Protein affecting K.sub.ATP channels

This invention describes the isolation and identification of a new protein, p56, useful for the identification of drugs that will selectively open or close K channels. The protein p56 has a molecular weight of about 56,000 daltons and the N-terminal peptide sequence is: Glu-Pro-Arg-Ala-Pro-Pro-Glu-Lys-Ile-Ala-Ile-Val-Gly-Ala-Gly-Ile.

FIELD OF THE INVENTION 
This invention relates to potassium channels and the proteins that comprise 
those channels. 
INFORMATION DISCLOSURE 
The following documents are related to the cyanopyridylguanidine compound 
used to isolate the protein disclosed herein. 
Petersen, Hans J., et. al., "Synthesis and Hypotensive Activity of 
N-Alkyl-N"-cyano-N'-pyridylguanidines," J. Med. Chem., 21, 8, pp. 773-781 
(1978). 
U.S. Pat. No. Re. 31,244, reissued May 17, 1983, "Antihypertensive 
Pyridylguanidine Compounds," H. J. Petersen. 
U.S. Pat. No. 4,057,636, issued Nov. 8, 1977, "Antihypertensive 
Pyridylguanidine Compounds," H. J. Petersen. 
WO 9211233-A1, published Dec. 19, 1990, "New aryl:cyano:guanidine potassium 
channel dilater--for treating hypertension," assigned to Kanebo Ltd. 
European Patent 405 525 A2, published Feb. 1, 1991, "Novel Cyanoguanidine 
Derivatives," M. Tominori. 
BACKGROUND 
Ionic channels of cell membranes are the basic sites where ionic fluxes 
take place. The modern era of the study of drug-channel interactions began 
when voltage clamp techniques were used to demonstrate the block of 
Sodium, (Na.sup.+), and potassium, (K.sup.+), channels of squid axons 
caused by procaine and cocaine. Narahashi, Ann Neurology (1984); 
16(suppl): S39-S51. 
This invention concerns proteins which regulate or constitute the pore 
region of potassium channels. Potassium channels appear to be ubiquitous, 
found even in bacteria. See, R. Milkman, "An Escherichia Coli homologue of 
eukaryotic potassium channel proteins" Proc. Natl. Acad. Sci. USA, Vol 91, 
pp. 3510-3514, (1994). Pharmacological, biophysical and molecular studies 
have revealed multiple subtypes for membrane ion channels that form 
potassium selective pores in the plasma membrane of many mammalian cells. 
One method of classifying K channels is based on what regulates channel 
activity or function. For example, one class can be defined as K channels 
modulated by transmembrane voltage, another class modulated solely by 
calcium and/or nucleotides, and yet a third class modulated by G protein 
involvement. However, in a more simplistic manner, one can classify the 
family of K channels simply by their respective gating properties. In 
other words, a comparison of the pharmacological and electrophysiological 
properties of potassium channels has given rise to an operational 
definition for grouping the various subtypes based largely on their gating 
properties. At present, potassium channels of known amino acid sequence 
comprise two distantly related protein families. One of these channel 
families is termed, "voltage-gated," the other channel family is termed 
"inward rectifying." 
The structure of the voltage-gated channel protein is known to be comprised 
of six membrane spanning domains in each subunit, each of which is 
regulated by changes in membrane potential. B. Hille. "Ionic Channels of 
Excitable Membranes" (Sinauer, Sunderland, Mass., 1992). Voltage-gated 
potassium channels sense changes in membrane potential and move potassium 
ions in response to this alteration in the cell membrane potential. 
Molecular cloning studies on potassium channel proteins has yielded 
information primarily for members of the voltage-gated family of potassium 
channels. Various genes encoding these voltage-gated family of potassium 
channel proteins have been cloned using Drosophila genes derived from both 
the Shaker, Shaw and Shab loci; Wei, A. et. al., Science (1990) Vol. 248 
pp. 599-603. 
Unlike the voltage-gated channel proteins with six membrane spanning 
regions, the inward rectifier channels have only two membrane spanning 
domains, each sensitive to changes in the net potassium concentration. 
Within this class of channels are the ATP-sensitive potassium channels. 
These channels are classified by their sensitivity to concentration fluxes 
in ATP. The ATP-sensitive, or ATP-gated, potassium channel is an important 
class of channels that links the bioenergetic situation of the cell to 
changes in cell function. These channels are blocked by high intracellular 
ATP concentrations and are open when ATP decreases. Lazdunski (1992); M. 
Lazdunski et al., "ATP-Sensitive K.sup.+ Channels", Renal Physiol. 
Biochem. Vol. 17: pp. 118-120 (1994). 
Although ATP-gated potassium channels were originally described in cardiac 
tissue; Noma, A. Nature (1983) Vol. 305 pp. 147-148, they have 
subsequently been described in pancreatic .beta.-cells; Cook et. al., 
Nature (1984) Vol. 311 pp. 271-273, vascular smooth muscle; Nelson, M. T. 
et. al., Am. J. Physiol. (1990) Vol. 259 pp. C3-C18 and in the thick 
ascending limb of the kidney; Wang, W. et. al. Am. J. Physiol. (1990) Vol. 
258, pp. F244-F-253. 
The ATP-sensitive, or ATP-gated potassium channels play an important role 
in human physiology. The ATP-sensitive potassium channel, like other 
potassium channels, selectively regulate the cell's permeability to 
potassium ions. These channels function to regulate the contraction and 
relaxation of the smooth muscle by opening or closing the channels in 
response to the modulation of receptors or potentials on the cell 
membrane. When ATP-sensitive potassium channels are opened, the increased 
permeability of the cell membrane allows more potassium ions to migrate 
outwardly so that the membrane potential shifts toward more negative 
values. When the membrane potential shifts toward more negative values the 
opening of the voltage-dependent calcium channels is reduced, this reduces 
the influx of calcium ions into the cell because the calcium channels 
become "increasingly less open" as the membrane potential becomes more 
negative. Consequently, drugs having ATP-sensitive potassium channel 
opening activity, drugs known as potassium channel openers, can relax 
vascular smooth muscle and are useful as hypotensive and coronary 
vasodilating agents. In contrast, drugs having ATP-sensitive potassium 
channel blocking activity, drugs known as potassium channel blockers, 
inhibit ATP-sensitive potassium channels by decreasing potassium efflux, 
leading to membrane depolarization which opens voltage-gated Ca.sup.2+ 
channels. Arkhammar et al. (1987) "Inhibition of ATP-regulated K.sup.+ 
channels precedes depolarization-induced increase in cytoplasmic free 
Ca.sup.2+ concentration in pancreatic B-cells", J. Biol. Chem. 262: 
5448-5454. These drugs find optimal use in the stimulation of insulin 
secretion in type II diabetes mellitus. 
A relatively large number of compounds are now known which open cell 
membrane ATP-sensitive potassium channels, particularly in smooth muscle: 
minoxidil sulfate, diazoxide and nicorandil are well known potassium 
channel openers. The target site for these agents is presumably on the 
potassium channel itself, but may also be on an associated regulatory 
protein. Isolation of the target site for the potassium channel openers 
would allow for protein sequence analysis and cloning of those potassium 
channel opener proteins. Similar analyses of drug binding proteins in 
K.sub.ATP channels have been performed for the class of K channel blockers 
such as glyburide. Sulfonylurea receptors have been analyzed on a variety 
of cell and tissue types using a photoactivable form of glyburide. 
Aguilar-Bryan, L., et al., "Photoaffinity Labeling and Partial 
Purification of the B Cell Sulfonylurea Receptor Using a Novel, 
Biologically Active Glyburide Analog", J. Biol. Chem. (May 15, 1990) Vol. 
265, pp. 8218-8224. 
Potassium channel openers represent a widely diverse series of compounds 
which all have the reported effect of opening only a subset of channels 
described as sensitive to ATP. As explained above, these compounds cause 
physiological responses by increasing membrane permeability to potassium, 
this leads to hyperpolarization of the cell membrane and temporal 
desensitization to electrical and chemical stimuli. 
Openers which target these channels have been synthesized as possible drugs 
in hypertension, angina pectoris, coronary heart disease, asthma, and 
urinary incontinence. Blockers which target these channels include the 
sulfonylureas, such as glyburide. The latter is an example of an important 
drug which targets K.sub.ATP channels in the pancreas, thus providing a 
treatment for non-insulin dependent diabetes mellitus. 
The rationale for the effectiveness of these drugs in targeting the 
K.sub.ATP channel resides in the fact that this channel constitutes the 
main resting conductance in the B-cell. Depolarization of the channel by 
the sulfonylurea blockers ultimately results in insulin release. 
Despite the apparent selectivity afforded by such drugs, it also appears 
true that openers have multiple effects on target cells as well as 
selective effects on several tissue types. K. Lawson and P. E. Hicks, 
"Potassium Channel Openers: Pharmacological Anomalies Suggest 
Heterogeneous Sites of Action", (1993) Curr. Opin. Invest. Drugs Vol 2 pp. 
1209-1216. It is the latter effect, that of multiple tissue targeting, 
that has reduced the importance of the K channel openers as selective 
marketable drugs. It is essential to understand what confers selectivity 
of drugs to specific organs before a systematic approach can be made 
towards drug design. 
The membrane proteins which bind to potassium channel openers are believed 
to be structurally related, although it isn't clear whether drug 
selectivity is imparted by the channel protein itself or by the 
contribution of accessory proteins. These proteins, which bind to 
selective drugs, may be novel K channels or they may be one of several K 
channel accessory proteins that act in concert with the primary K channel 
protein and that are needed by the system for the proper physiological 
response. 
An analogous system using the channel blocker, glyburide, has been explored 
for pancreatic B cell K.sub.ATP channels. Aguilar-Bryan, L., et al., 
"Co-Expression of Sulfonylurea Receptors and K.sub.ATP Channels in Hamster 
Insulinoma Tumor (HIT) Cells: Evidence for direct association of the 
receptor with the channel", J. Biol. Chem. (1992), Vol. 267 pp. 
14934-14940. 
This invention describes the isolation and identification of a new protein, 
p56, useful for the identification of selective drugs that will 
selectively open or close K channels. P56 is the first high affinity 
cyanoguanidine binding protein to be identified using a K channel opener 
photoactivable probe. Unexpectedly, this opener was shown to only bind to 
P56 in intact cells. 
SUMMARY OF THE INVENTION 
This invention comprises a glycoprotein of about 54,000 to 60,000 daltons 
and having an apparent core protein mass (free of sugars) of about 51,000 
daltons, capable of being isolated from rat A10 cells and capable of 
binding with 
N-(3-azido-5-iodophenyl)-N'-cyano-N"-(1,1-dimethylpropyl)-guanidine. A 
glycoprotein having K.sub.ATP channel activity either by itself or in 
membranes with other K.sub.ATP channel proteins. A glycoprotein of about 
56,000 daltons. A glycoprotein where the average mass of the individual 
sugars is about 2,500 daltons. A glycoprotein having at least three sites 
of N-linked glycosylation. A glycoprotein comprising the N terminal 
sequence of 
"Glu-Pro-Arg-Ala-Pro-Pro-Glu-Lys-Ile-Ala-Ile-Val-Gly-Ala-Gly-Ile-." A 
glycoprotein wherein the purified protein is the human homolog. A 
glycoprotein wherein the purified protein is the murine homolog. A 
glycoprotein having the characteristics of the p56 protein identified 
herein. An essentially pure glycoprotein as described herein.

ADDITIONAL DESCRIPTION OF THE INVENTION AND DESCRIPTION OF THE PREFERRED 
EMBODIMENTS 
The following definitions and equipment sources, are provided: 
Hour is h or hr. Minute is min. Milliliters is ml. DTT is dithiothreitol; 
it is purchased from any of several chemical suppliers. SDS is sodium 
dodecyl sulfate; the electrophoresis purity reagent supplier is Bio-Rad 
Laboratories, Richmond, Calif. All electrophoresis equipment was also 
purchased from the same vendor. NP40 is nonidet P40, a non-ionic detergent 
available from several chemical suppliers. HF is hydrogen 
fluoride/para-cresol (4-methylphenol) /para-thiocresol. 
All chromatography buffers and solvents were obtained from various chemical 
suppliers and were of the highest grade obtainable; all water used in 
experiments was purified using a Milli-Q water purification/filtration 
system obtained from Millipore Corporation. 
The YM10 or YM30 membranes used for ultrafiltration concentration were 
produced exclusively by Amicon, Danvers, Mass. X-OMAT AR scientific 
imaging films, are made by Eastman Kodak Company, Rochester, N.Y. 
Recombinant N-glycanase enzyme is obtained from Genzyme Corporation, 
Cambridge, Mass. The deglycosylation reactions were conducted as suggested 
by the manufacturer, who provide buyers a data sheet and suggested 
protocol. Peptide conjugation to KLH (Keyhole Limpet Hemocyanin), was 
conducted using the Inject Immunogen Kit obtained from Pierce, Rockford, 
Ill. 
Electrophoresis equipment was purchased from Bio-Rad Laboratories. (ABI) 
476A protein sequencer (Applied Biosystems, Inc., Foster City, Calif.). 
PVDF solid matrix is Immobilon-P Transfer Membrane obtained from Millipore 
Corporation, Bedford, Mass. SMART micropurification chromatography system 
(Pharmacia LKB Biotechnology, Uppsala, Sweden); chromatography columns 
used with this system are available through several manufacturers. 
Amino acid residues referred to in this application are listed below, they 
may also be given either three letter or single letter abbreviations, as 
follows: 
Alanine, Ala, A; Arginine, Arg, R; Asparagine, Asn, N; Aspartic acid, Asp, 
D; Cystein, Cys, C; Glutamine, Gln, Q; Glutamic Acid, Glu, E; Glycine, 
Gly, G; Histidine, His, H; Isoleucine, Ile, I; Leucine, Leu, L; Lysine, 
Lys, K; Methionine, Met, M; Phenylalanine, Phe, F; Proline, Pro, P; 
Serine, Ser, S; Threonine, Thr, T; Tryptophan, Trp, W; Tyrosine, Tyr, Y; 
Valine, Val, V; Aspartic acid or Asparagine, Asx, B; Glutamic acid or 
Glutamine, Glx, Z; Any amino acid, Xaa, X. 
All amino acids have a carboxyl group and an amino group. The amino group 
of the amino acid is also referred to as the "N-terminus" of the amino 
acid. The carboxyl group of an amino acid is also referred to as the 
"C-terminus" of the amino acid. The "N-terminus" of an amino acid may form 
a peptide bond with a carboxyl group of another compound. The carboxyl 
group that combines with the "N-terminus" of an amino acid may be the 
carboxyl group of another amino acid or it may be from another source. If 
several amino acids are linked into a polypeptide, then the polypeptide 
will have a "free" N-terminus and a "free" C-terminus. 
The materials and methods used to isolate, identify and characterize the 
protein are provided. 
Materials. The cyanoguanidine, 
N-(3-azido-5-iodophenyl)-N'-cyano-N"-(1,1-dimethylpropyl)-guanidine, a K 
channel opener utilized for photoaffinity labelling, is synthesized. The 
synthesis is according to the procedures described in U.S. Pat. No. 
5,525,742, issued Jun. 11, 1996, Gadwood, et. al., 
Azidophenylcyanoguanidine and Their Use as Photoaffinity Probes. The 
designated compound contained an aryl azide radiolabelled with .sup.125 
I! to a specific activity of 2200 Ci/mmole. (New England Nuclear). 
Electrophoresis. One-dimensional analytical SDS polyacrylamide gel 
electrophoresis is conducted using 10% gels in a mini Protean II system 
(Bio-Rad Laboratories) according to the method of Laemmli, U. K. Laemmli, 
(1970) Nature, Vol. 227, pp. 680-685, adjusted with minor modifications. 
Before electrophoresis samples are diluted 1:1 with denaturation buffer 
(2% SDS, 25% glycerol, 0.25 M Tris HCl, pH 6.8, and 1% 
.beta.-mercaptoethanol). Electrophoresis is conducted at constant power (5 
watts/gel) for 1 hour at room temperature and terminated when the dye 
front (bromphenol blue) reaches the bottom of the gel. The completed gels 
are either electroblotted onto nitrocellulose or PVDF, or fixed in 50% 
ethanol and 10% acetic acid, and stained with Coomassie Brilliant Blue 
G-250. 
Analysis of Radiolabelled Proteins (Assay Method). Radiolabelled fractions 
may be analyzed in several ways depending on the state of the sample. For 
solution samples, simply count a portion or all of the sample in a gamma 
counter (0.5 to 1.0 min per sample). For wet gels, incubate the gels in a 
-70.degree. C. freezer with XOMAT AR x-ray film, or cut the gel into 2 mm 
pieces and count the sections in a gamma counter. For dried gels or dried 
blot papers, a phosphorimager (Molecular Dynamics) for quantitation of the 
individual bands may be used. 
A10 Cell Growth, Photoaffinity Labelling, Membrane Preparation. A10 cell 
lines derived from embryonic rat aorta are obtained from the American Type 
Culture Collection (CRL-1476). The cells are subcultured and grown to 
confluence in Corning 150 mm tissue culture plates at 37.degree. C. in 
6.0% CO.sub.2 in Dulbecco's Modified Eagle's Medium supplemented with 20% 
(v/v) fetal bovine serum. The cells are then washed 2.times. with Earle's 
balanced salt solution buffered to pH 7.4 with 20 mM HEPES (EBSS-H). The 
cells are placed in EBSS-H containing 10 nM .sup.125 I!-U-97149. The A10 
cells are equilibrated with the radiolabeled compound for 15 min at 
37.degree. C. The A10 cells are then placed on ice for 2 min and exposed 
to 600 .mu.watts/cm.sup.2 of 254 nm UV light. 
After photolysis, the A10 cells are washed extensively with phosphate 
buffered saline. Membranes from the A10 cells are solubilized in a 
cocktail of 0.2% Triton X-100 detergent and 20 mM Tris pH 6.8 and protease 
inhibitors (10 .mu.g/ml leupeptin, 10 .mu.g/ml aprotinin, 10 .mu.g/ml 
pepstatin, and 5 mM benzamidine). The membranes are precipitated with 4 
volumes of ice cold acetone for 60 min on dry ice. The precipitated 
membrane proteins are collected by centrifugation at 20,000 rpm for 30 min 
at 4.degree. C. in a Beckman SW-28 rotor. After centrifugation the 
supernatant is discarded and the pellet is dissolved in 1% SDS and 10 mM 
.beta.ME. 
Purification of the protein p56. Preparative SDS PAGE--Triton solubilized 
.sup.125 I!-CG-labelled A10 membrane protein preparations from 24-48 
large culture plates are acetone precipitated and washed. The resulting 
pellets are resolubilized in 1% sodium dodecyl sulfate (SDS) containing 10 
mM dithiothreitol (DTT) (approximately 6-12 ml final volume) as described 
above. An equal volume of sample denaturation buffer is added (125 mM 
Tris, HCl pH 6.8, 1% SDS, 10 mM DTT), and after mild heating and mixing, 
the sample is distributed onto 4 preparative polyacrylamide gels (10% 
total; 37.5:1.0 acrylamide to bis-acrylamide ratio; 1.5 mm thickness). 
These gels are run according to the method of Laemmli with minor 
modifications in a Protean II cell at low voltage (25-30 V limited) 
overnight at room temperature. The upper tank buffer is supplemented with 
1 mM sodium thioglycollate to reduce possible protein modification due to 
oxidation or free radicals within the gel and to keep the proteins in 
reduced form during the run to prevent disulfide linked aggregation. 
Upon completion of electrophoresis, both the dye front, which consists 
mainly of free drug, and one of the major proteins, actin, are visualized 
by incubation of the completed gels in a solution of 0.1 M potassium 
chloride for about 5 min. Following brief washings in deionized water, the 
position of actin is marked and the bottom of the gel is cut to allow for 
recognition patterns on developing autoradiograms. Each gel is wrapped in 
saran wrap and sealed in plastic bags. Autoradiograms are made after 2-3 h 
exposure (-70.degree. C.) of the wet, frozen gels to X-OMAT AR films. 
These autoradiograms are then used as a guide for the excision of the 
target protein band; in this case, p56. The excised polyacrylamide gel 
strips are minced and fragmented using a mortar and pestle, and incubated 
in a large volume of 1% SDS containing 10 mM DTT (.about.500 ml). After a 
2 hour incubation with stirring, the solution is centrifuged at low speed 
in 50 ml tubes for 5 min. at room temperature. The supernatant is removed 
and poured through a 0.22 micron filter to remove remaining polyacrylamide 
fines. The resulting solution is concentrated by Amicon ultrafiltration 
(YM-30 membrane) to a final volume between 6 and 12 ml. 
Reverse Phase Chromatography. The concentrate from the above preparative 
SDS PAGE extract of p56 is injected onto a 0.46.times.15 cm (10.mu.) 
biphenyl HPLC column. The column profile is developed with a gradient of 
32% to 54.4% acetonitrile in 0.1% triflouroacetic acid over a period of 40 
min at a flow rate of 1 ml/min (1 ml fractions are collected). A 
radioactivity profile is obtained of individual fractions by gamma 
counting at 0.5 min each for .sup.125 I!. Appropriate fractions 
representing p56 as judged by SDS PAGE and blotting, followed by 
phosphorimaging to conclusively show the location of p56 in the resolved 
fractions, are pooled and dried by vacuum centrifugation (SpeedVac 
Evaporator/Concentrator System, Savant Instruments, Inc., Farmingdale, 
N.Y.). 
Deglycosylation of p56. The biphenyl reverse phase HPLC purified p56 sample 
is redissolved in minimal 1% SDS, and heated in boiling water for 2 min. 
After heating, the sample is treated with the non-ionic detergent, NP-40, 
such that for each 1% of SDS, a minimum of 1.5% of NP-40 is added. This 
solution is incubated in sodium phosphate, pH 7.0, and is mixed with 
N-glycanase (Genzyme) as suggested by the manufacturer. The 
deglycosylation is allowed to proceed overnight at 37.degree. C. 
Microbore (SMART) HPLC. In some instances, samples after deglycosylation 
are further resolved on a microbore biphenyl column (2.7 mm.times.15 cm) 
on a SMART system (Pharmacia-LKB) at 100 .mu.l/min. 
Electroblotting. Transferral of proteins from SDS gel to Immobilon-P 
Transfer Membrane (PVDF; Millipore Corp., Bedford, Mass.) is performed 
with a semi-dry blotter at .about.15 mA/cm.sup.2 for 15 min. 
Sequence analysis. N-terminal sequencing of the deglycosylated p56 protein 
electrophoretically transferred to PVDF, see, P. Matsudaira (1987) J. 
Biol. Chem. Vol. 262 pp. 10035-10038, after purification by SDS-PAGE, is 
performed on an Applied Biosystems Inc. (ABI) Model 476A protein 
sequencer. 
Synthesis of the N-Terminal Peptide. Solid phase peptide synthesis (Barany 
& Merrifield, 1979, in The Peptides, Vol. 2, pp. 1-284, E. Gross and J. 
Meienhofer, editors, Academic Press, New York) is performed at 0.5 mmole 
scale utilizing Boc-Cys(4-CH.sub.3 Bzl)OCH.sub.2 Pam resin (Applied 
Biosystems Inc., Foster City, Calif.) on an Applied Biosystems Inc. 430A 
Peptide Synthesizer. Amino acids may be obtained from any commercially 
available source. In the examples shown here all amino acids were obtained 
from Applied Biosystems Inc. The t-butyloxycarbonyl (BOC) group is used as 
the N -amino protecting group during step-wise synthesis. Tri-functional 
amino acid side chains are protected as follows: Arg(Tos), Glu(OBzl), and 
Lys(Cl-z). Each residue is coupled twice, then capped with acetic 
anhydride before the next cycle of synthesis. Quantitative ninhydrin tests 
are performed at each cycle of the synthesis. After removing the 
N-terminal Boc group in the usual fashion, the peptide is cleaved from the 
resin by treatment with Hydrofluoric acid 
(HF)/.rho.-cresol/.rho.-thiocresol (10:0.5:0.5) for 1 hour at -20.degree. 
to -5.degree. C. 
The peptide resin is titrated with ether, the crude peptide dissolved in 
50% acetic acid and the resin removed by filtration. The filtrate is 
evaporated to dryness under reduced pressure and lyophilized from glacial 
acetic acid. The crude peptide is purified by preparative reverse phase 
chromatography on a Vydac C-18 column (250.times.22.5 mm) using a water 
acetonitrile gradient, with each phase containing 0.1% TFA. Clean 
fractions, as determined by analytical HPLC, are pooled and acetonitrile 
evaporated under reduced pressure; an aqueous solution of the pooled 
fractions is lyophilized. The purified peptide is characterized by time of 
flight mass spectroscopy. The anticipated (M+H)+ is 1878.9. 
Preparation of N-Terminal p56 Polyclonal Antibody. The N-terminal peptide, 
prepared above, is conjugated to KLH-maleimide (Pierce Chemical Co.), 
using procedures supplied by the manufacturer, to form the KLH-peptide 
conjugate at a final concentration of 4.0 mg/ml (KLH) and 2.7 mg/ml 
(peptide). Verification of coupling is made using Ellman's reagent. The 
KLH-peptide conjugate is separated from the free peptide by dialysis 
versus 1.times. PBS, pH 7.5. At the same time, a peptide conjugated to 
ovalbumin is prepared in an identical manner to provide for a sample which 
could be used to screen the test and production bleeds as they are 
produced. For the latter, the peptide-ovalbumin conjugate exhibited an 
apparent molecular weight of 60-65,000, compared to 45,000 for the 
unconjugated ovalbumin protein. 
To test whether the rabbit sera is immunoreactive with the peptide, Western 
blots are conducted on nitrocellulose strips containing the 
ovalbumin-peptide conjugates at various concentrations. The primary sera 
are tested at a 1:100 dilution in TN buffer (20 mM Tris HCl, pH 7.5, 0.5 M 
NaCl) containing 1% BSA as carrier. The secondary antibody consists of 
dilute solutions of alkaline phosphatase-conjugates of goat-anti-rabbit 
IgG in TN buffer, supplemented with 1% BSA as carrier. Positives on the 
blots are visualized using AP substrates, NBT (p-nitroblue tetrazolium 
chloride) and BCIP (5-bromo-4-chloro-3-indolyl phosphate). To test whether 
the sera are capable of detecting p56 on blots, subject crude, partially 
purified and purified p56 samples to SDS polyacrylamide gel 
electrophoresis (10%), and blot onto nitrocellulose, as defined above. The 
blots are then probed using primary sera which had either been 
pre-adsorbed with the free N-terminal synthetic peptide or left 
unchallenged. 
The following documents, incorporated by reference, also contain useful 
methods generally known to one skilled in the art: U. K. Laemmli (1970) 
Nature 227:680-685 and P. Matsudaira (1987) J. Biol. Chem. 
262:10035-10038. 
UTILITY OF THE INVENTION 
This invention describes the isolation and identification of a new protein, 
p56, useful for the identification of drugs that will selectively open or 
close K channels. The p56 protein is the first high affinity 
cyanoguanidine binding protein to be identified using a K channel opener 
photoactivable probe. Unexpectedly, this opener was shown to only bind to 
p56 in intact cells, supporting the role of this protein in native 
potassium channel activity. 
The p56 protein is likely to be a K.sub.ATP channel or an accessory protein 
that regulates K.sub.ATP channel activity. As an accessory protein in the 
channel, it would likely impart selectivity and specificity towards 
binding of potassium channel directed drug molecules. In either case, as a 
channel or accessory to a channel, p56 is an important and novel drug 
target. 
The identification of a larger portion of the amino acid sequence will lead 
to the design of oligonucleotide probes which will permit the cloning of 
p56 from various species and expression of the p56 protein in bacterial 
and mammalian cell systems. An analogous approach has been taken by Bryan 
et al. in the isolation, characterization, and cloning of the glyburide 
receptor in HIT cells. Bryan, J., Aguilar-Bryan, L., and Nelson, D., 
"Cloning of a Sulfonylurea Receptor (ATP-Sensitive K.sup.+ Channel ?)" 
from Rodent a- and B- Cells, First International Conference on 
ATP-Sensitive K.sup.+ Channels and Sulfonylurea Receptors (Sep. 30-Oct. 1, 
1993), Houston, Tex., pp. 149-153. 
Knowledge of the amino acid sequence for p56 will allow the design of 
appropriate oligonucleotide probes for determination of mRNA levels in 
cell and tissue preparations using in situ hybridization experiments. 
Knowledge of the sequence will also allow the examination of the structure 
of the protein by computational software programs, providing a direct 
method for primary and secondary structure comparison of p56 to known 
potassium channel proteins. 
The identification of this protein will allow the design of higher affinity 
polyclonal antibodies and/or monoclonal antibodies to be developed that 
recognize p56 in different species. Such antibodies will allow cell and 
tissue distribution of the protein to be determined. Antibodies to the p56 
protein will allow for immunocytochemistry and histological examination of 
p56 protein expression in cells and tissue sections to complement the 
analysis of mRNA levels by Northern blot analysis. 
This system can be used to study how potassium channel openers and blockers 
interact with the channel complex by competition studies, and to study and 
identify the other members of the potassium channel complex. 
Additional understanding of the utility of the invention can be found in 
the following documents, incorporated by reference: U. Quast (1993) "Do 
the K+ Channel Openers Relax Smooth Muscle by Opening K+ Channels ?", 
Trends In Pharmaceutical Sciences 14:332-337. U. Quast, K. M. Bray, H. 
Andres, P. W. Manley, Y. Baumlin, and J. Dosogne (1993) Binding of the K+ 
Channel Opener, .sup.3 H! P1075 in rat isolated aorta: Relationship to 
functional effects of openers and blockers. Molecular Pharmacology 
43:474-481. D. R. Howlett and S. D. Longman (1992) Identification of a 
binding site for .sup.3 H! cromakalim in vascular and bronchial smooth 
muscle cells. British J. Pharmacol. 107:396P. Barany, Merrifield (1979) in 
The Peptides (Gross, E., and Meienhofer, J., eds.), Vol. 2, pp.1-284, 
Academic press, New York. 
DETAILED DESCRIPTION OF THE INVENTION 
The present inventors have isolated and purified a unique protein called 
p56. This protein is either a true K channel protein or an accessory 
protein which might confer selectivity to a given channel. The N-terminus 
of the protein has been determined and a peptide representing the 
N-terminus of the N-deglycosylated p56 was synthesized. Polyclonal 
antibodies to the peptide were created which immunoreact with both the 
free peptide as well as authentic p56 protein. 
Radiochemically labelled and photoactivable K channel openers are used to 
identify cyanoguanidine binding proteins in a membrane preparation of A10 
cells. One suitable probe is 
N-(3-azido-5-iodophenyl)-N'-cyano-N"-(1,1-dimethylpropyl)-guanidine, see 
formula 1, below. 
##STR1## 
The proteins in the A10 cell preparation are first photolabelled, and then 
the cells are extracted with a cocktail of 0.2% Triton X-100 detergent and 
20 mM Tris HCl, pH 6.8, containing protease inhibitors (10 ug/ml 
leupeptin, 10 ug/ml aprotinin, 10 ug/ml pepstatin, and 5 mM benzamidine). 
A cold solution of acetone is added to the Triton extract to precipitate 
the proteins, and allow for the removal of the unreacted photoaffinity 
label in the supernatant. Proteins present in the acetone pellet, 
including those radiolabelled proteins which were photolabelled, were then 
extracted with 1% sodium dodecyl sulfate (SDS) containing 10 mM 
dithiothreitol or 10 mM 2-mercaptoethanol. 
Preliminary studies of the protein, p56, showed that the solubility of the 
protein was lost in the absence of SDS. However, use of SDS solutions of 
crude membrane proteins was found ineffective for purification using 
several reverse phase columns (C4 or C18). A common problem was the 
resolution of entire micellar products containing several different sized 
proteins, without significant purification afforded. To avoid these 
problems, we chose a biphenyl HPLC chromatography step, since this column 
accommodates solutions of 1% SDS, allows for resolution of SDS-dissolved 
proteins, and doesn't result in broadening effects noted in C4 and C18 
column profiles. 
Therefore, since the presence of SDS in the sample could be tolerated prior 
to HPLC resolution, we preferred to first select for the size range of 
proteins which are approximately 56 kilodaltons by preparative SDS PAGE as 
described in the Methods section. This method effectively removes the 
contaminating radiolabelled proteins which are either lower or higher 
molecular weight than p56. A resulting autoradiogram of a typical wet gel 
after 2 hours incubation with X ray film at -70.degree. C. shows a complex 
pattern of radiolabelled proteins, with a major labelled protein easily 
detected at 56,000 daltons, see FIG. 1. The Y-axis of this illustration 
depicts the molecular weight, with the bottom representing the location 
where the smallest proteins migrate and the top representing the location 
where the largest proteins migrate (the relationship of migration distance 
to molecular weight is a logarithmic function). For these studies we 
commonly utilized 10% polyacrylamide (37.5:1.0 acrylamide:bis-acrylamide), 
resulting in an effective range of separation of proteins having masses 
from 15 kilodaltons through 200 kilodaltons. 
The relative molecular weight of p56 was determined by comparison of its 
migration to that of a series of proteins having known masses. The 
predicted mass is determined following a linear regression analysis of the 
migration distances of the known standard proteins versus their molecular 
masses. When the mass of p56 is calculated by this method, an average mass 
of 56 kD is measured, with variability of the measurement limiting the 
size from 54 to 60 kD. 
FIG. 1 shows that there are several radiolabelled bands present on the gel 
making it difficult to discern specific from non-specific radiolabelling. 
However, we know that the p56 band is specific since excess cold drug 
competition results in loss of detectable radioactivity at this position 
(data not shown). The major band noted at 56,000 daltons on the gel (as 
located by the autoradiogram) was excised and extracted out by passive 
diffusion in a 1% solution of sodium dodecyl sulfate containing 10 mM 
dithiothreitol. The resulting solution was concentrated to a small volumes 
(less than 8 ml) by ultrafiltration (Amicon Corporation, Lexington, 
Mass.). 
A determination of whether p56 is a glycoprotein was made by digesting the 
SDS extracted protein solution with N-glycanase, endoglycosidase H, 
endoglycosidase F, and a combination treatment of neuraminidase and 
O-glycanase. The results of experiments with a partially purified 
preparation of .sup.125 I! p56 using these enzymes are shown in FIG. 2. 
FIG. 2 shows the results of digestion of p56 (left to right) with: 
N-glycanase (Lane 1), O-glycanase+Neuraminidase (Lane 2), Endo H (Lane 3), 
Endo F (Lane 4), and Control (Lane 5). 
Results show that p56 is sensitive only to Endo H and N-glycanase (as 
judged by a demonstrated change in migration of the radiolabelled band on 
the gel), suggesting that the protein contains one or more sites of 
N-linked glycosylation of a high mannose type. The negative results with 
neuraminidase and O-glycanase suggested no O-linked glycosylation or 
sialic acid residues present in the glycoprotein. 
The deglycosylation pattern of p56 was examined with variable 
concentrations of N-glycanase. Analysis indicates that in addition to p56 
and its fully deglycosylated product (p52), there are at least two 
intermediate forms of glycosylated p56. We thus conclude that there are at 
least three sites of N-linked glycosylation on the p56 protein isolated 
from A10 membranes. 
Purification and Characterization of p56. 
Starting with a concentrate of the sodium dodecyl sulfate extraction of p56 
from gel slices, radiolabelled as a consequence of reacting with the 
photoactivatable K channel opener, as explained above, the sample is 
applied onto a biphenyl reverse phase column. 
A typical column profile is shown in FIGS. 3a and 3B. FIG. 3A shows the 
radioactivity profile (X axis is retention time as column fractions; Y 
axis=CPM), and the profile of the absorbance of the sample is shown in 
FIG. 3B (X axis=Retention Time; Y axis=Absorbance at 215 nm A.sub.215 !). 
The latter absorbance represents the most sensitive region of the 
absorption profile of proteins, indicative of peptide bonds. An 
examination of the selected fractions from this step by SDS PAGE is shown 
in FIGS. 4A and 4B. FIG. 4A shows the stained polyacrylamide gel, while an 
autoradiogram of the same gel is shown in FIG. 3B. The data shows the 
protein and radiochemical and protein purity, respectively, at this stage 
of the purification process. The p56 protein, defined by radioactivity and 
size, is noted in fractions 33-41. Appropriate fractions are selected and 
dried by vacuum centrifugation. The dried sample is redissolved in SDS 
containing buffer and subjected to deglycosylation with N-glycanase. The 
product of this step results in a p56 protein which is now devoid of 
N-linked oligosaccharides. 
The pool is then subjected to microbore biphenyl reverse phase HPLC (Vydac) 
to resolve the deglycosylated p56 from N-glycanase and other contaminating 
A10 proteins, as shown in FIG. 5. FIG. 5 shows the absorbance (unshaded) 
and the radioactivity (shaded) of the fractions (Y axis) versus the 
retention time (X axis) resulting from this final HPLC resolution step. 
This figure shows that additional resolution of p56 is obtained since the 
net radioactivity (fractions 25-32, shaded plot) is resolved from the 
major contaminating proteins (depicted by absorbance at 215 nm, unshaded 
plot). Individual fractions containing deglycosylated p56 (same figure, 
fractions 25-32) were subjected to SDS PAGE and blotted onto PVDF. The 
results of this experiment are shown following staining the proteins on 
the blot with Coomassie Brilliant Blue R-250, see FIG. 6. The arrow in the 
figure depicts the location of deglycosylated p56, whose identity was 
confirmed by detection of radioactivity using phosphorimaging (data not 
shown). To prepare the segment of the blot containing deglycosylated p56 
for microsequencing, the section representing the radiolabelled band was 
cut out of the PVDF paper. To verify that the band was indeed excised 
correctly, the remaining PVDF paper was reanalyzed by phosphorimaging to 
confirm that the radioactive band had indeed been selected precisely. 
N-Terminal Sequence Analysis of p56. 
Following deglycosylation and PVDF blotting, see P. Matsudaira (1987) J. 
Biol. Chem. Vol. 262, pp. 10035--10038, a peptide sequence was obtained 
(Glu-Pro-Arg-Ala-Pro-Pro-Glu-Lys-Ile-Ala-Ile-Val-Gly-Ala-Gly-Ile-) SEQ. 
ID. NO. 1 for a sample that was clearly in the picomolar range, see Table 
I below for yields per sequencing cycle. Thus, the amount of protein which 
was sequenced is estimated at 0.68 picomoles (from a minimum of 48 plates 
of A10 cells) based on the first cycle of sequencing. For a protein having 
a molecular mass of 52,000 daltons, this represents a yield of 0.7 
nanograms from each plate of A10 cells. This value for the protein yield 
is based on the accumulation of all purification steps, using as an assay 
the radiolabelled p56 protein, for which an efficiency of labelling by the 
photoactivatable cyanoguanidine was estimated at 0.05%. The yield, then, 
does not necessarily represent the actual amount of p56 expressed in A10 
cells. 
The peptide sequence obtained is not only the putative amino terminal of 
p56, but also a "unique" sequence, not observed in protein sequence 
databases. In a general search of proteins showing identity to the p56 
N-terminal peptide, no homology was noted to any mammalian potassium 
channel protein. A polyclonal antibody against the N-terminus of the 
protein was created to verify the conclusion that the sequence of the 
polypeptide was the same as the sequence of the N-terminus of the protein. 
Table I, next page, 
TABLE I 
______________________________________ 
N-Terminal Sequencing of Deglycosylated p56 
Residue # Amino Acid 
Quantity (pmoles) 
______________________________________ 
1 Glu 0.68 
2 Pro 0.39 
3 Arg 1.50 
4 Ala 1.40 
5 Pro 1.46 
6 Pro 1.54 
7 Glu 0.93 
8 Lys 0.46 
9 Ile 1.06 
10 Ala 1.29 
11 Ile 1.14 
12 Val 1.60 
13 Gly 1.13 
14 Ala 1.11 
15 Gly 1.02 
16 Ile 0.62 
______________________________________ 
Development of a Polyclonal Antibody to the N-Terminus of p56. 
The peptide representing the N-terminus of the N-deglycosylated p56 was 
synthesized, EPRAPPEKIAIVGGC SEQ. ID. NO. 2 (see Formula 2 below), 
terminal GGC added to aid conjugation to KLH, the sequence without the 
terminal GGC is SEQ. ID. NO. 3, and used for immunization of a single 
rabbit. 
EQU H-Glu-Pro-Arg-Ala-Pro-Pro-Glu-Lys-Ile-Ala-Ile-Val-Gly-Gly-Cys-OH 
EQU .circle-solid.3CF.sub.3 --COOH Formula 2 
The first antigen dose was administered subcutaneously with Freunds 
complete adjuvant. After three weeks, an observable titer against the 
peptide (as measured by peptide conjugated to ovalbumin) was detected, 
although no response to the p56 protein was noted by Western blotting at 
any dilution of the serum. At this point, the antigen was administered 
subcutaneously with Freunds incomplete adjuvant, a process continued for 
at least half a year. 
After the second bleed (6 weeks), a response was detected against p56 on 
Western blots (using partially purified as well as crude lysate material). 
Subsequently, all production bleeds of this single rabbit have yielded 
antibody with high titers against rat A10 p56. We have termed this serum 
antibody, which is defined as containing one or more IgG's specific for 
the N-terminal 12 amino acids of rat A10 p56, UP76. 
Importantly, the bands immunodetected on blots were exactly coincident with 
the radioactivity profile noted by phosphorimaging. To demonstrate that 
the peptide sequence was derived from p56, purified samples of p56 were 
deglycosylated with N-glycanase and analyzed by Western blotting. 
Conclusively, both before and after deglycosylation, the radiolabelled 
protein was exactly coincident with the immunodetected band. This 
observation eliminated the possibility that the sequence obtained was from 
a 50-52 kD contaminating protein rather than from p56 itself. 
To verify that the bands detected on Western blots were not due to a 
non-specific binding phenomenon, the response on blots was blocked by 
pre-incubation of the serum with the peptide as shown in FIG. 7. This 
Figure shows two Western blots of gels containing resolved 
photoaffinity-labelled crude A10 proteins. The left figure shows the 
results of probing one of the two duplicate blots with the anti-p56 serum. 
In this result one detects the presence of bands at 56 kD, at 30 kD, and 
at 35 kD. The right figure shows the results of probing one of the blots 
with peptide-competed anti-p56 serum. An analysis of the latter blot 
probed with peptide pre-adsorbed serum shows that the 56 and 30 kD bands 
are definitively peptide competed (since they are no longer detected on 
the blot), and thus contain the sequence shown in Formula 2. The band at 
35 kD is not related to p56 since it is not competed by peptide (Formula 
2), and is therefore the result of a non-specific interaction of the 
antibody with this unknown antigen. The above data indicate that the band 
at 30 kD, which contains an epitope which is competed with peptide 
(Formula 2), probably originated either through proteolytic truncation of 
p56 or as a separate gene product. 
The N-terminus of p56 is species-specific since no cross-reactivity is 
observed, using the rabbit anti-rat p56 antibody defined above, versus 
murine p56. A murine cell line derived from brain smooth muscle was 
labelled with the photoprobe. When the solubilized membrane pool was 
examined by Western blotting, no band was observed at p56. Similarly, 
following dissection of various tissues from a mouse, Western blotting was 
used to locate p56. Again, no signal was detected for non-rat p56 samples. 
In an analogous manner, COS cell extracts also failed to exhibit a p56 
protein which cross-reacts with the UP76 antibody. However, photolabelling 
of murine brain smooth muscle cells with 
N-(3-azido-5-iodophenyl)-N'-cyano-N"-(1,1-dimethylpropyl)-guanidine 
indicated the presence of a radiolabelled band in the p56 area. This 
suggests the N-terminus of p56 is species-specific as reflected by Western 
blotting with UP76, but that p56 is probably present in other species as 
suggested by the results of photoaffinity labelling. 
The p56 protein is present in the kidney, brain, trachea, and pancreas of 
rats. Utilizing the UP76 polyclonal antibody, described above, the 
extracts of various tissues from a dissected rat were examined by Western 
blotting. Both the soluble pools as well as the membrane pools (detergent 
solubilized) were used for these experiments. By Western blotting, bands 
at 56 kD and 30 kD were observed for pancreas, brain, trachea and kidney, 
each of which was eliminated when the serum had been preadsorbed with the 
competing peptide, shown in Formula 2. 
These data demonstrate, for the first time, the presence of p56 in tissues 
other than aortic smooth muscle. The presence of p56 in these tissues was 
also established by examination of the sensitivity of the p56 bands to 
deglycosylation with N-glycanase. In all cases, a shift from p56 to p52 
was observed as expected and as noted for authentic p56 from A10 cell 
membranes. Immunoreactive p56 was also observed in commercially obtained 
frozen rat kidney and brain tissue extracts. 
We have also purified the specific IgG fraction from the crude serum of 
UP76, the rabbit anti-rat p56 antibody using an immobilized form of the 
peptide antigen, (see Formula 2). Following binding of the total IgG pool 
to Protein A-Agarose, the IgG was eluted by dissociation at low pH. 
Following adjustment of the pH to neutrality, the IgG pool was incubated 
with an immobilized form of the peptide (formula 2). Following extensive 
washing to remove undesired IgG proteins, the specific peptide-binding IgG 
was eluted by low pH dissociation, neutralized to pH 7.5, and concentrated 
by ultrafiltration (Amicon). This specific antibody is being used for 
expression cloning attempts and to determine tissue and cell specificity. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 3 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 16 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: N-terminal 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- Glu Pro Arg Ala Pro Pro Glu Lys Ile Ala Il - #e Val Gly Ala Gly Ile 
# 15 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 15 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: N-terminal 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- Glu Pro Arg Ala Pro Pro Glu Lys Ile Ala Il - #e Val Gly Gly Cys 
# 15 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 12 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (v) FRAGMENT TYPE: N-terminal 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- Glu Pro Arg Ala Pro Pro Glu Lys Ile Ala Il - #e Val 
# 10 
__________________________________________________________________________