in the northern blot of mRNA from 
neonatal rat brain (FIG. 1B). These results may indicate that in the 
neonatal rat brain, there are two distinct mRNA species which hybridize to 
Kcal 1.6, possibly arising from the alternate splicing of mRNA. Next, an 
EcoRI cut-genomic southern blot is probed with Kcal 1.6 cDNA. As shown in 
FIG. 2, after repeated washing of the blot at high stringency, the Kcal 
1.6 probe detects a single 14 Kb band in human (lane 1) and in monkey 
(lane 2). However, there are variable patterns of hybridization in the rat 
(lane 3), mouse (lane 4), canine (lane 5), bovine (lane 6), rabbit (lane 
7) and chicken (lane 8) ranging from 14 Kb to 3.0 Kb. There is no 
detectable hybridization with the yeast DNA (lane 9). These results 
indicate that there are notable homologies among the genes encoding p80 in 
various species. 
2. Sequencing of Kcal 1.6 
DNA sequencing is performed using the "Taq-Track" sequencing system 
(Promega Corp.) or the "Sequenase" system (U.S. Biochemical, Cleveland, 
Ohio). The nucleotide sequence obtained indicates that the clone is not 
full length, and lacks an initiation methionine residue. To obtain a 
full-length clone, Kcal 1.6 is used as a probe to screen the original 
porcine aorta cDNA library. Positive clones are analyzed by restriction 
mapping and electrophoresis for relatedness and insert size. One cDNA 
clone (designated Kcal 1.8) which is slightly longer than Kcal 1.6 is 
isolated and sequenced. The nucleotide and amino acid sequence of Kcal 1.8 
is shown in FIGS. 3A-E. The cDNA encodes 437 amino acids, the hydropathy 
plot (FIG. 3F) indicates four strongly hydrophobic putative transmembrane 
domains. There is a putative calcium binding domain which closely matches 
that of the cloned cDNA slo encoding a putative calcium activated 
K-channel in Drosophila. However, there is no significant sequence 
homology between Kcal 1.8 and slo in other regions. 
There is one strong consensus sequence in Kcal 1.8 for the cAMP dependent 
protein kinase, as well as those putative casein kinase phosphorylation 
sites. The Kcal 1.8 sequence has no significant homologies with any known 
voltage gated K-channels, sodium channels or calcium channels. 
3. Expression of Kcal 1.8 
CV-1 cells (ATCC CCL70) stably expressing the Kcal 1.8 gene product are 
produced by introducing the cDNA in the stable mammalian expression 
plasmid, pRc/CMV (InVitrogen) which contains a Neo.sup.r marker. The Kcal 
1.8 sequence is extracted from the pBluescript vector by digestion, with 
EcoRI and XhoI, and ligated into the corresponding sites of pRc/CMV. To 
transfect the cells, confluent 100 mm dishes of CV-1 cells are split and 
replated the day before the transfection, to ensure the cells are in 
log-growth phase. For electropotation, cells are harvested with trypsin, 
washed once with phosphate-buffered saline, and twice with an isotonic, 
low ionic strength buffer containing 272 mM sucrose, 7 mM sodium 
phosphate, pH 7.4 and 1 mM MgCl.sub.2 (buffer E). The cells are 
resuspended in this same buffer to a final concentration of 
1.5.times.10.sup.6 cells/ml. Twenty .mu.g of the appropriate vector are 
digested with 40 units of ScaI for 2 hours at 37.degree. C. to linearize 
the plasmid. The linearized plasmid is phenol/chloroform extracted, EtOH 
precipitated, and resuspended in 400 .mu.l of Buffer E. The resuspended 
DNA is mixed with 400 ul of Cv-1 cells (1.times.10.sup.6 cells) and 
incubated at room temperature for 2 minutes prior to electroporation. 
Electroporation is accomplished using a Bio-Rad gene pulser with a 300-V 
pulse at 25 .mu.Farads. Transfections are done in duplicate. The 
suspension is allowed to further incubate for 5 minutes at room 
temperature, and then plated onto 100 mm tissue culture dishes with 10 mls 
of Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Two 
days following transfection, G418 is added to a final concentration of 200 
ug/ml. When isolated G418-resistant colonies are identified, they are 
selected with cloning cylinders and amplified. 
Transfected cells are harvested and washed. They are incubated with 
[.sup.125 I]apamin in the binding buffer "B": Tris-HCl 10 mM, KCl 10 mM, 
pH 7.4, in the presence or absence of 1 uM cold apamin. The incubation is 
at 4.degree. C. for 30 minutes with cold apamin, followed by 1 hour 
incubation at 4.degree. C. with [.sup.125 I]apamin (20,000 cpm/well). 
Target cells are then filtered and washed with the binding buffer plus 
BSA. The filters are counted in a gamma counter. 
As shown in FIG. 4, Transfectant #1, 3, 10 and 12 show significantly 
enhanced binding of [.sup.125 I]apamin, compared to other transfectants 
shown. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1730 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Pig 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 140..1456 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AGCAGCTCCATAGGCCCAGCCCCGGCGTACAAGGATCACTTCCGGTGGTACTTCACTACC60 
AAGAAGCTGCGATTGGGCGAGCGTGGAAG GGGCATTTCCGGTGTCCACCTGCTTGGGTTC120 
TTTGGACAGAAGTAGGAAGATGGAGCTCGGCGCCGCGGCCCGTGCTTGGTCG172 
MetGluLeuGlyAlaAlaAlaArgAlaTrpSer 
1510 
CTCTTGTGGCTGCTGCTTCCCTTGCTTGGCCTGGTCGGCGCCAGCGGT220 
LeuLeuTrpLeuLeuLeuProLeuLeuGlyLeuValGlyAlaSerGly 
1 52025 
CCCCGTACCTTAGTGCTTCTGGACAACCTCAACCTGCGGGAGACGCAT268 
ProArgThrLeuValLeuLeuAspAsnLeuAsnLeuArgGluThrHis 
30 3540 
TCACTTTTCTTCCGGAGCCTAAAGGATCGGGGCTTCGTACTCACATTC316 
SerLeuPhePheArgSerLeuLysAspArgGlyPheValLeuThrPhe 
45 5055 
AAGACCGCAGATGACCCCAGCCTGTCCCTGATTAAGTACGGAGAGTTC364 
LysThrAlaAspAspProSerLeuSerLeuIleLysTyrGlyGluPhe 
6065 7075 
CTCTATGACAATCTCATCGTCTTTTCACCTTCGGTAGAAGATTTTGGA412 
LeuTyrAspAsnLeuIleValPheSerProSerValGluAspPheGly 
80 8590 
GGAAATATCAACGTGGAGACCATCAGTACCTTTATCGACGGCGGAGGC460 
GlyAsnIleAsnValGluThrIleSerThrPheIleAspGlyGlyGly 
95 100105 
AGTGTCCTGGTAGCTGCCAGCTCAGACATCGGTGACCCTCTCCGCGAG508 
SerValLeuValAlaAlaSerSerAspIleGlyAspProLeuArgGlu 
110115 120 
CTGGGCAGTGAGTGTGGGATTGAGTTTGACGAGGAGAAAACGGCCGTC556 
LeuGlySerGluCysGlyIleGluPheAspGluGluLysThrAlaVal 
125130 135 
ATTGACCATCACAACTATGATGTCTCAGACCTCGGCCAGCACACGCTC604 
IleAspHisHisAsnTyrAspValSerAspLeuGlyGlnHisThrLeu 
140145150 155 
ATTGTGGCCGACACTGAGAACCTGCTGAAGGCCCCGACCATCGTCGGG652 
IleValAlaAspThrGluAsnLeuLeuLysAlaProThrIleValGly 
160165 170 
AAGTCATCTCTGAATCCCATCCTCTTCCGAGGTGTTGGGATGGTGGCT700 
LysSerSerLeuAsnProIleLeuPheArgGlyValGlyMetValAla 
175180 185 
GATCCTGACAATCCTTTGGTGCTGGACATCCTGACCGGCTCTTCTACC748 
AspProAspAsnProLeuValLeuAspIleLeuThrGlySerSerThr 
190195 200 
TCTTACTCCTTCTTCCCAGATAAACCCATCACGCAGTACCCGCACGCG796 
SerTyrSerPhePheProAspLysProIleThrGlnTyrProHisAla 
205210215 
GTG GGGAAGAACACGCTGCTCATCGCGGGGCTGCAGGCCCGGAACAAC844 
ValGlyLysAsnThrLeuLeuIleAlaGlyLeuGlnAlaArgAsnAsn 
220225230235 
GCCCGTGTCATCTTCAGCGGCTCCCTCGACTTCTTCAGCGATGCCTTC892 
AlaArgValIlePheSerGlySerLeuAspPhePheSerAspAlaPhe 
240245250 
TTCAACTCCGCGGTGCAGAAGGCCACGCCTGGCTCCCAGAGGTATCCC940 
PheAsnSerAlaValGlnLysAlaThrProGlySerGlnArgTyrPro 
255260265 
C AGACAGGCAACTATGAGCTCGCCGTGGCCCTCTCCCGCTGGGTGTTC988 
GlnThrGlyAsnTyrGluLeuAlaValAlaLeuSerArgTrpValPhe 
270275280 
AAGGAG GAGGGTGTCCTCCGAGTGGGGCCTGTGTCCCACCATCGGGTG1036 
LysGluGluGlyValLeuArgValGlyProValSerHisHisArgVal 
285290295 
GGCGAGAAAGCCCCA CCCAACGCCTACACCGTCACTGACCTAGTCGAG1084 
GlyGluLysAlaProProAsnAlaTyrThrValThrAspLeuValGlu 
300305310315 
TACAGCATCGT GATTGAGCAGCTCTCACAGGGCAGATGGGTCCCCTTT1132 
TyrSerIleValIleGluGlnLeuSerGlnGlyArgTrpValProPhe 
320325330 
GATGGCGACG ACATTCAGCTGGAGTTTGTCCGCATCGATCCTTTCGTG1180 
AspGlyAspAspIleGlnLeuGluPheValArgIleAspProPheVal 
335340345 
AGGACCTTCTTG AAGAGGAAAGGCGGCAAGTACAGCGTCCAGTTCAAG1228 
ArgThrPheLeuLysArgLysGlyGlyLysTyrSerValGlnPheLys 
350355360 
TTGCCGGACGTGTACGGC GTGTTCCAGTTCAAAGTGGACTACAACCGG1276 
LeuProAspValTyrGlyValPheGlnPheLysValAspTyrAsnArg 
365370375 
CTGGGCTACACGCACCTGTACTCCTC CACTCAGGTGTCCGTGCGGCCC1324 
LeuGlyTyrThrHisLeuTyrSerSerThrGlnValSerValArgPro 
380385390395 
CTGCAGGCACACGCAGTACGAG CGCTTCATCCCCTCGGCTTACCCCTA1372 
LeuGlnAlaHisAlaValArgAlaLeuHisProLeuGlyLeuProLeu 
400405410 
CTACGCCAGCGCCTTCTCCAT GATGGTCGGGCTCTTCATCTTCAGCGT1420 
LeuArgGlnArgLeuLeuHisAspGlyArgAlaLeuHisLeuGlnArg 
415420425 
CGTCTTCTTGCACATGAAGGAGAA GGAGAAGTCTGACTGAGGGGCCGGGCCGG1473 
ArgLeuLeuAlaHisGluGlyGluGlyGluVal 
430435 
GCCCCAGGACTCCTTACAACACACAGGGAGGGTTTTTATAGGCTTGCCTTCCCCCCCCTT1533 
TAT GGTGGGCTTTGTTTGTTTTTAAAGCCACGGACAATGGCACAGCTTACCTCAGTGGGA1593 
GATGCAAGATGAGTACCAGGGGGTGGTTAGGAATAATTTCTAAGTTTTTCCACCTTGAAT1653 
GCTGAGTGGTATTTTTCATATGTAAAGTCAACTGATTTCTAAAATAAAA GAAAAACATCA1713 
CCCTCAGAAAAAAAAAA1730 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 438 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetGluLeuGlyAlaAlaAlaArgAlaTrpSerLeuLeuTrpLeuLeu 
151015 
LeuProLeuLeuGlyLeuValGlyAlaSerGlyProArgThrLeuVal 
202530 
LeuLeuAspAsnLeuAsnLeuArgGluThrHisSerLeuPhePheArg 
354045 
SerLeuLysAspArgGlyP heValLeuThrPheLysThrAlaAspAsp 
505560 
ProSerLeuSerLeuIleLysTyrGlyGluPheLeuTyrAspAsnLeu 
657075 80 
IleValPheSerProSerValGluAspPheGlyGlyAsnIleAsnVal 
859095 
GluThrIleSerThrPheIleAspGlyGlyGlySerVal LeuValAla 
100105110 
AlaSerSerAspIleGlyAspProLeuArgGluLeuGlySerGluCys 
115120125 
GlyIleGl uPheAspGluGluLysThrAlaValIleAspHisHisAsn 
130135140 
TyrAspValSerAspLeuGlyGlnHisThrLeuIleValAlaAspThr 
145150 155160 
GluAsnLeuLeuLysAlaProThrIleValGlyLysSerSerLeuAsn 
165170175 
ProIleLeuPheArgGlyValGlyMetV alAlaAspProAspAsnPro 
180185190 
LeuValLeuAspIleLeuThrGlySerSerThrSerTyrSerPhePhe 
1952002 05 
ProAspLysProIleThrGlnTyrProHisAlaValGlyLysAsnThr 
210215220 
LeuLeuIleAlaGlyLeuGlnAlaArgAsnAsnAlaArgValIlePhe 
225 230235240 
SerGlySerLeuAspPhePheSerAspAlaPhePheAsnSerAlaVal 
245250255 
GlnLysAlaThrProGl ySerGlnArgTyrProGlnThrGlyAsnTyr 
260265270 
GluLeuAlaValAlaLeuSerArgTrpValPheLysGluGluGlyVal 
275280 285 
LeuArgValGlyProValSerHisHisArgValGlyGluLysAlaPro 
290295300 
ProAsnAlaTyrThrValThrAspLeuValGluTyrSerIleValIle 
305310315320 
GluGlnLeuSerGlnGlyArgTrpValProPheAspGlyAspAspIle 
325330335 
GlnLeu GluPheValArgIleAspProPheValArgThrPheLeuLys 
340345350 
ArgLysGlyGlyLysTyrSerValGlnPheLysLeuProAspValTyr 
355 360365 
GlyValPheGlnPheLysValAspTyrAsnArgLeuGlyTyrThrHis 
370375380 
LeuTyrSerSerThrGlnValSerValArgProLeuGl nAlaHisAla 
385390395400 
ValArgAlaLeuHisProLeuGlyLeuProLeuLeuArgGlnArgLeu 
405410 415 
LeuHisAspGlyArgAlaLeuHisLeuGlnArgArgLeuLeuAlaHis 
420425430 
GluGlyGluGlyGluVal 
435 
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