DNA encoding ATP-sensitive potassium channel genes

This invention relates to DNA and protein compositions useful in the diagnosis and treatment of diabetes, heart disease and skeletal muscle disease. More specifically, this invention relates to DNA and protein compositions for ATP-sensitive potassium channel proteins, and methods of using these compositions.

FIELD OF THE INVENTION 
This invention relates to DNA and protein compositions useful in the 
diagnosis and treatment of diabetes, heart disease and skeletal muscle 
disease. More specifically, this invention relates to DNA and protein 
compositions for ATP-sensitive potassium channel proteins, and methods of 
using these compositions. 
BACKGROUND OF THE INVENTION 
The concentration of potassium ions is maintained at a relatively high 
concentration intracellularly, primarily by the action of a 
sodium-potassium pump present in the cell membrane. The transport of 
potassium across the cell membrane is also regulated by a variety of 
potassium channel proteins which are present in the cell membranes of 
various tissues. One type of potassium channel is inhibited by ATP and has 
been termed the ATP-sensitive potassium channel. (See Ashcroft, S. M. 
(1988) Ann Rev. Neurosci. 11:97-118 and Edwards, G., et al. (1993) Ann. 
Rev. Pharmacol. Toxicol. 33:597-637 for a description of ATP-sensitive 
potassium ion channels.) 
ATP-sensitive potassium channels are inhibited by ATP. The physiology, 
pharmacology, and tissue distribution of the ATP-sensitive potassium 
channels has been extensively studied by the membrane patch-clamp 
technique (see Ashcroft, S. M., supra). Potassium channels are known to be 
present in cardiac and skeletal muscle as well as in the insulin-secreting 
.beta.-cells of the pancreas. In addition, there is evidence that 
ATP-sensitive potassium channels are also present in smooth muscle and in 
neurons. 
The ATP-sensitive potassium channel has important physiological functions 
in the pancreas. The ATP-sensitive potassium channel plays a key role in 
mediating glucose-stimulated insulin release from pancreatic .beta.-cells. 
Modulation of the pancreatic ATP-sensitive potassium channel is also 
important in treatment of diabetes. For example, sulfonylurea drugs, such 
as glyburide, that are used in the treatment of non-insulin dependent 
diabetes are known to stimulate insulin secretion by inhibiting the 
opening of the ATP-sensitive potassium channel. 
The ATP-sensitive potassium channel is also important in the physiology and 
pathophysiology of the heart. For instance, activation of the 
ATP-sensitive potassium channel in anoxia appears to be responsible for 
shortening the ventricular action potential and reducing heart muscle 
contraction. Activation of the potassium channel also increases the 
threshold for electrical excitation thereby slowing pacemaker activity. 
The ATP-sensitive potassium channel appears to be the target for drugs 
used as potassium channel openers in heart muscle. 
In addition to its role in cardiac muscle, the ATP-sensitive potassium 
channel is also involved in regulation of potassium ion transport in 
skeletal muscle. Potassium channel openers that target the ATP-sensitive 
potassium channel may be useful in skeletal muscle diseases such as 
myotonia congentia and hyperkalemic paralysis (see Edwards, G., et al., 
supra). 
Many of the potential uses of ATP-sensitive potassium channel proteins 
require isolation of the proteins or isolation of DNA encoding the 
proteins. The sequence of the potassium channel proteins and the genes 
encoding them have not been described in the prior art. Isolation of 
ATP-sensitive potassium channel proteins and DNA encoding these proteins 
facilitates the design and selection of improved potassium channel 
inhibitors and potassium channel openers useful in treatment of diabetes, 
heart disease, and skeletal muscle disease. Isolation of these proteins 
and genes also allows for development of in vitro diagnostic methods for 
detection and diagnosis of disorders involving the ATP-sensitive potassium 
channel. These and other needs are addressed by the present invention. 
SUMMARY OF THE INVENTION 
The present invention provides for isolated ATP-sensitive potassium channel 
proteins. These proteins specifically bind to antibodies generated against 
an immunogen which is a protein of Seq. ID No. 2. Preferably, these 
ATP-sensitive potassium channel proteins are of human origin. An example 
of a human ATP-sensitive potassium channel protein is the protein of Seq. 
ID No. 2. The ATP-sensitive potassium channel proteins may also be of 
non-human origin, for example, of rat origin. An example of a rat 
ATP-sensitive potassium channel protein is the protein of Seq. ID No. 4. 
The ATP-sensitive potassium channel proteins can be recombinantly produced 
and can be full-length. 
In addition to providing for ATP-sensitive potassium channel proteins, the 
present invention also provides for isolated nucleic acids encoding these 
proteins. Thus, the invention provides for nucleic acids which encode the 
ATP-sensitive potassium channel proteins described above. These nucleic 
acids can selectively hybridize to a nucleic acid encoding a human heart 
ATP-sensitive potassium protein of Seq. ID No. 1 in the presence of a 
genomic library under hybridization wash conditions of 50% formamide at 
42.degree. C. Preferably these nucleic acids are of human origin. An 
example of a nucleic acid encoding a human ATP-sensitive potassium channel 
protein is the nucleic acid of Seq. ID No. 1. These nucleic acids can also 
be of non-human origin, for example, of rat origin. An example of a 
nucleic acid encoding a rat ATP-sensitive potassium channel protein is the 
nucleic acid of Seq. ID No. 3. 
The invention further provides for host cells stably transfected with 
nucleic acids that encode ATP-sensitive potassium channel proteins. For 
example, host cells may be transfected with a nucleic acid of Seq. ID No. 
1 or Seq. ID No. 3. 
In addition to providing for host cells stably transfected with nucleic 
acids encoding ATP-sensitive potassium channel proteins, this invention 
also uses these transfected host cells to detect compounds that are 
capable of inhibiting or that are capable of accelerating the movement of 
potassium through ATP-sensitive potassium channels in the cell membrane. 
In these methods, the electrical potential is measured across a cell 
membrane of the transfected host cell. Preferably, the transfected host 
cell is a eukaryotic cell. Examples of such cells are HEK293 and BHK21 
cells. An example of a compound that is detected in this method is 
pinacidil. 
The invention further provides for nucleic acid probes that are capable of 
selectively hybridizing to a nucleic acid encoding an ATP-sensitive 
potassium channel protein. For example, the nucleic acid probe can be the 
nucleic acid of Seq. ID No. 1 or the nucleic acid of Seq. ID No. 3. As an 
additional example, the nucleic acid probe can be capable of hybridizing 
to a nucleic acid encoding the protein of Seq. ID No. 2 or Seq. ID No. 4. 
These nucleic acid probes can be used to measure or detect nucleic acids 
encoding ATP-sensitive potassium channel proteins. The probes are 
incubated with a biological sample to form a hybrid of the probe with 
complementary nucleic acid sequences present in the sample. The extent of 
hybridization of the probe to these complementary nucleic acid sequences 
is then determined. Preferably the biological sample is human. 
The invention further provides for antibodies specifically immunoreactive 
with the protein of Seq. ID No. 2. Methods of measuring or detecting 
ATP-sensitive potassium channel proteins and antibodies reactive with 
these proteins are also provided. ATP-sensitive potassium channel proteins 
can be detected by incubating a biological sample with a binding agent 
having an affinity for these proteins to form a binding 
agent:ATP-sensitive potassium channel protein complex and detecting the 
complex. Preferably, the binding agent is an antibody and the biological 
sample is human. 
Antibodies reactive to ATP-sensitive potassium channel proteins present in 
biological samples can be detected by incubating a recombinant or isolated 
ATP-sensitive potassium channel protein with a biological sample to form 
an antibody:ATP-sensitive potassium channel protein complex, and detecting 
the complex. Preferably, the biological sample is human. 
DEFINITIONS 
Abbreviations for the twenty naturally occurring amino acids follow 
conventional usage. In the polypeptide notation used herein, the left-hand 
direction is the amino terminal direction and the right-hand direction is 
the carboxy-terminal direction, in accordance with standard usage and 
convention. Similarly, unless specified otherwise, the left hand end of 
single-stranded polynucleotide sequences is the 5' end; the left hand 
direction of double-stranded polynucleotide sequences is referred to as 
the 5' direction. The direction of 5' to 3' addition of nascent RNA 
transcripts is referred to as the transcription direction; sequence 
regions on the DNA strand having the same sequence as the RNA and which 
are 5' to the 5' end of the RNA transcript are referred to as "upstream 
sequences"; sequence regions on the DNA strand having the same sequence as 
the RNA and which are 3' to the 3' end of the RNA transcript are referred 
to as "downstream sequences". 
The term "nucleic acids", as used herein, refers to either DNA or RNA. 
"Nucleic acid sequence" or "polynucleotide sequence" refers to a single- 
or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases 
read from the 5' to the 3' end. It includes both self-replicating 
plasmids, infectious polymers of DNA or RNA and nonfunctional DNA or RNA. 
"Nucleic acid probes" may be DNA or RNA fragments. DNA fragments can be 
prepared, for example, by digesting plasmid DNA, or by use of PCR, or 
synthesized by either the phosphoramidite method described by Beaucage and 
Carruthers, Tetrahedron Lett. 22:1859-1862 (1981), or by the triester 
method according to Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), 
both incorporated herein by reference. A double stranded fragment may then 
be obtained, if desired, by annealing the chemically synthesized single 
strands together under appropriate conditions or by synthesizing the 
complementary strand using DNA polymerase with an appropriate primer 
sequence. Where a specific sequence for a nucleic acid probe is given, it 
is understood that the complementary strand is also identified and 
included. The complementary strand will work equally well in situations 
where the target is a double-stranded nucleic acid. 
The phrase "selectively hybridizing to" refers to a nucleic acid probe that 
hybridizes, duplexes or binds only to a particular target DNA or RNA 
sequence when the target sequences are present in a preparation of total 
cellular DNA or RNA. "Complementary" or "target" nucleic acid sequences 
refer to those nucleic acid sequences which selectively hybridize to a 
nucleic acid probe. Proper annealing conditions depend, for example, upon 
a probe's length, base composition, and the number of mismatches and their 
position on the probe, and must often be determined empirically. For 
discussions of nucleic acid probe design and annealing conditions, see, 
for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd 
ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989) or Current 
Protocols in Molecular Biology, F. Ausubel et al., ed. Greene Publishing 
and Wiley-Interscience, New York (1987). 
The phrase "nucleic acid sequence encoding" refers to a nucleic acid which 
directs the expression of a specific protein or peptide. The nucleic acid 
sequences include both the DNA strand sequence that is transcribed into 
RNA and the RNA sequence that is translated into protein. The nucleic acid 
sequences include both the full length nucleic acid sequences as well as 
non-full length sequences derived from the full length protein. It being 
further understood that the sequence includes the degenerate codons of the 
native sequence or sequences which may be introduced to provide codon 
preference in a specific host cell. 
The phrase "isolated" or "substantially pure" when referring to nucleic 
acid sequences encoding ATP-sensitive potassium channel proteins refers to 
isolated nucleic acids that do not encode proteins or peptides other than 
ATP-sensitive potassium channel proteins or peptides. 
The phrase "expression cassette", refers to nucleotide sequences which are 
capable of affecting expression of a structural gene in hosts compatible 
with such sequences. Such cassettes include at least promoters and 
optionally, transcription termination signals. Additional factors 
necessary or helpful in effecting expression may also be used as described 
herein. 
The term "operably linked" as used herein refers to linkage of a promoter 
upstream from a DNA sequence such that the promoter mediates transcription 
of the DNA sequence. 
The term "vector", refers to viral expression systems, autonomous 
self-replicating circular DNA (plasmids), and includes both expression and 
nonexpression plasmids. Where a recombinant microorganism or cell culture 
is described as hosting an "expression vector," this includes both 
extrachromosomal circular DNA and DNA that has been incorporated into the 
host chromosome(s). Where a vector is being maintained by a host cell, the 
vector may either be stably replicated by the cells during mitosis as an 
autonomous structure, or is incorporated within the host's genome. 
The term "plasmid" refers to an autonomous circular DNA molecule capable of 
replication in a cell, and includes both the expression and nonexpression 
types. Where a recombinant microorganism or cell culture is described as 
hosting an "expression plasmid", this includes both extrachromosomal 
circular DNA molecules and DNA that has been incorporated into the host 
chromosome(s). Where a plasmid is being maintained by a host cell, the 
plasmid is either being stably replicated by the cells during mitosis as 
an autonomous structure or is incorporated within the host's genome. 
The phrase "recombinant protein" or "recombinantly produced protein" refers 
to a peptide or protein produced using non-native cells that do not have 
an endogenous copy of DNA able to express the protein. The cells produce 
the protein because they have been genetically altered by the introduction 
of the appropriate nucleic acid sequence. The recombinant protein will not 
be found in association with proteins and other subcellular components 
normally associated with the cells producing the protein. 
The following terms are used to describe the sequence relationships between 
two or more nucleic acids or polynucleotides: "reference sequence", 
"comparison window", "sequence identity", "percentage of sequence 
identity", and "substantial identity". A "reference sequence" is a defined 
sequence used as a basis for a sequence comparison; a reference sequence 
may be a subset of a larger sequence, for example, as a segment of a 
full-length cDNA or gene sequence given in a sequence listing, such as the 
nucleic acid sequence of Seq. ID No. 2, or may comprise a complete cDNA or 
gene sequence. 
Optimal alignment of sequences for aligning a comparison window may be 
conducted by the local homology algorithm of Smith and Waterman (1981) 
Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman 
and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity 
method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA) 85:2444, 
or by computerized implementations of these algorithms (GAP, BESTFIT, 
FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, 
Genetics Computer Group, 575 Science Dr., Madison, Wis.). 
The terms "substantial identity" or "substantial sequence identity" as 
applied to nucleic acid sequences and as used herein and denote a 
characteristic of a polynucleotide sequence, wherein the polynucleotide 
comprises a sequence that has at least 85 percent sequence identity, 
preferably at least 90 to 95 percent sequence identity, and more 
preferably at least 99 percent sequence identity as compared to a 
reference sequence over a comparison window of at least 20 nucleotide 
positions, frequently over a window of at least 25-50 nucleotides, wherein 
the percentage of sequence identity is calculated by comparing the 
reference sequence to the polynucleotide sequence which may include 
deletions or additions which total 20 percent or less of the reference 
sequence over the window of comparison. The reference sequence may be a 
subset of a larger sequence, for example, as a segment of the human heart 
ATP-sensitive potassium channel protein disclosed herein. 
As applied to polypeptides, the terms "substantial identity" or 
"substantial sequence identity" mean that two peptide sequences, when 
optimally aligned, such as by the programs GAP or BESTFIT using default 
gap weights, share at least 70 percent sequence identity, preferably at 
least 80 percent sequence identity, more preferably at least 90 percent 
sequence identity, and most preferably at least 95 percent amino acid 
identity or more. "Percentage amino acid identity" or "percentage amino 
acid sequence identity" refers to a comparison of the amino acids of two 
polypeptides which, when optimally aligned, have approximately the 
designated percentage of the same amino acids. For example, "95% amino 
acid identity" refers to a comparison of the amino acids of two 
polypeptides which when optimally aligned have 95% amino acid identity. 
Preferably, residue positions which are not identical differ by 
conservative amino acid substitutions. For example, the substitution of 
amino acids having similar chemical properties such as charge or polarity 
are not likely to effect the properties of a protein. Examples include 
glutamine for asparagine or glutamic acid for aspartic acid. 
The phrase "substantially purified" or "isolated" when referring to an 
ATP-sensitive potassium channel peptide or protein, means a chemical 
composition which is essentially free of other cellular components. It is 
preferably in a homogeneous state although it can be in either a dry or 
aqueous solution. Purity and homogeneity are typically determined using 
analytical chemistry techniques such as polyacrylamide gel electrophoresis 
or high performance liquid chromatography. A protein which is the 
predominant species present in a preparation is substantially purified. 
Generally, a substantially purified or isolated protein will comprise more 
than 80% of all macromolecular species present in the preparation. 
Preferably, the protein is purified to represent greater than 90% of all 
macromolecular species present. More preferably the protein is purified to 
greater than 95%, and most preferably the protein is purified to essential 
homogeneity, wherein other macromolecular species are not detected by 
conventional techniques. 
The phrase "specifically binds to an antibody" or "specifically 
immunoreactive with", when referring to a protein or peptide, refers to a 
binding reaction which is determinative of the presence of the protein in 
the presence of 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 the human heart ATP-sensitive potassium channel protein 
immunogen with the amino acid sequence depicted in Seq. ID No. 2 can be 
selected to obtain antibodies specifically immunoreactive with 
ATP-sensitive potassium channel proteins and not with other proteins. 
These antibodies recognize proteins homologous to the human heart 
ATP-sensitive potassium channel protein. Homologous proteins encompass the 
family of ATP-sensitive potassium channel proteins, but do not include 
other potassium channel proteins which are not inhibited by ATP. 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 monoclonal antibodies 
specifically immunoreactive with a protein. See Harlow and Lane (1988) 
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New 
York, for a description of immunoassay formats and conditions that can be 
used to determine specific immunoreactivity. 
The term "binding agent:ATP-sensitive potassium channel protein complex", 
as used herein, refers to a complex of a binding agent and an 
ATP-sensitive potassium channel protein that is formed by specific binding 
of the binding agent to the ATP-sensitive potassium channel protein. 
Specific binding of the binding agent means that the binding agent has a 
specific binding site that recognizes a site on the ATP-sensitive 
potassium channel protein. For example, antibodies raised to an 
ATP-sensitive potassium channel protein and recognizing an epitope on the 
ATP-sensitive potassium channel protein are capable of forming a binding 
agent:ATP-sensitive potassium channel protein complex by specific binding. 
Typically, the formation of a binding agent:ATP-sensitive potassium 
channel protein complex allows the measurement of ATP-sensitive potassium 
channel protein in a mixture of other proteins and biologics. The term 
"antibody:ATP-sensitive potassium channel protein complex" refers to a 
binding agent:ATP-sensitive potassium channel protein complex in which the 
binding agent is an antibody. 
"Biological sample" as used herein refers to any sample obtained from a 
living organism or from an organism that has died. Examples of biological 
samples include body fluids and tissue specimens. 
DETAILED DESCRIPTION 
This invention provides for isolated ATP-sensitive potassium channel 
proteins and for isolated nucleic acids encoding these proteins. These 
isolated DNA and protein compositions can be used in a number of 
applications. For instance, they can be used for the design and selection 
of potassium channel openers and inhibitors that act on the ATP-sensitive 
potassium channel. These compositions can also be used in in vitro 
diagnostic methods for the detection and diagnosis of diseases, such as 
diabetes and heart disease, which involves ATP-sensitive potassium 
channels. Compositions and methods for using the DNA and protein sequences 
of the ATP-sensitive potassium channel proteins are described below. 
A. ATP-sensitive Potassium Channel Proteins 
As described above, ATP-sensitive potassium channel proteins are known to 
be active in heart, skeletal muscle and pancreatic .beta.-cells. In 
addition, there is evidence that these proteins are present in neurons and 
smooth muscle tissue as well. 
The ATP-sensitive potassium channel proteins present in different tissues 
appear to be the product of different genes. For example, the pancreatic 
.beta.-cell ATP-sensitive potassium channel protein is a different gene 
product from the heart ATP-sensitive potassium channel protein. Thus, the 
ATP-sensitive potassium channel proteins represent a family of highly 
homologous proteins with the same functional characteristics. The 
predicted amino acid sequence of the human heart ATP-sensitive potassium 
channel protein and the rat heart ATP-sensitive potassium channel protein 
is shown as Seq. ID No. 2 and Seq. ID No. 4, respectively. The predicted 
amino acid sequence of the rat pancreatic .beta.-cell ATP-sensitive 
potassium channel protein is shown as Seq. ID No. 13, and a full-length or 
nearly full-length predicted amino acid sequence of the human pancreatic 
.beta.-cell ATP-sensitive potassium channel protein is shown as Seq. ID 
No. 15. 
The amino acid sequences listed for the rat and human heart ATP-sensitive 
potassium channel proteins are full-length sequences, as is the amino acid 
sequence listed for the rat .beta.-cell channel protein. The human 
.beta.-cell ATP-sensitive potassium channel protein is a full-length 
sequence or a nearly full-length sequence. When the initiator methione 
designated in the cDNA sequence of Seq. ID No. 14 is used in a 
heterologous expression system, functional ATP channel proteins with the 
amino acid sequence of Seq. ID No. 15 are produced. 
The percent amino acid identity of these proteins was determined by the GAP 
computer program (version 7.3.1, Genetics Computer Group, 575 Science 
Drive, Madison, Wis.). The Needleman and Wunsch homology alignment 
algorithm was used with the default settings. Using this procedure, there 
is 95.95% amino acid identity between the amino acid sequences for the rat 
and human heart ATP-sensitive potassium channel proteins. By comparison, 
there is 98.2% amino acid identity between the amino acid sequences for 
the rat and human pancreatic .beta.-cell ATP-sensitive potassium channel 
proteins. In contrast, there is 72.3% amino acid identity when the rat 
heart ATP-sensitive potassium channel protein sequence is compared to that 
of the rat pancreatic .beta.-cell ATP-sensitive potassium channel protein. 
Lastly, there is 74.9% amino acid identity between the amino acid 
sequences of the human heart ATP-sensitive potassium channel protein and 
the human pancreatic .beta.-cell ATP-sensitive potassium channel protein. 
The term "ATP-sensitive potassium channel protein" refers to a family of 
proteins that form a potassium channel in the cell membrane which is 
inhibited by high intracellular concentrations of ATP. ATP-sensitive 
potassium channel proteins are known to be present and active in certain 
vertebrate tissues such as heart, skeletal muscle and the pancreas. The 
physiological and pharmacological characteristics of ATP-sensitive 
potassium channels have been characterized by the membrane patch-clamp 
technique (see Hamil, O . P., et al. (1981) Pflugers Arch. 351:85-100. 
Accordingly, the proteins are defined by their functional characteristics 
when present in active form in the cell membrane. For instance, 
ATP-sensitive potassium channels are inhibited by ATP with a half maximal 
inhibition in the range of 10-100 .mu.M. They have a unitary conductance 
of from 40-80 pS when measured under high symmetrical potassium 
concentrations, and are calcium- and voltage-independent and potassium 
selective. They are inhibited by agents such as tolbutamide and glyburide. 
For a detailed description of the properties of ATP-sensitive potassium 
channels, see Ashcroft, F. M, supra and Edwards, G., et al. supra. 
ATP-sensitive potassium channel proteins typically show substantial 
sequence identity (as defined above) to the amino acid sequence of the 
human heart ATP-sensitive potassium channel protein as depicted in Seq. ID 
No. 2. ATP-sensitive potassium channel proteins from different tissues and 
from different mammalian species are all specifically immunoreactive with 
antibodies raised to the human heart ATP-sensitive potassium channel 
protein described herein and consisting of the amino acid sequence of Seq. 
ID. No. 2. 
An ATP-sensitive potassium channel protein that specifically binds to or 
that is specifically immunoreactive to an antibody generated against a 
defined immunogen, such as an immunogen consisting of the amino acid 
sequence of Seq. ID No. 2, is determined in an immunoassay. The 
immunoassay uses a polyclonal antiserum which was raised to the protein of 
Seq. ID No. 2. This antiserum is selected to have low crossreactivity 
against other (non-ATP-sensitive) potassium channel proteins and any such 
crossreactivity is removed by immnuoabsorbtion prior to use in the 
immunoassay. 
In order to produce antisera for use in an immunoassay, the protein of Seq. 
ID No. 2 is isolated as described herein. For example, recombinant protein 
is produced in a mammalian cell line. An inbred strain of mice such as 
balb/c is immunized with the protein of Seq. ID No. 2 using a standard 
adjuvant, such as Freund's adjuvant, and a standard mouse immunization 
protocol (see Harlow and Lane, supra). Alternatively, a synthetic peptide 
derived from the sequences disclosed herein and conjugated to a carrier 
protein can be used an immunogen. For instance, the peptides of Seq. ID 
Nos. 10 and 11 may be used. 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 10.sup.4 or greater are selected and 
tested for their cross reactivity against non-ATP-sensitive potassium 
channel proteins, using a competitive binding immunoassay such as the one 
described in Harlow and Lane, supra, at pages 570-573. Three non-ATP 
sensitive potassium channel proteins are used in this determination: the 
IRK protein (Kubo, et al. (1993) Nature 362:127), the G-IRK protein (Kubo, 
et al. (1993) Nature 364:802) and the ROM-K protein (Ho, et al. (1993) 
Nature 362:127. These non-ATP sensitive potassium channel proteins can be 
produced as recombinant proteins and isolated using standard molecular 
biology and protein chemistry techniques as described herein. 
Immunoassays in the competitive binding format can be used for the 
crossreactivity determinations. For example, the protein of Seq. ID No. 2 
can be immobilized to a solid support. Proteins added to the assay 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 the protein of Seq. ID No. 2. 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 then removed from the pooled antisera by 
immunoabsorbtion with the above-listed proteins. 
The immunoabsorbed and pooled antisera are then used in a competitive 
binding immunoassay as described above to compare a second protein to the 
immunogen protein (the ATP-sensitive potassium channel protein of Seq. ID 
No. 2). 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 is 
less than 10 times the amount of the protein of Seq. ID No. 2 that is 
required, then the second protein is said to specifically bind to an 
antibody generated to an immunogen consisting of the protein of Seq. ID 
No. 2. 
It is understood that ATP-sensitive potassium channel proteins refer to a 
family of homologous proteins that are encoded by two or more genes. For a 
particular gene product, such as the human heart ATP-sensitive potassium 
channel protein, the term refers not only to the amino acid sequences 
disclosed herein, but also to other proteins that are allelic, non-allelic 
or species variants. It also understood that the term "ATP-sensitive 
potassium channel proteins" includes nonnatural mutations introduced by 
deliberate mutation using conventional recombinant technology such as 
single site mutation or by excising short sections of DNA encoding 
ATP-sensitive potassium channel proteins or by substituting new amino 
acids or adding new amino acids. Such minor alterations must substantially 
maintain the immunoidentity of the original molecule and/or its biological 
activity. Thus, these alterations include proteins that are specifically 
immunoreactive with a designated naturally occurring ATP-sensitive 
potassium channel protein, for example, the human heart protein shown in 
Seq. ID No. 2. The biological properties of the altered proteins can be 
determined by expressing the protein in an appropriate cell line and using 
the membrane patch-clamp technique to determine the function of the 
ATP-sensitive potassium channel in a membrane patch (see example 4, 
herein). Particular protein modifications considered minor would include 
substitution of amino acids of similar chemical properties, e.g., glutamic 
acid for aspartic acid or glutamine for asparagine. By aligning a protein 
optimally with the protein of Seq. ID No. 2 and by using the conventional 
immunoassays described herein to determine immunoidentity, or by using 
patch-clamp membrane techniques to determine biological activity, one can 
readily determine the protein compositions of the invention. 
ATP-sensitive potassium channel proteins designated by their tissue of 
origin refer to the gene-product from this family that is predominantly 
expressed in that tissue. For instance, the term "heart ATP-sensitive 
potassium channel protein" refers to the ATP-sensitive potassium channel 
protein that is expressed in heart tissue. As another example, the term 
"pancreatic .beta.-cell ATP-sensitive potassium channel protein" refers to 
the ATP-sensitive protein that is expressed in the pancreatic .beta.-cell. 
Since ATP-sensitive potassium channel proteins represent a family of 
homologous proteins, the proteins expressed in different tissues can be 
the product of different genes in the family. 
B. Nucleic Acids Encoding ATP--sensitive Potassium Channel Proteins 
This invention relates to isolated nucleic acid sequences encoding 
ATP-sensitive potassium channel proteins. The nucleic acid compositions of 
this invention, whether RNA, cDNA, genomic DNA, or a hybrid of the various 
combinations, may be isolated from natural sources or may be synthesized 
in vitro. The nucleic acids claimed may be present in transformed or 
transfected whole cells, in a transformed or transfected cell lysate, or 
in a partially purified or substantially pure form. 
The nucleic acid sequences of the invention are typically identical to or 
show substantial sequence identity (determined as described above) to the 
nucleic acid sequence of SEQ ID. No. 1. Nucleic acids encoding mammalian 
ATP-sensitive potassium channel proteins will typically hybridize to the 
nucleic acid sequence of Seq. ID No. 1 under stringent conditions. For 
example, nucleic acids encoding ATP-sensitive potassium channel proteins 
will hybridize to the nucleic acid of sequence ID No. 1 under the 
hybridization and wash conditions of 50% formamide at 42.degree. C. Other 
stringent hybridization conditions may also be selected. Generally, 
stringent conditions are selected to be about 5.degree. C. lower than the 
thermal melting point (Tm) for the specific sequence at a defined ionic 
strength and pH. The Tm is the temperature (under defined ionic strength 
and pH) at which 50% of the target sequence hybridizes to a perfectly 
matched probe. Typically, stringent conditions will be those in which the 
salt concentration is at least about 0.02 molar at pH 7 and the 
temperature is at least about 60.degree.0 C. As other factors may 
significantly affect the stringency of hybridization, including, among 
others, base composition and size of the complementary strands, the 
presence of organic solvents and the extent of base mismatching, the 
combination of parameters is more important than the absolute measure of 
any one. 
Techniques for nucleic acid manipulation of genes encoding the 
ATP-sensitive potassium channel proteins such as subcloning nucleic acid 
sequences encoding polypeptides into expression vectors, labelling probes, 
DNA hybridization, and the like are described generally in Sambrook, et 
al., Molecular Cloning--A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold 
Spring Harbor Laboratory, Cold Spring Harbor, N.Y, 1989, which is 
incorporated herein by reference. This manual is hereinafter referred to 
as "Sambrook, et al." 
There are various methods of isolating the DNA sequences encoding 
ATP-sensitive potassium channel proteins. For example, DNA is isolated 
from a genomic or cDNA library using labelled oligonucleotide probes 
having sequences complementary to the sequences disclosed herein (Seq. ID 
Nos. 1, 3, 8, 12 and 14). For example, full-length probes may be used, or 
oligonucleotide probes may also be generated by comparison of the 
sequences of Seq. ID Nos. 1, 3, 8, 12 and 14. Such probes can be used 
directly in hybridization assays to isolate DNA encoding ATP-sensitive 
potassium channel proteins. Alternatively probes can be designed for use 
in amplification techniques such as PCR, and DNA encoding ATP-sensitive 
potassium channel proteins may be isolated by using methods such as PCR 
(see below). 
To prepare a cDNA library, MRNA is isolated from tissue such as heart or 
pancreas which expresses an ATP-sensitive potassium channel protein. cDNA 
is prepared from the mRNA and ligated into a recombinant vector. The 
vector is transfected into a recombinant host for propagation, screening 
and cloning. Methods for making and screening cDNA libraries are well 
known. See Gubler, U. and Hoffman, B . J. Gene 25:263-269, 1983 and 
Sambrook, et al. 
For a genomic library, the DNA is extracted from 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, as described in Sambrook, et al. 
Recombinant phage are analyzed by plaque hybridization as described in 
Benton and Davis, Science, 196:180-182 (1977). Colony hybridization is 
carried out as generally described in M. Grunstein et al. Proc. Natl. 
Acad. Sci. USA., 72:3961-3965 (1975). 
DNA encoding an ATP-sensitive potassium channel protein is identified in 
either cDNA or genomic libraries by its ability to hybridize with nucleic 
acid probes, for example on Southern blots, and these DNA regions are 
isolated by standard methods familiar to those of skill in the art. See 
Sambrook, et al. 
Various methods of amplifying target sequences, such as the polymerase 
chain reaction, can also be used to prepare DNA encoding ATP-sensitive 
potassium channel protein. Polymerase chain reaction (PCR) technology is 
used to amplify such nucleic acid sequences directly from mRNA, from cDNA, 
and from genomic libraries or cDNA libraries. The isolated sequences 
encoding ATP-sensitive potassium channel protein may also be used as 
templates for PCR amplification. 
In PCR techniques, oligonucleotide primers complementary to the two 3' 
borders of the DNA region to be amplified are synthesized. The polymerase 
chain reaction is then carried out using the two primers. See PCR 
Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D., 
Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990). 
Primers can be selected to amplify the entire regions encoding a 
full-length ATP-sensitive potassium channel protein or to amplify smaller 
DNA segments as desired. 
PCR can be used in a variety of protocols to isolate cDNA's encoding the 
ATP-sensitive potassium channel proteins. In these protocols, appropriate 
primers and probes for amplifying DNA encoding ATP-sensitive potassium 
channel proteins are generated from analysis of the DNA sequences listed 
herein. For example, the oligonucleotides of Seq. ID Nos. 5 and 6 can be 
used in a PCR protocol as described in example 2 herein to amplify regions 
of DNA's encoding potassium channel proteins. Once such regions are 
PCR-amplified, they can be sequenced and oligonucleotide probes can be 
prepared from sequence obtained. These probes can then be used to isolate 
DNA's encoding ATP-sensitive potassium channel proteins, similar to the 
procedure used in example 2 herein. ATP-sensitive potassium channel 
proteins can be isolated from a variety of different tissues using this 
procedure. Other oligonucleotide probes in addition to those of Seq. ID 
No. 5 and 6 and which are obtained from the sequences described herein can 
also be used in PCR protocols to isolate cDNA's encoding the ATP-sensitive 
potassium channel proteins. 
Oligonucleotides for use as probes are chemically synthesized according to 
the solid phase phosphoramidite triester method first described by 
Beaucage, S. L. and Carruthers, M. H., 1981, Tetrahedron Lett., 
22(20):1859-1862 using an automated synthesizer, as described in 
Needham-VanDevanter, D. R., et al., 1984, Nucleic Acids Res., 
12:6159-6168. Purification of oligonucleotides is by either native 
acrylamide gel electrophoresis or by anion-exchange HPLC as described in 
Pearson, J. D. and Regnier, F. E., 1983, J. Chrom., 255:137-149. The 
sequence of the synthetic oligonucleotide can be verified using the 
chemical degradation method of Maxam, A. M. and Gilbert, W. 1980, in 
Grossman, L. and Moldave, D., eds. Academic Press, New York, Methods in 
Enzymology, 65:499-560. 
Other methods known to those of skill in the art may also be used to 
isolate DNA encoding the ATP-sensitive potassium channel protein. See 
Sambrook, et al. for a description of other techniques for the isolation 
of DNA encoding specific protein molecules. 
C. Expression of ATP-sensitive potassium channel proteins 
Once DNA encoding ATP-sensitive potassium channel proteins is isolated and 
cloned, one can express the ATP-sensitive potassium channel proteins in a 
variety of recombinantly engineered cells. It is expected that those of 
skill in the art are knowledgeable in the numerous expression systems 
available for expression of DNA encoding ATP-sensitive potassium channel 
proteins. No attempt to describe in detail the various methods known for 
the expression of proteins in prokaryotes or eukaryotes is made here. 
In brief summary, the expression of natural or synthetic nucleic acids 
encoding ATP-sensitive potassium channel proteins will typically be 
achieved by operably linking the DNA or cDNA to a promoter (which is 
either constitutive or inducible), followed by incorporation into an 
expression vector. The vectors can be suitable for replication and 
integration in either prokaryotes or eukaryotes. Typical expression 
vectors contain transcription and translation terminators, initiation 
sequences, and promoters useful for regulation of the expression of 
polynucleotide sequence encoding ATP-sensitive potassium channel proteins. 
To obtain high level expression of a cloned gene, such as those 
polynucleotide sequences encoding ATP-sensitive potassium channel 
proteins, it is desirable to construct expression plasmids which contain, 
at the minimum, a strong promoter to direct transcription, a ribosome 
binding site for translational initiation, and a transcription/translation 
terminator. The expression vectors may also comprise generic expression 
cassettes containing at least one independent terminator sequence, 
sequences permitting replication of the plasmid in both eukaryotes and 
prokaryotes, i.e., shuttle vectors, and selection markers for both 
prokaryotic and eukaryotic systems. See Sambrook et al. Examples of 
expression of ATP-sensitive potassium channel proteins in both prokaryotic 
and eukaryotic systems are described below. 
1. Expression in Prokaryotes 
A variety of procaryotic expression systems may be used to express 
ATP-sensitive potassium channel proteins. Examples include E. coli, 
Bacillus, Streptomyces, and the like. For example, ATP-sensitive potassium 
channel proteins may be expressed in E. coli. 
It is essential to construct expression plasmids which contain, at the 
minimum, a strong promoter to direct transcription, a ribosome binding 
site for translational initiation, and a transcription/translation 
terminator. Examples of regulatory regions suitable for this purpose in E. 
coli are the promoter and operator region of the E. coli tryptophan 
biosynthetic pathway as described by Yanofsky, C., 1984, J. Bacteriol., 
158:1018-1024 and the leftward promoter of phage lambda (P.lambda.) as 
described by Herskowitz, I. and Hagen, D., 1980, Ann. Rev. Genet., 
14:399-445. The inclusion of selection markers in DNA vectors transformed 
in E. coli is also useful. Examples of such markers include genes 
specifying resistance to ampicillin, tetracycline, or chloramphenicol. See 
Sambrook et al. for details concerning selection markers for use in E. 
coli. 
ATP-sensitive potassium channel proteins produced by prokaryotic cells may 
not necessarily fold properly. During purification from E. coli, the 
expressed protein may first be denatured and then renatured. This can be 
accomplished by solubilizing the bacterially produced proteins in a 
chaotropic agent such as guanidine HCl and reducing all the cysteine 
residues with a reducing agent such as beta-mercaptoethanol. The protein 
is then renatured, either by slow dialysis or by gel filtration. See U.S. 
Pat. No. 4,511,503. 
Detection of the expressed antigen is achieved by methods known in the art 
as radioimmunoassay, or Western blotting techniques or 
immunoprecipitation. Purification from E. coli can be achieved following 
procedures described in U.S. Pat. No. 4,511,503. 
2. Expression in Eukaryotes 
A variety of eukaryotic expression systems such as yeast, insect cell 
lines, bird, fish, and mammalian cells, are known to those of skill in the 
art. As explained briefly below, ATP-sensitive potassium channel proteins 
may be expressed in these eukaryotic systems. 
Synthesis of heterologous proteins in yeast is well known. Methods in Yeast 
Genetics, Sherman, F., et al., Cold Spring Harbor Laboratory, (1982) is a 
well recognized work describing the various methods available to produce 
the protein in yeast. 
Suitable vectors usually have expression control sequences, such as 
promoters, including 3-phosphoglycerate kinase or other glycolytic 
enzymes, and an origin of replication, termination sequences and the like 
as desired. For instance, suitable vectors are described in the literature 
(Botstein, et al., 1979, Gene, 8:17-24; Broach, et al., 1979, Gene, 
8:121-133). 
Two procedures are used in transforming yeast cells. In one case, yeast 
cells are first converted into protoplasts using zymolyase, lyticase or 
glusulase, followed by addition of DNA and polyethylene glycol (PEG). The 
PEG-treated protoplasts are then regenerated in a 3% agar medium under 
selective conditions. Details of this procedure are given in the papers by 
J. D. Beggs, 1978, Nature (London), 275:104-109; and Hinnen, A., et al., 
1978, Proc. Natl. Acad. Sci. USA, 75:1929-1933. The second procedure does 
not involve removal of the cell wall. Instead the cells are treated with 
lithium chloride or acetate and PEG and put on selective plates (Ito, H., 
et al., 1983, J. Bact., 153:163-168). 
ATP-sensitive potassium channel proteins, once expressed, can be isolated 
from yeast by lysing the cells and applying standard protein isolation 
techniques to the lysates. The monitoring of the purification process can 
be accomplished by using Western blot techniques or radioimmunoassay of 
other standard immunoassay techniques. 
The sequences encoding ATP-sensitive potassium channel proteins can also be 
ligated to various expression vectors for use in transforming cell 
cultures of, for instance, mammalian, insect, bird or fish origin. 
Illustrative of cell cultures useful for the production of the 
polypeptides are mammalian cells. Mammalian cell systems often will be in 
the form of monolayers of cells although mammalian cell suspensions may 
also be used. A number of suitable host cell lines capable of expressing 
intact proteins have been developed in the art, and include the HEK293, 
BHK21, and CHO cell lines, and various human cells such as COS cell lines, 
HeLa cells, myeloma cell lines, Jurkat cells, etc. Expression vectors for 
these cells can include expression control sequences, such as an origin of 
replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk 
(phosphoglycerate kinase) promoter), an enhancer (Queen et al. (1986) 
Immunol. Rev. 89:49), and necessary processing information sites, such as 
ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an 
SV40 large T Ag poly A addition site), and transcriptional terminator 
sequences. Other animal cells useful for production of ATP-sensitive 
potassium channel proteins are available, for instance, from the American 
Type Culture Collection Catalogue of Cell Lines and Hybridomas (7th 
edition, 1992). 
Appropriate vectors for expressing ATP-sensitive potassium channel proteins 
in insect cells are usually derived from the SF9 baculovirus. Suitable 
insect cell lines include mosquito larvae, silkworm, armyworm, moth and 
Drosophila cell lines such as a Schneider cell line (See Schneider J. 
Embryol. Exp. Morphol. 27:353-365 (1987). 
As indicated above, the vector, e.g., a plasmid, which is used to transform 
the host cell, preferably contains DNA sequences to initiate transcription 
and sequences to control the translation of the protein. These sequences 
are referred to as expression control sequences. 
As with yeast, when higher animal host cells are employed, polyadenlyation 
or transcription terminator sequences from known mammalian genes need to 
be incorporated into the vector. An example of a terminator sequence is 
the polyadenlyation sequence from the bovine growth hormone gene. 
Sequences for accurate splicing of the transcript may also be included. An 
example of a splicing sequence is the VP1 intron from SV40 (Sprague, J. et 
al., 1983, J. Virol. 45: 773-781). 
Additionally, gene sequences to control replication in the host cell may be 
incorporated into the vector such as those found in bovine papilloma virus 
type-vectors. Saveria-Campo, M., 1985, "Bovine Papilloma virus DNA a 
Eukaryotic Cloning Vector" in DNA Cloning Vol. II a Practical Approach Ed. 
D. M. Glover, IRL Press, Arlington, Va. pp. 213-238. 
The host cells are competent or rendered competent for transformation by 
various means. There are several well-known methods of introducing DNA 
into animal cells. These include: calcium phosphate precipitation, fusion 
of the recipient cells with bacterial protoplasts containing the DNA, 
treatment of the recipient cells with liposomes containing the DNA, DEAE 
dextran, electroporation and micro-injection of the DNA directly into the 
cells. 
The transformed cells are cultured by means well known in the art. 
Biochemical Methods in Cell Culture and Virology, Kuchler, R. J., Dowden, 
Hutchinson and Ross, Inc., (1977). The expressed polypeptides are isolated 
from cells grown as suspensions or as monolayers. The latter are recovered 
by well known mechanical, chemical or enzymatic means. 
D. Purification of ATP-sensitive potassium channel proteins 
The polypeptides produced by recombinant DNA technology may be purified by 
standard techniques well known to those of skill in the art. Recombinantly 
produced polypeptides can be directly expressed or expressed as a fusion 
protein. The protein is then purified by a combination of cell lysis 
(e.g., sonication) and affinity chromatography. For fusion products, 
subsequent digestion of the fusion protein with an appropriate proteolytic 
enzyme releases the desired polypeptide. 
The polypeptides of this invention may be purified to substantial purity by 
standard techniques well known in the art, including selective 
precipitation with such substances as ammonium sulfate, column 
chromatography, immunopurification methods, and others. See, for instance, 
R. Scopes, Protein Purification: Principles and Practice, Springer-Verlag: 
New York (1982), incorporated herein by reference. For example, antibodies 
may be raised to the ATP-sensitive potassium channel proteins as described 
herein. Cell membranes are isolated from a cell line expressing the 
recombinant protein, the protein is extracted from the membranes and 
immunoprecipitated. The proteins may then be further purified by standard 
protein chemistry techniques as described above. 
E. Assays for Biologically Active ATP-sensitive Potassium Channel Proteins 
and for DNA encoding Such Proteins 
The presence of ATP-sensitive potassium channel proteins may be measured by 
a variety of techniques. For example, the proteins may be measured in 
immunoassays as described below. In addition, biologically active 
ATP-sensitive potassium channel proteins or DNA encoding such proteins can 
be measured by the membrane patch-clamp technique (see Hamil, O. P. et al. 
(1981) Pflugers Arch. 351:85-100). In order to use this technique, DNA or 
cDNA encoding ATP-sensitive potassium channel proteins is first isolated, 
inserted into a suitable expression vector and transfected into a cell 
line, as described herein. Expression of recombinant ATP-sensitive 
proteins in an appropriate cell line results in the incorporation of the 
protein into the cell membrane. Cell-free membrane patches are prepared 
and single channel currents are measured by the membrane patch-clamp 
technique. (See Ashcroft, F. M., et al. supra for a review of the 
measurement of ATP-sensitive potassium channels by the patch-clamp 
technique.) An example of the use of the membrane patch-clamp technique to 
detect DNA encoding ATP-sensitive potassium channel proteins is described 
in example 4, herein. 
F. Assays for Compounds that Inhibit or Open the ATP-sensitive Potassium 
Channel 
DNA encoding ATP-sensitive potassium channel proteins or recombinantly 
produced proteins can be used in a variety of assays to detect compounds 
that are inhibitors or openers of the ATP-sensitive potassium channel. For 
example, the membrane patch-clamp technique can be used for this purpose. 
Isolated DNA encoding an ATP-sensitive potassium channel protein can be 
inserted into an expression vector, transfected into an appropriate cell 
line and expressed in the cell line as described herein. Single channel 
currents are measured in cell free membrane patches as described above 
(see Ashcroft, F. M., et al. supra). Assays for compounds capable of 
opening the A of opening the ATP-sensitive potassium channel can be 
performed by application of the compounds to a bath solution including ATP 
as described by Fan, Z., et al. (1993) Pflugers Arch. 415:387-394. (See 
example 5 herein for an illustration of the use of the patch-clamp 
technique to measure an ATP-sensitive potassium channel opener.) Assays 
for compounds that are inhibitors of the ATP-sensitive potassium channel 
can be measured under similar conditions (see Ashcroft, F. M., supra). 
In addition to assaying for compounds with unknown activity, the 
compositions of the invention can also be used to determine the 
concentration of known ATP-sensitive potassium channel openers and 
inhibitors. For example, the membrane patch-clamp technique can be used 
with transfected cell lines as described above. However, different 
concentrations of known ATP-sensitive potassium channel openers or 
inhibitors can be applied under designated conditions. Concentrations of 
biologically active compounds can be expressed as activity units under 
standardized conditions or can by expressed in mass of the compound by 
reference to a standard preparation of the compound. A threshold level for 
opening or inhibiting the ATP-sensitive potassium channel is used. For 
instance, the patch-clamp measurement conditions and the threshold level 
as described in Fan, Z. et al., supra, could be used. The determination of 
the concentration of pinacidil, an ATP-sensitive potassium channel opener 
is illustrated in example 5, herein. Other potassium channel openers may 
also be measured by this method. 
The concentration of potassium channel inhibitors such as sulfonylurea 
drugs can also be measured by similar methods. For instance, the assay 
described in example 5 can readily be modified to measure a compound that 
inhibits rather than activates the ATP-sensitive potassium channel. 
Examples of ATP-sensitive potassium channel inhibitors include glyburide 
and tolbutamide (both obtained from Upjohn, Kalamazoo, Mich., U.S.A.). 
Examples of ATP-sensitive potassium channel openers include pinacidil 
(Upjohn), diazide, nicorandil, cromakalim, and a variety of other 
compounds. (See Edwards, G., et al., supra for a discussion of 
ATP-sensitive potassium channel openers and inhibitors.) 
G. In Vitro Diagnostic Methods: Detection of Nucleic Acids Encoding 
ATP-sensitive Potassium Channel Proteins and Detection of ATP-sensitive 
Potassium Channel Proteins by Immunoassay 
The present invention provides methods for detecting DNA or RNA encoding 
ATP-sensitive potassium channel proteins and for measuring the proteins by 
immunoassay techniques. These methods are useful for two general purposes. 
First, assays for detection of nucleic acids encoding ATP-sensitive 
potassium channel proteins are useful for the isolation of these nucleic 
acids from a variety of vertebrate species according to the methods 
described in section (B) above and by use of the nucleic acid 
hybridization assays described below. The immunoassays described below may 
be useful for isolation of nucleic acids encoding ATP-sensitive potassium 
channel proteins by expression cloning methods (see section (B) above and 
Sambrook, et al.). 
The nucleic acid hybridization assays and the immunoassays described below 
are also useful as in vitro diagnostic assays for disorders in which 
alterations in ATP-sensitive potassium channel proteins play a role. These 
diseases include diabetes, heart disease, and certain skeletal muscle 
disorders. 
1. Nucleic Acid Hybridization Assays 
A variety of methods for specific DNA and RNA measurement using nucleic 
acid hybridization techniques are known to those of skill in the art. See 
Sambrook, et al. For example, one method for evaluating the presence or 
absence of DNA encoding ATP-sensitive potassium channel proteins in a 
sample involves a Southern transfer. Briefly, the digested genomic DNA is 
run on agarose slab gels in buffer and transferred to membranes. 
Hybridization is carried out using the nucleic acid probes discussed 
above. As described above, nucleic acid probes are designed based on the 
nucleic acid sequences encoding the human heart and rat heart 
ATP-sensitive potassium channel proteins or the pancreatic .beta.-cell 
protein. (See Seq. ID. Nos. 1, 3, 8, 12 and 14.) The probes can be full 
length or less than the full length of the nucleic acid sequence encoding 
the potassium channel protein. Shorter probes are empirically tested for 
specificity. Preferably nucleic acid probes are 20 bases or longer in 
length. (See Sambrook, et al. for methods of selecting nucleic acid probe 
sequences for use in nucleic acid hybridization.) Visualization of the 
hybridized portions allows the qualitative determination of the presence 
or absence of DNA encoding ATP-sensitive potassium channel proteins. 
Similarly, a Northern transfer may be used for the detection of mRNA 
encoding ATP-sensitive potassium channel proteins. In brief, the mRNA is 
isolated from a given cell sample using an acid 
guanidinium-phenol-chloroform extraction method. The MRNA is then 
electrophoresed to separate the mRNA species and the MRNA is transferred 
from the gel to a nitrocellulose membrane. As with the Southern blots, 
labeled probes are used to identify the presence or absence of 
ATP-sensitive potassium channel proteins. 
A variety of nucleic acid hybridization formats are known to those skilled 
in the art. For example, common formats include sandwich assays and 
competition or displacement assays. Hybridization techniques are generally 
described in "Nucleic Acid Hybridization, A Practical Approach," Ed. 
Hames, B. D. and Higgins, S. J., IRL Press, 1985; Gall and Pardue (1969), 
Proc. Natl. Acad. Sci., U.S.A., 63:378-383; and John, Burnsteil and Jones 
(1969) Nature, 223:58-587. 
For example, sandwich assays are commercially useful hybridization assays 
for detecting or isolating nucleic acid sequences. Such assays utilize a 
"capture" nucleic acid covalently immobilized to a solid support and a 
labelled "signal" nucleic acid in solution. The clinical sample will 
provide the target nucleic acid. The "capture" nucleic acid and "signal" 
nucleic acid probe hybridize with the target nucleic acid to form a 
"sandwich" hybridization complex. To be effective, the signal nucleic acid 
cannot hybridize with the capture nucleic acid. 
Typically, labelled signal nucleic acids are used to detect hybridization. 
Complementary nucleic acids or signal nucleic acids may be labelled by any 
one of several methods typically used to detect the presence of hybridized 
polynucleotides. The most common method of detection is the use of 
autoradiography with .sup.3 H, .sup.125 I, .sup.35 S, .sup.14 C, or.sup.32 
P-labelled probes or the like. Other labels include ligands which bind to 
labelled antibodies, fluorophores, chemiluminescent agents, enzymes, and 
antibodies which can serve as specific binding pair members for a labelled 
ligand. 
Detection of a hybridization complex may require the binding of a signal 
generating complex to a duplex of target and probe polynucleotides or 
nucleic acids. Typically, such binding occurs through ligand and 
anti-ligand interactions as between a ligand-conjugated probe and an 
anti-ligand conjugated with a signal. 
The label may also allow indirect detection of the hybridization complex. 
For example, where the label is a hapten or antigen, the sample can be 
detected by using antibodies. In these systems, a signal is generated by 
attaching fluorescent or enzyme molecules to the antibodies or, in some 
cases, by attachment to a radioactive label. (Tijssen, P., "Practice and 
Theory of Enzyme Immunoassays," Laboratory Techniques in Biochemistry and 
Molecular Biology, Burdon, R. H., van Knippenberg, P. H., Eds., Elsevier 
(1985), pp. 9-20.) 
The sensitivity of the hybridization assays may be enhanced through use of 
a nucleic acid amplification system which multiplies the target nucleic 
acid being detected. Examples of such systems include the polymerase chain 
reaction (PCR) system and the ligase chain reaction (LCR) system. Other 
methods recently described in the art are the nucleic acid sequence based 
amplification (NASBA.TM., Cangene, Mississauga, Ontario) and Q Beta 
Replicase systems. 
An alternative means for determining the level of expression of a gene 
encoding an ATP-sensitive potassium channel protein is in situ 
hybridization. In situ hybridization assays are well known and are 
generally described in Angerer, et al., Methods Enzymol., 152:649-660 
(1987). In an in situ hybridization assay, cells are fixed to a solid 
support, typically a glass slide. If DNA is to be probed, the cells are 
denatured with heat or alkali. The cells are then contacted with a 
hybridization solution at a moderate temperature to permit annealing of 
labeled probes specific to ATP-sensitive potassium channel proteins. The 
probes are preferably labelled with radioisotopes or fluorescent 
reporters. 
2. Production of Antibodies and Development of Immunoassays 
In addition to detecting expression of ATP-sensitive potassium channel 
proteins by nucleic acid hybridization, one can also use immunoassays to 
detect the proteins. Immunoassays can be used to qualitatively or 
quantitatively analyze for the proteins. A general overview of the 
applicable technology can be found in Harlow and Lane, Antibodies: A 
Laboratory Manual, Cold Spring Harbor Pubs., New York (1988), incorporated 
herein by reference. 
a. Antibody Production 
A number of immunogens may be used to produce antibodies specifically 
reactive with ATP-sensitive potassium channel proteins. Recombinant 
protein is the preferred immunogen for the production of monoclonal or 
polyclonal antibodies. Naturally occurring protein may also be used either 
in pure or impure form. Synthetic peptides made using the human heart or 
rat heart ATP-sensitive potassium channel protein sequences described 
herein may also used as an immunogen for the production of antibodies to 
the protein. Recombinant protein can be expressed in eukaryotic or 
prokaryotic cells as described above, and purified as generally described 
above. 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. In brief, an immunogen, preferably a purified protein, is 
mixed with an adjuvant and animals are immunized. The animal's immune 
response to the immunogen preparation is monitored by taking test bleeds 
and determining the titer of reactivity to the ATP-sensitive potassium 
channel protein. 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 and 
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 and Milstein, Eur. J. Immunol. 6:511-519 (1976), 
incorporated herein by reference). Alternative methods of immortalization 
include transformation with Epstein Barr Virus, oncogenes, or 
retroviruses, or other methods well 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. (1989) Science 246:1275-1281. 
Methods of production of synthetic peptides are known to those of skill in 
the art. Briefly, the predicted immunogenic regions of the ATP-sensitive 
potassium channel protein sequences described herein are identified. 
Peptides preferably at least 10 amino acids in length are synthesized 
corresponding to these regions and the peptides are conjugated to larger 
protein molecules for subsequent immunization. Preferably, peptide 
sequences corresponding to unique regions of an ATP-sensitive potassium 
channel protein are used to generate antibodies specifically 
immunoreactive with the potassium channel proteins. Examples of such 
peptides are depicted in Seq. ID Nos. 10 and 11. Production of monoclonal 
or polyclonal antibodies is then carried out as described above. 
b. Immunoassays 
A particular protein can be measured by a variety of immunoassay methods. 
For a review of immunological and immunoassay procedures in general, see 
Basic and Clinical Immunology 7th Edition (D. Stites and A. Terr ed.) 
1991. Moreover, the immunoassays of the present invention can be performed 
in any of several configurations, which are reviewed extensively in Enzyme 
Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Fla. (1980); 
"Practice and Theory of Enzyme Immunoassays," P. Tijssen, Laboratory 
Techniques in Biochemistry and Molecular Biology, Elsevier Science 
Publishers B. V. Amsterdam (1985); and, Harlow and Lane, Antibodies, A 
Laboratory Manual, supra, each of which is incorporated herein by 
reference. 
Immunoassays for measurement of ATP-sensitive potassium channel proteins 
can be performed by a variety of methods known to those skilled in the 
art. In brief, immunoassays to measure the protein can be either 
competitive or noncompetitive binding assays. In competitive binding 
assays, the sample analyte competes with a labeled analyte for specific 
binding sites on a capture agent bound to a solid surface. Preferably the 
capture agent is an antibody specifically reactive with ATP-sensitive 
potassium channel proteins produced as described above. The concentration 
of labeled analyte bound to the capture agent is inversely proportional to 
the amount of free analyte present in the sample. 
In a competitive binding immunoassay, the ATP-sensitive potassium channel 
protein present in the sample competes with labelled protein for binding 
to a specific binding agent, for example, an antibody specifically 
reactive with the ATP-sensitive potassium channel protein. The binding 
agent may be bound to a solid surface to effect separation of bound 
labelled protein from the unbound labelled protein. Alternately, the 
competitive binding assay may be conducted in liquid phase and any of a 
variety of techniques known in the art may be used to separate the bound 
labelled protein from the unbound labelled protein. Following separation, 
the amount of bound labeled protein is determined. The amount of protein 
present in the sample is inversely proportional to the amount of labelled 
protein binding. 
Alternatively, a homogenous immunoassay may be performed in which a 
separation step is not needed. In these immunoassays, the label on the 
protein is altered by the binding of the protein to its specific binding 
agent. This alteration in the labelled protein results in a decrease or 
increase in the signal emitted by label, so that measurement of the label 
at the end of the immunoassay allows for detection or quantitation of the 
protein. 
ATP-sensitive potassium channel proteins may also be determined by a 
variety of noncompetitive immunoassay methods. For example, a two-site, 
solid phase sandwich immunoassay is used. In this type of assay, a binding 
agent for the protein, for example an antibody, is attached to a solid 
phase. A second protein binding agent, which may also be an antibody, and 
which binds the protein at a different site, is labelled. After binding at 
both sites on the protein has occurred, the unbound labelled binding agent 
is removed and the amount of labelled binding agent bound to the solid 
phase is measured. The amount of labelled binding agent bound is directly 
proportional to the amount of protein in the sample. 
Western blot analysis can also be done to determine the presence of 
ATP-sensitive potassium channel proteins in a sample. Electrophoresis is 
carried out, for example, on a tissue sample suspected of containing the 
protein. Following electrophoresis to separate the proteins, and transfer 
of the proteins to a suitable solid support such as a nitrocellulose 
filter, the solid support is then incubated with an antibody reactive with 
the protein. This antibody may be labelled, or alternatively may be it may 
be detected by subsequent incubation with a second labelled antibody that 
binds the primary antibody. 
The immunoassay formats described above employ labelled assay components. 
The label can be in a variety of forms. The label may be coupled directly 
or indirectly to the desired component of the assay according to methods 
well known in the art. A wide variety of labels may be used. The component 
may be labelled by any one of several methods. Traditionally a radioactive 
label incorporating .sup.3 H, .sup.125 I, .sup.35 S, .sup.14 C, or .sup.32 
P was used. Non-radioactive labels include ligands which bind to labelled 
antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies 
which can serve as specific binding pair members for a labelled ligand. 
The choice of label depends on sensitivity required, ease of conjugation 
with the compound, stability requirements, and available instrumentation. 
For a review of various labelling or signal producing systems which may be 
used, see U.S. Pat. No. 4,391,904, which is incorporated herein by 
reference. 
Antibodies reactive with a particular protein can also be measured by a 
variety of immunoassay methods. For a review of immunological and 
immunoassay procedures applicable to the measurement of antibodies by 
immunoassay techniques, see Basic and Clinical Immunology 7th Edition (D. 
Stites and A. Terr ed.) supra, Enzyme Immunoassay, E. T. Maggio, ed., 
supra, and Harlow and Lane, Antibodies, A Laboratory Manual, supra. 
In brief, immunoassays to measure antisera reactive with ATP-sensitive 
potassium channel proteins can be either competitive or noncompetitive 
binding assays. In competitive binding assays, the sample analyte competes 
with a labeled analyte for specific binding sites on a capture agent bound 
to a solid surface. Preferably the capture agent is a purified recombinant 
ATP-sensitive potassium channel protein produced as described above. Other 
sources of ATP-sensitive potassium channel proteins, including isolated or 
partially purified naturally occurring protein, may also be used. 
Noncompetitive assays are typically sandwich assays, in which the sample 
analyte is bound between two analyte-specific binding reagents. One of the 
binding agents is used as a capture agent and is bound to a solid surface. 
The second binding agent is labelled and is used to measure or detect the 
resultant complex by visual or instrument means. A number of combinations 
of capture agent and labelled binding agent can be used. A variety of 
different immunoassay formats, separation techniques and labels can be 
also be used similar to those described above for the measurement of 
ATP-sensitive potassium channel proteins. 
This invention also embraces kits for detecting the presence of 
ATP-sensitive potassium channel proteins in tissue or blood samples which 
comprise a container containing antibodies selectively immunoreactive to 
the protein and instructional material for performing the test. The kit 
may also contain other components such as ATP-sensitive potassium channel 
proteins, controls, buffer solutions, and secondary antibodies. Kits for 
detecting antibodies to ATP-sensitive potassium channel proteins comprise 
a container containing an ATP-sensitive potassium channel protein, 
instructional material and may comprise other materials such as secondary 
antibodies and labels as described herein. 
This invention further embraces diagnostic kits for detecting DNA or RNA 
encoding ATP-sensitive potassium channel proteins in tissue or blood 
samples which comprise nucleic probes as described herein and 
instructional material. The kit may also contain additional components 
such as labeled compounds, as described herein, for identification of 
duplexed nucleic acids.