JP2003527818A - Novel ESK potassium channel polypeptide and polynucleotide compositions - Google Patents

Abstract

  (57) [Summary] The present invention provides novel potassium channel subunits (ESKs), and polynucleotides that identify and encode ESKs. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding ESK. The present invention also provides antibodies to the ESKs, and methods for diagnosing and treating diseases associated with ESK expression, and screening assays using the polypeptide, nucleotide, and antibody compositions.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to novel human potassium channel polypeptide and polynucleotide compositions, the production of these compositions, and the use of these compositions in the diagnosis, prevention and treatment of disease states. Regarding

BACKGROUND OF THE INVENTION Potassium channels are a heterogeneous group of ion channels that allow the selective permeation of potassium ions across the plasma membrane, with details of the activation mechanism, the potential range of activity, and Different reaction rate characteristics (Hille, B. (1992) Ioni
c Channels of Excitable Membranes, 2nd
Ed., Sinauer, Sunderland, MA; Laterre, R .; And Miller, C.I. (1983) J. Memb. Biol. 7: 11-30)
. They have numerous physiological functions (eg action potential repolarization, cardiac rhythm regulation (c
ardiac pacemaking), neuron burst, muscle contraction, hormone secretion, vascular tone regulation, renal ion reabsorption,
Learning and memory, and cell proliferation and differentiation).

Most potassium channel genes isolated and characterized to date are polypeptides containing six putative transmembrane segments and one single pore-forming P domain (ie, 1P / 6TM subunit). Code Channels containing 1P / 6TM K + channels from different families (Drosophila
"Ether-a-go-go" (represented by an eag) K + channel) contains a putative cyclic nucleotide binding domain (cNBD) at the carboxyl terminus.
Although the eag superfamily is somewhat related to the shaker K + channel superfamily, the structural differences between the two are substantial, with less than 20% identity in the hydrophobic core region. The putative cNBDs in the eag members share substantial similarity to cNBDs present in cyclic nucleotide-gated cation channels (Warmker et al. (199).
1) Science 252: 1560-1564.

In the eag superfamily, three channel subfamilies are currently known, eag, erg (eag-related genes), and elk (eag-like K).
+ Channel). Generally, two members of the same subfamily from different species have about 65 in the region spanning the S1 to cNBD segment.
Share 70% amino acid sequence identity. In contrast, two different subfamily members from the same species share only about 40-50% amino acid sequence identity over the same region (Warmke and Ganetzky (1994) P.
． N. A. S. 91: 3438-3442).

Channels in the eag superfamily from different animal species are often
It develops channel currents with completely different properties. For example, when expressed in Xenopus oocytes, the Drosophila eag channel
Is permeable to both K + and Ca ++ ions, and cAMP
But the mouse eag channel is not permeable to Ca ++ and is not regulated by cAMP (Bruggemann et al. (1993).
Nature 365: 445-448; Ludwig et al. (1994), EMBO.
J. 13: 4451-4458; Trudea et al. (1995), Science.
e269: 92-95). Three known mammalian members of the erg subfamily (human erg1 (HERG), rat erg2, and rat erg3) are activated by membrane depolarization, but their inactivation is faster than their activation. .
Unlike erg1 and erg2, which are active above the activation threshold, erg3 is active below the activation threshold and is therefore expected to affect the basal activity of excitable cells. The erg1 channel is abundantly expressed in the heart and brain, whereas erg2 and erg3 are absent in the heart but have a broad expression pattern in the nervous system. All three are expressed in some sympathetic ganglia (Si et al. (1997) J. Neurosci. 17 (24) 9423.
~ 9432).

Mutations in channels in the eag superfamily have been found to cause profound defects in animal physiology. For example, mutations in Drosophila eag cause excessive membrane excitability in motor neurons resulting in foot-swing behavior in mutant flies. Mutations in the Drosophila erg can cause paralysis in affected flies. The mutation in HERG is
Associated with the fatal heart disease long QT type 2 (LQT2) (C
urran et al. (1995) Cell 80: 795-803; Sanguien.
tti et al. (1995) Cell 81: 299-301).

[0007] Potassium channels are associated with various disease states. In some diseases and disorders, abnormal ion channels are believed to be the causative factor, while other diseases appear to result from improper regulation of otherwise normal ion channels. Diseases that are believed to have particular association with potassium channels include neurological disorders, cardiovascular disorders, skeletal muscle disorders and proliferative disorders (eg, cancer).

The discovery of novel channel polypeptides representing novel subfamilies of the eagK + channel subfamily, as well as the polynucleotides encoding them, has been
The need in the art is met by providing novel compositions that are useful in the treatment of various diseases associated with ion channel dysfunction.

SUMMARY OF THE INVENTION The present invention is based on the discovery of the human K + channel, ESK1 (eag-like K + subfamily), which represents a novel ESK subfamily of the eagK + channel superfamily. The present invention is directed to the region corresponding to residues 212-668 of the polypeptide identified herein as SEQ ID NO: 2, which region is identified herein as SEQ ID NO: 5. It comprises an isolated ESK potassium channel polypeptide comprising an amino acid sequence having at least 60%, preferably at least 65-70%, more preferably at least 80% and most preferably 90% sequence identity. The invention also provides (i) fragments of ESK capable of interacting with other proteins, peptides or chemicals, such interactions altering the functional properties or cell localization / compartmental localization of ESK, And (ii) a pharmaceutical composition comprising ESK or ESK fragment. The invention also includes potassium channels that include one or more ESK polypeptides.

In a particular embodiment, the present invention comprises an isolated ESK1 polypeptide comprising an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 2. In another embodiment, the polypeptide is at least 70 relative to SEQ ID NO: 2.
Include sequences that are%, 80%, 90% or 95% identical. In a more particular embodiment, the polypeptide has the sequence SEQ ID NO: 2 or SEQ ID NO: 4.

In another aspect, the invention provides an isolated polynucleotide having a sequence encoding ESK as described above, or an isolated polynucleotide having a sequence that is complementary to the ESK coding sequence, and A composition containing the polynucleotide is included. This polynucleotide includes mRNA, cRNA, DNA, cDNA,
It can be genomic DNA, peptide nucleic acids, as well as their antisense analogs. The polypeptide can encode an ESK polypeptide that includes a sequence that has at least 60% sequence identity to SEQ ID NO: 5. In another embodiment,
This polynucleotide encodes an ESK1 polypeptide having at least 70% amino acid sequence identity to SEQ ID NO: 2. This polynucleotide is
For example, a polynucleotide sequence identified as SEQ ID NO: 1 or its complement may include a coding sequence that hybridizes under conditions of high stringency. In a particular embodiment, the polynucleotide is SEQ ID NO: 1 or SEQ ID NO: 3.
Has a sequence identified as The present invention also contemplates fragments of this polynucleotide, preferably at least 12 nucleotides in length, more preferably at least 20 or 30 nucleotides in length.

In another embodiment, the invention comprises a nucleic acid molecule of at least 12 nucleotides in length that specifically hybridizes under stringent conditions to a polynucleotide sequence encoding any of the above polypeptides. This nucleic acid molecule is
It can be NA, DNA, genomic DNA, peptide nucleic acids, and their antisense analogs. The invention also includes polynucleotide sequences that include the complements of polynucleotides that encode any of the above polypeptides.

Recombinant expression comprising a polynucleotide or fragment encoding the above ESK and a regulatory sequence operably linked to the polynucleotide and effective for expression of the protein in a selected host. Vectors are also disclosed. Preferably the coding sequences are provided above. In a related aspect,
The present invention includes a host cell containing this vector.

The present invention comprises the step of recombinantly culturing a recombinant prokaryotic or eukaryotic host cell containing a nucleic acid sequence encoding ESK under conditions which promote the expression of this protein, and subsequently, Further included is a method of producing ESK by recovering the protein from the host cell or cell culture medium.

In yet another aspect, the invention includes an antibody specific for ESK.
This antibody has diagnostic and therapeutic applications, especially in the treatment of neurological and neurodegenerative diseases. Treatment methods using antisense or coding sequence polynucleotides to inhibit or enhance the level of ESK are also contemplated, as are treatment methods using antibodies specific for ESK. The present invention also includes methods of altering the expression level of ESK by gene therapy techniques to achieve therapeutic benefit in a patient.

Diagnostic methods for detecting the level of ESK in a particular tissue sample, and diagnostic methods for detecting the level of expression of ESK in a tissue also form part of the invention, in one embodiment, biology. A method of detecting a polynucleotide encoding ESK in a biological sample comprises the steps of: (a) hybridizing a complement of a polynucleotide encoding ESK to a nucleic acid material of a biological sample, Forming a hybridization complex by
And (b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding ESK in the biological sample. Methods of detecting mutations in the coding region of ESK are also contemplated.

Screening methods that use ESK to identify candidate compounds that can bind to ESK and modulate the activity of ESK also form part of the invention.
Exemplary methods are: (a) contacting a test compound with ESK, (b) measuring the effect of this test compound on the activity of ESK, and (c) using this test compound as a candidate compound, the activity of ESK. Selecting whether the effect of this candidate compound on the selected threshold level is exceeded. The measured activity is
For example, it may be the establishment or regulation of potassium conductance. In one embodiment, the test compound is a component of a combinatorial library.
In another embodiment, the test compound is an antibody specific for ESK protein.

The invention also includes, in a related aspect, the compounds identified by the screening methods described above, including purified agonists and antagonists. The invention further includes a purified antibody that specifically binds to the above polypeptide.

These and other objects of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings.

(Short Description of Sequence) SEQ ID NO: 1 is a nucleic acid sequence encoding hESK1; SEQ ID NO: 2 is a deduced amino acid translation of SEQ ID NO: 1; SEQ ID NO: 3 codes for a putative hESK1 splice variant. SEQ ID NO: 4 is the deduced amino acid translation of the putative hESK1 splice variant; SEQ ID NO: 5 is the amino acid sequence of residues 212-668 of SEQ ID NO: 2; SEQ ID NO: 6 is the hERG ( GenBank PID g487738); and SEQ ID NO: 7 is human EST U69184 (GenBank NID g27).
39408; accession number U69184).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions Unless otherwise indicated, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Have. The practitioner is particularly aware of definitions and terminology in the art such as Sambrook et al. (1989) Molecular.
ar Cloning: A Laboratory Manual (2nd edition),
Cold Spring Harbor Press, Plainview, N
． Y. And Ausubel FM et al. (1989) Current Proto.
cols in Molecular Biology, John Wiley
& Sons, New York, N.M. Y. Be instructed to. It is to be understood that this invention is not limited thereto, as the particular methodology, protocols, and reagents described may vary.

As used herein, the term “polypeptide” refers to a compound composed of single chain amino acid residues linked by peptide bonds. As used herein, the term "protein" may be synonymous with the term "polypeptide" or may even refer to a complex of two or more polypeptides.

As used herein, “channel” or “channel protein” refers to a multi-subunit protein containing two or more P domain-containing polypeptide subunits, and from multimers of the same polypeptide ( “Homomeric” channels) or from different polypeptides (“Heteromeric” channels). Channel proteins may also include "accessory subunits" that regulate the activity of that channel.

“A polypeptide belonging to the eag superfamily” means a possible P domain, six putative transmembrane domains (S1 to S6), a cyclic nucleotide binding domain (
cNBD), and from S1 to this cNBD
It has about 40% or more sequence identity to the aligned corresponding regions of another polypeptide member of the eagK + superfamily (eg, eag or human erg1 (hERG)) in the region across the segment.

“Eag-like K + polypeptide” or “ESK polypeptide” refers to eag
A region spanning the S1 to cNBD segment that corresponds to residues 212-668 of the polypeptide that belongs to the superfamily and is identified herein as SEQ ID NO: 2, which region is referred to herein as SEQ ID NO: 2. 5)), further comprising a sequence having a sequence identity of at least 60%, preferably at least 65-70%, more preferably at least 80%, and most preferably at least 90%. Say.

“ESK1” includes a sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity to SEQ ID NO: 2, Refers to ESK polypeptide.

As used herein, the designation “ESK” or “ESK1” refers to
Unless otherwise indicated by context, full-length polypeptides and fragments thereof are intended to be included.

The term “ESK channel” refers to a multimeric potassium channel that comprises at least one ESK polypeptide.

The term “mature ESK” refers to an ESK polypeptide that is present in a cell after post-translational processing, eg, after removal of the signal sequence.

The term “altered” when referring to a polypeptide of the present invention, refers to a natural process (eg, processing or other post-translational modification) or chemical modification techniques well known in the art. It means a polypeptide which is modified by either. Among the many known modifications that may exist are acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of lipids or lipid derivatives, methylation, myristylation, pegylation. (Pegylation
), Prenylation, phosphorylation, ubiquitination or any similar process.

The term “biologically active” refers to the structural conformation of a naturally occurring ESK polypeptide,
Regulatory or biochemical functions, including the ability to support potassium ion conductance when it self-associates into homomeric channels or when it associates with other channel polypeptides into heteromeric channels. (Not limited to). Similarly, "immunologically active" means that a natural, recombinant, or synthetic ESK, or any fragment thereof, elicits a specific immune response in a suitable animal or cell and is directed to specific antibodies. It defines the ability to bind.

The term “fragment,” when referring to ESK, is a polypeptide having an amino acid sequence that is the same as some but not all of the amino acid sequence of ESK and retains at least one function or activity of ESK. Or, it means a polypeptide that can interact with ESK, other proteins, peptides, or other molecules to alter the function or activity of ESK channels or cell / cell compartment localization. Contemplated fragments include ESK fragments that retain the ability of an ESK channel to bind a ligand, ESK fragments that block the binding of a ligand to an ESK channel, or ESK fragments that retain the immunological activity of an ESK. , But not limited to these. The fragment preferably comprises at least 20, more preferably at least 50 consecutive amino acid residues of ESK.

The term “portion”, when referring to a polypeptide of the invention, is a polypeptide having an amino acid sequence which is the same as part of the amino acid sequence of the invention or variants thereof, By a polypeptide is meant not necessarily possessing any biological function or activity.

“Conservative substitution” refers to the replacement of an amino acid of one class by an amino acid of the same class, where the class is common physicochemical amino acid side chain properties and (eg, standard Dayhoff frequency exchange matrix or BLOSU
It is defined by a high substitution frequency in homologous proteins found in nature (as determined by the M matrix). When classified as above, the six general classes of amino acid side chains include: Class I (Cys)
Class II (Ser, Thr, Pro, Ala, Gly); Class III (A
sn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Ty).
r, Trp). For example, a substitution with Asp for another Class III residue (eg Asn, Gln or Glu) is a conservative substitution.

“Non-conservative substitution” refers to the substitution of an amino acid from one class with an amino acid from another class; eg, Asp, Asn, of the Class II residue Ala,
Substitution with class III residues such as Glu or Gln.

“Optimal alignment” is defined as the alignment that produces the highest percent identity score. Such alignments can be performed using various commercially available sequence analysis programs (eg, ktup
= 1, the local alignment program LALIGN) with default parameters and default PAM). The preferred alignment is the default parameters (open gap penalty = 10.0, extended ga).
p penalty = 0.1, and BLOSUM similarity m
CLUS in MacVector, operated using (including atrix)
Alignment performed using the TAL-W program.

“Percent sequence identity” with respect to two amino acid or polynucleotide sequences refers to the percentage of residues that are identical in the two sequences when the two sequences are optimally aligned. Thus, 80% amino acid sequence identity means 80% amino acid identity in two or more optimally aligned polypeptide sequences. If it is necessary to insert a gap in the first sequence in order to optimally align the first sequence with the second sequence, the percent identity is calculated as the residue paired with the corresponding amino acid residue. Is calculated using only (ie, this calculation is
Does not consider residues in the second sequence that are present in "gaps" of the first sequence).

The first polypeptide region is essential for these regions when compared to the second polypeptide region, when the sequences containing these regions are aligned using a sequence alignment program as described above. If they have the same spread, they are said to correspond. Corresponding polypeptide regions typically contain a similar number of residues, if not identical. However, it is understood that the corresponding regions may contain insertions or deletions of residues with respect to each other, as well as some differences in their sequence.

“Corresponding” polynucleotide or polypeptide fragments typically contain a similar number of residues, if not identical. However, it is understood that the corresponding fragments may contain insertions or deletions of residues with respect to each other, as well as some differences in their sequence.

The term “sequence identity” means the identity of nucleic acid sequences or amino acid sequences in two or more aligned sequences aligned as described above.

“Sequence similarity” between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with its conservative amino acid substitutions on the sequence of a second polypeptide. Thus, 80% protein sequence similarity means that 80% of amino acid residues in two or more aligned protein sequences are conservative amino acid residues, ie, conservative substitutions. .

“Hybridization” includes any process by which a strand of nucleic acid joins with a complementary nucleic acid strand through base pairing. Thus, strictly speaking, the term refers to the ability of the complement of the target sequence to bind the test sequence, or vice versa.

“Hybridization conditions” are based on the melting temperature (Tm) of the nucleic acid binding complex or probe, and are typically classified by the degree of “stringency” at which hybridization is measured. . For example, "maximal stringency" typically occurs at about Tm-5 [deg.] C (5 [deg.] Below the Tm of the probe); "high stringency" is about 5-10 below its Tm.
Occurs at <RTIgt; 0 C </ RTI>;"intermediatestringency" is about 1 <Tm of the probe.
It occurs at 0-20 ° C lower; and "low stringency" is about 2 below its Tm.
It occurs at 0-25 ° lower. For practical purposes, maximum stringency conditions may be used to identify nucleic acid sequences that have strict or near strict identity with the hybridization probe; while high stringency conditions are used with the probe. Nucleic acid sequences having a sequence identity of greater than about 80% are identified.

An example of “high stringency” conditions is hybridization at about 65 ° C. in about 5 × SSPE, and washing at about 65 ° C. in about 0.1 × SSPE (where 1 ×. SSPE = 0.15 sodium chloride, 0.010M sodium phosphate, and 0.001M disodium EDTA).

As used herein, the term “gene” means the segment of DNA involved in the production of polypeptide chains; a gene is a region before and after the coding region (eg, 5 ′). Untranslated (5'UTR) or "leader" sequence, and 3 '
UTRs or "trailer" sequences), as well as intervening sequences (introns) between individual coding segments (exons).

“Isolated polynucleotide having a sequence encoding ESK” refers to ES
The coding sequence of K is (i) isolated and (ii) combined with a further coding sequence (eg a fusion protein or signal peptide), wherein the ESK coding sequence is the dominant coding sequence, (iii) In combination with non-coding sequences (eg introns and control elements (eg promoter and terminator elements) or 5 ′ untranslated region and / or 3 ′ untranslated region) effective for expression of the coding sequence in a suitable host, and / Or (iv)
A polynucleotide that includes an ESK coding sequence in a vector or host environment in which it is a heterologous gene.

The terms “heterologous DNA” and “heterologous RNA” refer to nucleotides that are not endogenous to the part of the cell or genome in which they are present. Generally, such nucleotides are used for transfection, microinjection,
It is added by electroporation. Such nucleotides generally include at least one coding sequence, but this coding sequence need not be expressed.

The term “isolated” refers to the substance in which it is in its original environment (
For example, if it is naturally occurring, it means that it has been removed from its natural environment). For example, a naturally occurring polynucleotide or polypeptide present in a living animal has not been isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting substances in the natural system is It has been isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, and such a vector or composition is part of its natural environment. It is still isolated in that it is not.

The term “fragment”, when referring to an ESK coding sequence, refers to its E
By a polynucleotide having the same nucleic acid sequence as the nucleic acid sequence portion of the SK coding sequence, but not all the same. This fragment preferably contains the ESK coding sequence of at least 12 consecutive bases.

The term “expression vector” refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of the appropriate expression vector is
It is within the knowledge of a person skilled in the art.

The term “substantially purified” is removed from its natural environment,
A molecule, either polynucleotide or polypeptide, that is isolated or separated and is free of at least 60%, preferably 75%, and most preferably 90% free of other components with which it is naturally associated. Say.

A “variant” polynucleotide sequence can encode a “variant” amino acid sequence that has been altered by one or more amino acids from the reference polypeptide sequence. The variant polynucleotide sequence may encode a variant amino acid sequence containing "conservative" substitutions, wherein the substituted amino acid has similar structural or chemical properties to the amino acid it replaces. Have. Additionally or alternatively, the variant polynucleotide sequence may encode a variant amino acid sequence that includes "non-conservative" substitutions, wherein the substituted amino acid differs from the amino acid that it replaces by a structural characteristic or Has chemical properties. Variant polynucleotides can also encode variant amino acid sequences that include amino acid insertions or deletions, or both. Further, the variant polynucleotide may encode the same polypeptide as the reference polynucleotide sequence, but which has one or more bases changed from that of the reference polynucleotide sequence due to the degeneracy of the genetic code. Has an array.

An “allelic variant (variant)” is an alternative form of a polynucleotide sequence that may have one or more nucleotide substitutions, deletions or additions, the function of the encoded polypeptide. Is substantially unchanged.

“Alternative splicing” is the process by which multiple polypeptide isoforms are produced from a single gene, and during processing of some (but not all) transcripts of that gene. Including splicing that brings together non-consecutive exons. Thus, a particular exon can be linked to any one of several alternative exons to form a messenger RNA. This alternatively spliced mRNA produces polypeptides ("splice variants") that are common in some parts but different in others.

An ESK “splice variant” when referred to in the context of an mRNA transcript
It is an mRNA produced by alternative splicing of the coding region (ie, exon) from the ESK gene.

“Splice variants” of ESK, when referred to in the context of the protein itself,
E encoded by an alternatively spliced ESK mRNA transcript, E
It is a translation product of SK.

A “variant” amino acid sequence or polynucleotide sequence is a variant amino acid sequence, or a variant polynucleotide sequence encoding a variant amino acid sequence, which is the biological activity of a naturally occurring protein. Has a biological activity that is significantly altered from.

A “deletion” is defined as a change in either the nucleotide or amino acid sequence, where each lacks one or more nucleotide or amino acid residues.

“Insertion” or “addition” is a change in a nucleotide or amino acid sequence that results in the addition of one or more nucleotide or amino acid residues, respectively, when compared to the naturally occurring sequence. .

“Substitution” results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.

The term “modulate”, as used herein, refers to a change in the activity of a polypeptide of the invention. Modulation can be associated with an increase or decrease in the biological activity, binding characteristics, or any other biological, functional, or immunological properties of the molecule.

The term “agonist” as used herein, when bound to a channel of the invention, induces, increases, or extends the duration of the biological activity mediated by this channel. Refers to a molecule that regulates the activity of this channel. An agonist can itself be a polypeptide, nucleic acid, carbohydrate, lipid, or derivative thereof, or it can be any other ligand that binds to this channel and modulates its activity.

The term “antagonist” as used herein, when bound to a channel of the invention, blocks, reduces, or reduces the duration of biological activity mediated by this channel. Refers to a molecule that regulates the activity of this channel. An antagonist may itself be a polypeptide, nucleic acid, carbohydrate, lipid, or derivative thereof, or any other ligand that binds to this channel and modulates its activity.

The term “humanized antibody” refers to a human antibody that has one or more amino acids substituted in its non-antigen binding region in order to more closely resemble it, but still retain the original binding activity of the antibody. Refers to an antibody molecule.

“Treatment of a disease” refers to the administration of a therapeutic substance that is effective to reduce the symptoms of the disease and / or reduce the severity of the disease.

II. Polynucleotide Encoding ESK Polypeptide The present invention provides an isolated ESK polypeptide, and an isolated polynucleotide encoding this polypeptide. As more fully defined in Section III below, ESK represents (i) a novel subfamily of the eag potassium channel superfamily, and (ii) to the region identified as SEQ ID NO: 5, It includes amino acid sequences that have at least 60% sequence identity. An exemplary ESK polypeptide, human ESK1 (hESK1), has the sequence identified as SEQ ID NO: 2.

As shown in FIG. 1, SEQ ID NO: 1 is a nucleic acid sequence of 3829 bases. It contains an open reading frame encoding a 1080 amino acid polypeptide identified herein as the hESK1 polypeptide having the sequence of SEQ ID NO: 2. Putative hESK1 splice variant (nucleotide 2 of SEQ ID NO: 1)
Containing a 33 nucleotide insert between 819 and 2820, resulting in the 3862 base sequence identified herein as SEQ ID NO: 3) is amino acids 855 and 85.
A translation product is produced which is identical to SEQ ID NO: 2 except for an 11 amino acid insert between 6 and 6, resulting in a 1091 amino acid polypeptide SEQ ID NO: 4.

A polynucleotide encoding hESK1 was found in a cDNA library derived from human brain. The coding sequence was identified, cloned, and sequenced substantially as described in Example 1. Briefly, a biotin-labeled human probe based on the human EST sequence (GenBank accession number U69184; SEQ ID NO: 7) was used to assay brain cDNA by solution hybridization.
The target cDNA molecule was captured from the A library. After secondary and tertiary screening with radiolabeled oligonucleotide probes or cDNA probes, positive colonies were cultured and the plasmid cDNA was isolated and sequenced and identified herein as SEQ ID NO: 1. Resulting in the construction and identification of nucleic acid sequences.

Northern analysis, performed as described in Example 2, showed that in the tissues tested hESK1 transcript expression was observed only in the brain, predominantly the forebrain.

A. Polynucleotide Compositions The polynucleotides of the invention include ESK coding sequences, and sequences complementary to the coding coding sequences, as well as novel fragments of the polynucleotides. These polynucleotides can be in the form of RNA or in the form of DNA, and include mRNA, cRNA, synthetic RNA and synthetic DNA and their analogs, cDNA, peptide nucleic acids, and genomic DNA. These polynucleotides can be double-stranded or single-stranded, and if single-stranded can be the coding strand or non-coding (antisense, complementary) strand.

In a general embodiment, the polynucleotide of the invention is SEQ ID NO: 1, SEQ ID NO: 3 under stringent conditions, preferably under high stringency conditions.
, Or to sequences identified as their complements.
Exemplary hybridization conditions are described below in Section IIB. In another embodiment, the polynucleotide of the invention comprises SEQ ID NO: 5 and at least 60
Encodes an ESK polypeptide that comprises a sequence with% sequence identity. In another embodiment, the polynucleotide encodes an ESK1 polypeptide having at least 70% amino acid sequence identity with SEQ ID NO: 2 or SEQ ID NO: 4. In another embodiment, the polynucleotide of the invention is SEQ ID NO: 1, SEQ ID NO: 3,
Or it has at least 70%, preferably at least 80% or 90% sequence identity with the sequences identified as their complements. In a more particular embodiment, the polynucleotide of the invention has the sequence identified as SEQ ID NO: 1 or SEQ ID NO: 3.

The polynucleotide of the present invention comprises (i) isolating the coding sequence of ESK to (i)
i) additional coding sequences (eg fusion proteins, or signal peptides)
(Where the ESK coding sequence is the dominant coding sequence),
(Iii) non-coding sequences (eg, introns and control elements (eg, promoter and terminator elements), or 5 ′ untranslated region and / or 3 ′ untranslated region) effective for expression of the coding sequence in a suitable host. And / or (iv) in the vector or host environment where the ESK coding sequence is a heterologous gene.

Polynucleotides of the invention include polypeptide fragments of ESK (eg,
Extracellular fragment or intracellular fragment, cleaved from the transmembrane domain of ESK.

The polynucleotides of the present invention may also have a protein coding sequence fused in frame to a marker sequence that allows for purification of ESK. The marker sequence can be, for example, a hexahistidine tag that provides for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a mammalian host (eg, COS-7). If cells are used, it can be a hemagglutinin (HA) tag. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I. et al.
1984) Cell 37: 767).

Of a polynucleotide (typically also referred to herein as an oligonucleotide) having at least 12 bases, preferably 20 or 30 bases, corresponding to a region of the polynucleotide of the coding sequence or its complement. New uses are also contemplated. This polynucleotide can be used as a probe, a primer, an antisense agent, etc. according to a known method.

(B. Preparation of Polynucleotide) The polynucleotide of the present invention uses an oligonucleotide probe capable of hybridizing to the polynucleotide encoding the ESK and the fragment disclosed above, or capable of PCR-amplifying the polynucleotide. And can be obtained by screening a cDNA library. CDNA libraries prepared from various tissues are commercially available, and procedures for screening and isolating cDNA clones are well known to those of skill in the art. Such a technique is, for example, S
ambrook et al. (1989) Molecular Cloning: A La.
laboratory Manual (2nd edition), Cold Spring Har
bor Press, Plainview, N.M. Y. And Ausubel F
M et al. (1989) Current Protocols in Molecule
ar Biology, John Wiley & Sons, New York,
N. Y. It is described in.

Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe, and are typically classified by the degree of “stringency” under the conditions under which hybridization is measured. For example, "maximum stringency" is typically about Tm-5 ° C (5 than the Tm of the probe.
"High stringency" occurs about 5-10 ° below its Tm; "intermediate stringency" occurs about 10-2 below the Tm of the probe.
It occurs at 0 ° below; and "low stringency" is about 20-2 below its Tm.
It occurs at 5 ° lower. In practice, maximum stringency conditions can be used to identify nucleic acid sequences that have exact or near-exact identity with the hybridization probe: while high stringency conditions are used with that probe. Used to identify nucleic acid sequences having a sequence identity of greater than about 80%. Examples of high stringency conditions include hybridization in about 5 × SSPE at about 65 ° C., and about 0.1 × SSPE (where 1 × SSPE is
= 0.15 sodium chloride, 0.010 M sodium phosphate, and 0.0
Washing conditions of about 65 ° C. in 01 M disodium EDTA) are included.

The polynucleotides of the invention include promoters, regulatory elements, and 5 ′
It can be extended to obtain upstream and downstream sequences, such as the untranslated region and the 3'untranslated region (UTR). Extension of this available transcript sequence can be accomplished by a number of methods known to those of skill in the art, such as PCR or primer extension (Sambrook et al., Sambrook et al.
(See above)) or, for example, the Marathon RACE kit (Clontec).
h, catalog number K1802-1).

Alternatively, the technique of “restriction site” PCR using universal primers to recover flanking sequences flanking a known locus (Gobinda et al. (1993)
) PCR Methods Appl. 2: 318-22) can be used. First, genomic DNA is amplified in the presence of a primer for the linker sequence and a primer specific for a known region. This amplified sequence was cloned using the same linker primer and another specific primer inside the first primer,
Subject to the second PCR. The product of each round of PCR is transcribed with an appropriate RNA polymerase and sequenced with reverse transcriptase.

Inverse PCR is based on known primers based on divergent primers.
mer) can be used to amplify or extend the sequence (Trigl).
ia T et al. (1988) Nucleic Acids Res 16: 8186.
). This primer is based on OLIGO (R) 4.06 Primer Analysis.
sis Software (1992; National Bioscience)
es Inc. , Plymouth, Minn. ) Or another suitable program, having a length of 22 to 30 nucleotides and a GC content of 50% or more,
And can be designed to anneal to the target sequence at a temperature of about 68-72 ° C. This method uses several restriction enzymes to generate appropriate fragments within the known region of the gene. This fragment is then circularized by intramolecular ligation and used as a PCR template.

Capture PCR (Lagerstrom M et al. (19
91) PCR Methods Appl. 1: 111-19) is a method for PCR amplification of DNA fragments flanking known sequences in human and yeast artificial chromosomal DNA. The capture PCR can also be performed by
Multiple restriction enzyme digests and ligations are required to place the engineered double-stranded sequence within adjacent portions of the A molecule.

Another method that can be used to recover flanking sequences is Parker, JD et al.
1991; Nucleic Acids Res 19: 3055-60). Further, those skilled in the art will walk through the genomic DNA.
in), PCR, nested primer
er) and PromoterFinder ^™ libraries can be used (C
lontech, Palo Alto, CA). This process avoids the need to screen the library and is useful in finding intron / exon junctions. A preferred library for screening full-length cDNAs is a library size-selected to contain larger cDNAs. Also, a randomly primed library is preferred in that it contains more sequences that include the 5'region and the upstream region of the gene. Randomly primed library is oligo d (T) library full length cd
It may be particularly useful when not producing NA. Genomic libraries are useful for extension into 5'untranslated regulatory regions.

The polynucleotides and oligonucleotides of the invention can also be prepared by solid phase methods according to known synthetic methods. Typically, fragments of up to about 100 bases are individually synthesized and then ligated to form a continuous sequence of up to several hundred bases.

C. Application of Polynucleotides The polynucleotide coding sequences and novel oligonucleotides of the invention have various uses in: (1) ESK synthesis, (2) diagnostics, (3) gene mapping. , And (4) treatment.

C1. Synthesis of ESK A polynucleotide sequence encoding ESK, a splice variant, a fragment of this polypeptide, a fusion protein, or their functional equivalents according to the invention (summarized herein). Referred to as "ESK") in recombinant DNA molecules that direct the expression of ESK in a suitable host cell. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences that encode substantially the same amino acid sequence or that encode functionally equivalent amino acid sequences have been used to clone and express ESK. obtain.

As will be appreciated by those in the art, it may be advantageous to generate nucleotide sequences encoding ESK that carry non-naturally occurring codons. Preferred Codons for Certain Prokaryotic or Eukaryotic Hosts (Murray, E. et al. (1989) N
uc Acids Res 17: 477-508), for example, to increase the rate of ESK polypeptide expression, or to have desired properties (eg, longer half-life than transcripts generated from naturally occurring sequences). It can be selected to produce a recombinant RNA transcript.

The polynucleotide sequences of the present invention may be engineered to alter the ESK coding sequence for a variety of reasons, including but not limited to alterations that alter cloning, processing and / or expression of gene products. Can be done. For example, alterations may be made using techniques well known in the art (eg, site-directed mutagenesis) to insert new restriction sites, alter glycosylation patterns, and to change codon preference. ), To generate splice variants, and so on.

The present invention also includes recombinant constructs that include one or more of the sequences broadly described above. This construct comprises a vector, such as a plasmid vector or a viral vector, into which the sequences of the invention have been inserted in the forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences operably linked to the sequences of the invention (eg, including a promoter). Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are also described in Sambrook et al. (Supra).

The present invention also relates to host cells genetically engineered with the vectors of the invention, and the production of proteins and polypeptides of the invention by recombinant techniques. Host cells are genetically engineered (ie, transduced, transformed, or transfected) with a vector of the invention, which can be, for example, a cloning vector or an expression vector. This vector can be in the form of, for example, a plasmid, virus particle, phage or the like. The engineered host cells can be cultured in conventional nutrient media modified as appropriate to activate the promoter, select for transformants, or amplify the ESK gene. The culture conditions (eg, temperature, pH, etc.) are those previously used with the host cell selected for expression, and will be apparent to those of skill in the art.

The polynucleotides of the present invention can be included in any one of a variety of expression vectors for expressing the polypeptide. Such vectors include chromosomal sequences, non-chromosomal sequences and synthetic DNA sequences (for example, derivatives of SV40; bacterial plasmids; phage DNA: baculovirus; yeast plasmids; vectors derived from a combination of plasmid and phage DNA, viral DNA. (Eg, vaccinia, adenovirus, fowlpox virus, and pseudorabies)). However, any other vector may be used, so long as it is replicable and viable in the host. The appropriate DNA sequence may be inserted into this vector by a variety of procedures. Generally, this DNA sequence is inserted into the appropriate restriction endonuclease site by procedures known in the art. Such procedures and related subcloning procedures are considered to be within the skill of those in the art.

The DNA sequence in this expression vector is operably linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis. Examples of such promoters include: LTR promoter or SV40 promoter, E. coli. E. coli lac promoter or trp promoter, lambda phage PL promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation, and a transcription terminator. The vector may also include appropriate sequences for amplifying expression. In addition, the expression vector preferably has phenotypic characteristics for selection of transformed host cells (eg, dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or in E. coli, for example). , Tetracycline resistance or ampicillin resistance).

Vectors containing the appropriate DNA sequences as described above, as well as the appropriate promoter or control sequences, can be used to transform an appropriate host and allow the host to express the protein. . Examples of suitable expression hosts include bacterial cells such as E. coli, Streptomyces, and Salmone.
fungal cells (eg, yeast); insect cells (eg, Drosophila and Spodoptera Sf9); mammalian cells (eg, CHO, COS, BHK, HEK293, or Bowes melanoma); adenovirus; plant cells and the like. To be Not all cells or cell lines are capable of producing a fully functional ESK (eg bacterial expression is intended for the production of fragments of ESK which may not retain all functions of ESK). To be understood). Selection of the appropriate host is considered to be within the skill of those in the art given the teachings herein. The present invention is not limited by the host cell used.

In bacterial systems, a number of expression vectors may be chosen depending on the intended use for ESK. For example, if large amounts of ESK or fragments thereof are required for the induction of antibodies, a vector that directs high levels of expression of the fusion protein that is easily purified may be desired. Such vectors include multifunctional E
． coli cloning and expression vectors (eg, Bluescript (
(Stratagene), in which the ESK coding sequence can be ligated into the vector in frame with the amino-terminal Met and subsequent 7-residue sequence of β-galactosidase, resulting in a hybrid protein. PIN vector (Van Heeke & Schuster (1
989) J. Biol. Chem. 264: 5503-5509); pET vector (Novagen, Madison WI) and the like, but are not limited thereto.

In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For an overview, see A
usubel et al. (supra) and Grant et al. (1987; Methods in
Enzymology 153: 516-544).

If a plant expression vector is used, expression of the ESK coding sequence may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al. (1984) Nature 310: 511-514), alone or from the TMV derived ω leader sequence (Takamatsu et al. (19).
87) EMBO J 6: 307-311). Alternatively, a plant promoter (eg, the small subunit of RUBISCO (Coru
zzi et al. (1984) EMBO J 3: 1671-1680; Brogie.
(1984) Science 224: 838-843); or heat shock promoters (Winter J and Sinibaldi RM (1991).
Results. Probl. Cell Differ. 17: 85-105)
) Can be used. These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. For a review of such techniques, see Hobbs S or Murry LE (1992), McGr.
aw Hill Yearbook of Science and Tech
Noology, McGraw Hill, New York, N .; Y. , 191
-196; or Weissbach and Weissbach (1988) M.
methods for Plant Molecular Biology, A
academic Press, New York, N.C. Y. , 421-463.

ESK can also be expressed in insect systems. In one such system,
Autographa californica nuclear polyhedrosis virus (AcNP
V) is used as a vector, Spodoptera frugiperda
Express foreign genes in Sf9 cells or Trichoplusia larvae. The ESK coding sequence is cloned into a nonessential region of the virus (eg the polyhedrin gene) and placed under control of the polyhedrin promoter. Successful insertion of the ESK coding sequence inactivates this polyhedrin gene and produces recombinant virus lacking coat protein coat. This recombinant virus is then used to express ESK. frugipe
rda cells or Trichoplusia larvae are infected (Smith et al.
1983) J Virol 46: 584; Engelhard EK et al. (19).
94) Proc Nat Acad Sci 91: 3224-3227).

In mammalian host cells, a number of viral-based expression systems may be utilized.
When an adenovirus is used as an expression vector, the ESK coding sequence can be linked to an adenovirus transcription / translation complex consisting of a late promoter and tripartite leader sequence. Insertions in the nonessential El or E3 regions of this viral genome result in a viable virus capable of expressing ESK in infected host cells (Logan and Shenk (198).
4) Proc Natl Acad Sci 81: 3655-3659). In addition, transcription enhancers, such as the Rous Sarcoma Virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

Specific initiation signals may also be required for efficient translation of ESK coding sequences. These signals include the ATG initiation codon and adjacent sequences. E
If the SK coding sequence, its start codon, and upstream sequences are inserted into a suitable expression vector, additional translation control signals may not be needed. However, if only the coding sequence or part thereof is inserted, an exogenous transcriptional control signal (including the ATG start codon) must be provided. Moreover, this start codon must be in the correct reading frame to ensure transcription of the complete insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. Expression efficiency can be enhanced by including an enhancer appropriate for the cell line used (Sch
arf D et al. (1994) Results Probl Cell Diffe
r 20: 125-62; Bittner et al. (1987) Methods in.
Enzymol 153: 516-544).

In a further embodiment, the present invention relates to a host cell containing the construct described above. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryote, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. . Introduction of the construct into this host cell can be effected by calcium phosphate transfection, DEAE dextran mediated transfection, or electroporation (Davis, L. et al.
, Dibner, M .; , And Battey, I .; (1986) Basic M
methods in Molecular Biology). Cell-free translation systems can also be used to produce polypeptides using RNA derived from the DNA constructs of the invention. ESK cRNA is used in cells (eg, Xenopus laev) for the production of ESK for electrophysiological measurements or other assays.
is oocytes).

A host cell strain may be chosen for its ability to regulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the protein include acetylation, carboxylation, glycosylation,
Phosphorylation, lipidation, and acylation include, but are not limited to. Post-translational processing which cleaves a "prepro form" of the protein also results in correct insertion,
It may be important for folding and / or function. Different host cells (eg, CHO, HeLa, BHK, MDCK, 293, WI38, etc.) have specific cellular and characteristic mechanisms for such post-translational activity, and correct modification of the introduced foreign protein. And can be selected to ensure processing. In order to carry out certain aspects of the invention (eg, electrophysiological measurements as described below), the host cell may have an endogenous functionally expressed potassium channel (described herein). It will be appreciated that it may be desirable to lack a current characteristic similar to that exhibited or regulated by the ESK channel.

For long-term, high-yield recombinant protein production, stable expression is preferred. For example, a cell line that stably expresses ESK can be transformed with an expression vector containing a viral origin of replication or an endogenous expression element and a selectable marker gene. After the introduction of this vector, the cells are allowed to reach 1-2 in the enriched medium.
It can be grown for days and then the cells are transferred to selective medium. The purpose of this selectable marker is to confer resistance to selection, and its presence
Allows growth and recovery of cells that successfully express the introduced sequences. The resistant mass of stably transformed cells can be grown using tissue culture techniques appropriate to the cell type.

Host cells transformed with a nucleotide sequence encoding ESK can be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein or fragment thereof produced by a recombinant cell may be secreted, membrane bound or contained intracellularly depending on the sequence and / or the vector used. As will be appreciated by those in the art, expression vectors containing ESK-encoding polynucleotides can be designed with signal sequences that direct secretion of ESK through prokaryotic or eukaryotic cell membranes.

ESK can also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Domains that facilitate such purification include metal chelating peptides (eg,
Histidine-tryptophan module allowing purification on immobilized metal, protein A domain allowing purification on immobilized immunoglobulin, and FLAGS deployment / affinity purification system (Immunex Corp)
． , Seattle, Wash. ), But is not limited to these. Inclusion of a protease cleavable polypeptide linker sequence between this purification domain and ESK is useful to facilitate purification. One such expression vector provides for expression of a fusion protein comprising ESK (eg, soluble ESK fragment) fused to a polyhistidine region separated by an enterokinase cleavage site. This histidine residue
IMIAC (immobilized metal affinity chromatography, Porath et al.
1992) Protein Expression and Purifica
(described in Tion 3: 263-281), while the enterokinase cleavage site provides a means for isolating ESK from the fusion protein. pGEX vector (Promega, Madison, Wi
s. Can also be used to express foreign polypeptides as a fusion protein with glutathione S-transferase (GST). Typically,
Such fusion proteins are soluble and can be removed from lysed cells by adsorption to ligand-agarose beads (eg glutathione-agarose in the case of GST-fusion) followed by elution in the presence of free ligand. It can be easily purified.

After transformation of the appropriate host strain and growth of this host strain to the appropriate cell density, the selected promoter is induced by appropriate means (eg temperature shift or chemical induction) and the cells Incubate for a further period. The cells are typically
The crude extract collected by centrifugation, disrupted by physical or chemical means, and obtained is retained for further purification. Microbial cells used in the expression of proteins can be disrupted by any convenient method. These methods include freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysing agents, or other methods well known to those of skill in the art.

ESK can be recovered and purified from recombinant cell culture by any of a number of methods well known in the art. These methods include ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion exchange or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin. Chromatography can be mentioned.
If necessary, protein refolding steps can be used in completing the configuration of the mature protein. Finally, high performance liquid chromatography (H
PLC) can be used for the final purification step.

C2. Diagnostic Applications The polynucleotides of the present invention can be used for a variety of diagnostic purposes. This polynucleotide enhances expression of ESK in cells of a patient (eg, biopsy tissue).
By detecting the presence of mRNA encoding SK, it can be used to detect and quantify. The assay typically involves obtaining total RNA from the tissue and contacting the mRNA with a nucleic acid probe. The probe is a nucleic acid molecule of at least 12 nucleotides, preferably of at least 20 nucleotides or at least 30 nucleotides, which under hybridization conditions specifically binds to a sequence contained in the sequence of the nucleic acid molecule encoding ESK. The presence of mRNA that can hybridize and hybridize to this probe can be detected, and thereby the expression of ESK can be detected. This assay is based on E
It can be used to distinguish between absence, presence and overexpression of SK, and to monitor the level of ESK expression during therapeutic intervention.

The present invention also contemplates the use of this polynucleotide as a diagnostic agent for diseases resulting from genetically deficient ESK genes. These genes are defective (
That is, it can be detected by comparing the sequence of the mutant) ESK gene with the sequence of the normal gene. The relationship between mutant ESK genes and aberrant ESK activity (eg, aberrant channel activity) can be verified. Furthermore, the mutant ESK gene
In a functional assay system, it may be inserted into a suitable vector for expression as yet another means to verify or identify mutations. Once the mutant gene is identified, one of skill in the art can screen a population of interest for carriers of the mutant gene.

The present invention also includes methods of determining whether a subject is at risk for a disorder associated with, for example, ESK channel activity. The method comprises detecting at least one of the following: a) aberrant modification or mutation of the polynucleotide sequence encoding ESK, b) misregulation of ESK and c) aberrant post-translational modification. In one embodiment, the step of determining the genetic damage comprises determining the presence of at least one of the following: deletion of one or more nucleotides from the ESK gene, 1 to the ESK gene. Addition of one or more nucleotides, substitution of one or more nucleotides of this gene, gross chromosomal arrangement of this gene, altered level of mRNA transcript of this gene; mRNA of this gene
Presence of aberrant splicing pattern of transcripts; non-wild type level ESK protein expression. In a preferred embodiment, the ESK polynucleotide of the invention is combined with the nucleic acid of a cell and the hybridization of this polynucleotide to this nucleic acid is determined. The non-hybridization of the polynucleotide or the reduction of the hybridisation signal is indicative of a mutation in the ESK gene.

Individuals carrying mutations in the gene of the present invention can be detected at the DNA level by a variety of techniques. Nucleic acids used for diagnosis can be obtained from cells of a patient, including, but not limited to, blood, urine, saliva, placenta, tissue biopsy, and autopsy material. Genomic DNA can be used directly for detection or can be enzymatically amplified by using PCR prior to analysis (Saiki et al.
1986) Nature 324: 163-166). RNA or cDNA may also be used for the same purpose. As an example, a PC complementary to the nucleic acid of the invention
The R primer can be used to identify and analyze mutations in the genes of the invention. Deletions and insertions can be detected by a change in the size of the amplification product compared to the normal genotype.

Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA of the invention, or, alternatively, radiolabeled antisense DNA sequences of the invention. Sequence changes at specific positions may also result in nuclease protection assays (eg, RNase and S1 protection or chemical cleavage methods (eg, Cotton et al. (1985) Proc. Natl. Acad. Sci. US).
A 85: 4397-4401)) or by differences in melting temperatures. A "molecular beacon" (Kostrikis LG et al. (1998) Scien) containing a probe sequence that is complementary to a nucleic acid of the invention.
279: 1228-1229), hairpin, single-stranded synthetic oligonucleotides can also be used to detect point mutations or other sequence changes as well as to monitor the expression level of ESK. Such diagnostic agents are particularly useful, for example, for prenatal testing.

Another method for detecting mutations uses two DNA probes containing flanking bases that are designed to hybridize to flanking regions of the target. In this probe, the region of known mutation or the region of suspected mutation is at or near its adjacent bases. The two probes may be ligated at their adjacent bases, eg, in the presence of a ligase enzyme, provided that both probes form an exact base pair in the probe junction region. . The presence or absence of the mutation can then be detected by the presence or absence of the linked probe.

[0112] To detect mutations in the ESK coding sequence, hybridization sequencing (SBH) -based oligonucleotide array methods (described, for example, in US Pat. No. 5,547,839) are also provided. May be appropriate. In a typical method, a DNA target analyte is hybridized with an array of oligonucleotides formed on a microchip. The target sequence can then be "read" from the pattern of target binding to the array.

C3. Gene Mapping The sequences of the present invention are also valuable for chromosome identification. This sequence may be specifically targeted to and hybridize to a particular location on an individual human chromosome. In addition, there currently exists a need to identify specific sites on the chromosome. Chromosome marking reagents based on actual sequence data (repeated polymorphisms) for marking chromosomal locations are currently scarcely available. The mapping of DNA to chromosomes, according to the present invention, is the first important step in correlating these sequences with genes involved in disease.

Briefly, PCR primers (preferably
The sequence can be mapped to the chromosome by preparing 12-30 bp). Computer analysis of the 3'untranslated region is used to rapidly select primers that do not span more than one exon in genomic DNA. This complicates the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only the hybrid containing the human gene corresponding to that primer will produce an amplified fragment.

PCR mapping of somatic cell hybrids showed that specific DN for specific chromosomes
A quick procedure for assessing A. Using the present invention with the same oligonucleotide primers, sublocalization can be accomplished with a panel of fragments from a particular chromosome or pool of large genomic clones in a similar fashion. Other mapping strategies that may also be used to map to that chromosome include in situ hybridization, prescreening with labeled flow-identified chromosomes, and for constructing chromosome-specific cDNA libraries. Pre-selection by hybridization is mentioned.

Fluorescence in situ hybridization (FISH) of cDNA clones to metaphase chromosome spreads can be used to provide accurate chromosomal location in one step. This technique can be used with cDNAs as short as 50 or 60 bases. For a review of this technology, see Verma et al. (1988) Human.
Chromosomes: a Manual of Basic Techni
See ques, Pergamon Press, New York.

Once a sequence has been mapped to a precise chromosomal location, the physical location of that sequence on that chromosome can be correlated with genetic map data. Such data can be obtained, for example, from the OMIM database (Center for Medica).
l Genetics, Johns Hopkins University,
Baltimore, MD and National Center for B
iotechnology Information, National Li
B. of of Medicine, Bethesda, MD). This OMIM genetic map represents the cytogenetic map location of disease genes and other expressed genes. The OMIM database provides information for diseases associated with chromosomal location. Such associations include the results of linkage analysis mapped to this interval, as well as the correlation of translocations and other chromosomal abnormalities in this region with the development of polygenic disorders (eg, cancer).

C4. Therapeutic Applications The polynucleotides encoding ESK, or the complements of the polynucleotides, can also be used for therapeutic purposes. Expression of ESK can be regulated via antisense technology. This technique uses a polynucleotide complementary to the regulatory region, 5 ′ region or regulatory region of the gene encoding ESK (ie, antisense DN).
A or RNA) to control gene expression. For example, using the 5'coding portion of the polynucleotide sequence encoding the protein of the invention, about 10-40
Design antisense oligonucleotides of base pair length. Oligonucleotides from the transcription start site (eg, between -10 and +10 from the start site) are preferred. Antisense DNA oligonucleotides are designed to be complementary to regions of the gene involved in transcription (Lee et al. (1979) Nucl. Ac).
ids Res. 6: 3073; Cooney et al. (1988) Science.
241: 456; and Dervan et al. (1991) Science 251:
1360), thereby preventing ESK transcription and production. Antisense R
The NA oligonucleotide hybridizes to the mRNA in vivo and E
Preventing translation of mRNA molecules into SK proteins (Okano (1991) J. et al.
Neurochem. 56: 560). The antisense construct can be delivered to cells by procedures known in the art so that the antisense RNA or DNA can be expressed in vivo.

The therapeutic polynucleotides of the present invention can be used in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of this compound and a pharmaceutically acceptable carrier or excipient. Such carriers include saline,
Included but not limited to buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The polypeptides, as well as agonist and antagonist compounds that are polypeptides, can also be used according to the invention by the expression of such polypeptides in vivo, which is often referred to as "gene therapy." The patient-derived cell is a polynucleotide (DNA or RNA) encoding a polypeptide.
) Is used ex vivo, and the engineered cells are then provided to the patient to be treated with the polypeptide. Such methods are well known in the art. For example, cells can be engineered by procedures known in the art by using retroviral particles that include RNA that encodes a polypeptide of the invention.

Similarly, cells may be engineered in vivo for expression of the polypeptide in vivo by procedures known in the art. As is known in the art,
Producer cells for producing retroviral particles containing RNA encoding a polypeptide of the invention may be administered to a patient for engineering cells in vivo and expression of this polypeptide in vivo. . By such methods, these and other methods for administering the polypeptides of the invention can be understood by those of skill in the art from the teachings of the invention, e.g., expression vehicles for manipulating cells include, for example, It can be an adenovirus, which can be used to engineer cells in vivo after combination with a suitable delivery vehicle (Yeh P. et al. (19
97) FASEB J 11: 615-623).

The retrovirus derived from the above retrovirus plasmid vector includes Moloney murine leukemia virus, spleen necrosis virus, the following: Rous sarcoma virus, Harvey sarcoma virus, chicken leukemia virus, gibbons leukemia virus, adenovirus, myeloproliferative property. Examples include, but are not limited to, retroviruses such as sarcoma virus, and breast cancer virus.

This retroviral plasmid vector is used to transduce a packaging cell line to form a producer cell line. Examples of packaging cells that can be transfected are PE501, PA317, psi-2, psi-.
AM, PA12, T19-14X, VT-19-17-H2, psi-CRE,
psi-CRIP, GP + E-86, GP + envAm12, and Mille
r (1990; Human Gene Therapy, Vol. 1, pp. 5-14)
DAN cell lines described in, but are not limited to. The vector can transduce the packaging cells by any means known in the art. Such means, electroporation, the use of liposomes, and including CaPO ₄ precipitation, is not limited thereto. In one alternative, the retroviral plasmid vector can be encapsulated in liposomes or can be lipid bound and then administered to a host.

This producer cell line produces infectious retroviral vector particles,
The particle comprises a nucleic acid sequence encoding the polypeptide. Such retroviral vector particles can then be used to transduce eukaryotic cells either in vitro or in vivo. Transduced eukaryotic cells express a nucleic acid sequence encoding this polypeptide. Eukaryotic cells that can be transduced include embryonic stem cells, embryonal cancer cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,
Including but not limited to keratinocytes, endothelial cells, and bronchial epithelial cells.

The gene introduced into the cell may be an inducible promoter (eg, radiation-induced Eg
r-1 promoter (Maceri, H. J. et al. (1996) Center R
es 56 (19): 4311) to stimulate ESK production or antisense inhibition in response to radiation, eg, radiation therapy to treat tumors.

III. ESK and ESK Channels The present invention is based, in part, on the structural similarities between ESK and other known subunits of the eag potassium channel superfamily (hERG). An exemplary ESK polypeptide sequence, SEQ ID NO: 2, has 1080 residues, six potential transmembrane domains (approximately residues 212-239 (S1), 259-277 (S.
2) 297-320 (S3), 329-349 (S4), 356-378 (S
5), and 477-501 (S6)), and potential N-linked glycosylation sites (residues Asn418, Asn425, Asn433, Asn467,
And Asn 496). The hESK sequence SEQ ID NO: 2 also contains a potential pore-forming P domain (approximately residues 449-468 (P1)), and a putative cyclic nucleotide binding domain (cNBD) (over approximately residues 601-668). These structural motifs are characteristic of the eag channel subunit.

The hESK amino acid sequence SEQ ID NO: 2 and the hERG sequence SEQ ID NO: 6 are:
MacVector ^™ software (ver. 6.01; Oxford Mol
e.c. Ltd., Oxford Ltd., UK) using the CLUSTAL-W pairwise alignment program with default pairwise parameters. This protein has about 37% overall amino acid sequence identity, which
About 47% identity in the region spanning the S1 to cNBD segment (corresponding to residues 212-668 of SEQ ID NO: 2 and identified herein as SEQ ID NO: 5) (
216/457 identical residues). This level of sequence identity in the S1-cNBD region indicates that ESK represents a novel subfamily of the eag superfamily. Generally, two members of the same subfamily from different species have about 65-70% in the region spanning the S1 to cNBD segment.
Share the amino acid sequence identity of. In contrast, two different subfamily members of the same species share only about 40-50% amino acid sequence identity over the same region (Warmke and Ganetzky (1994) PN.
A. S. 91: 3438-3442).

The substantially purified ESK of the invention has at least 60 relative to SEQ ID NO: 5.
%, Preferably at least 65-70%, more preferably at least 80%, and most preferably 90% or 95% sequence identity. In one embodiment, the invention provides at least 70%, at least 80%, at least 90% to SEQ ID NO: 2 or SEQ ID NO: 4,
Or a polypeptide having at least 95% sequence identity, or having the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. The polypeptide may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptide may be in mature and / or modified form, which is also as described above. Also, fragments derived from ESK that are capable of interacting with other polypeptides, proteins, or other molecules are also contemplated. Such interactions alter the functional properties or cellular / compartmental localization of this ESK channel. The present invention also includes a substantially purified potassium channel that comprises at least one ESK subunit.

Variations of this polypeptide sequence may include those that are considered conservative and non-conservative substitutions, as described above. Therefore, for example, SEQ ID NO: 2 (10
A polypeptide having a sequence having at least 80% sequence identity with a polypeptide identified as 80 amino acids), when optimally aligned as described above,
Contains up to 16 amino acid substitutions. In a more particular embodiment, the polypeptide has a sequence that is substantially identical (97% -100% identical) to SEQ ID NO: 2 or SEQ ID NO: 4. ESK is (i) in the sequences listed above:
A polypeptide in which one or more amino acid residues are substituted with conservative amino acid residues or non-conservative amino acid residues (preferably conservative amino acid residues), or (
ii) a polypeptide in which one or more amino acid residues contains a substituent, or (iii)
A polypeptide in which ESK is fused to another compound (eg, a compound that increases the half-life of the polypeptide (eg, polyethylene glycol (PEG))),
Or (iv) a polypeptide in which additional amino acids are fused to ESK, or (
v) an isolated fragment of the polypeptide. Such fragments, variants and derivatives are considered to be within the skill of those in the art given the teachings herein. In particular, splice variants of this polypeptide are also contemplated.

A. Preparation of ESK Recombinant methods for generating and isolating ESK and fragments are described above.

In addition to recombinant production, fragments and portions of ESK have been synthesized by solid phase techniques (eg, Stewart et al.
synthesis, WH Freeman Co, San Francisco
Merrifield J (1963) J Am Chem Soc 85:
2149-2154). In vitro peptide synthesis can be performed using manual techniques or by automation.
Automated synthesis is described, for example, in Applied Biosystems 431A P.
eptide Synthesizer (Perkin Elmer, Fast
er City, Calif. ) According to the instructions provided by the manufacturer. Portions of the ESK can be chemically synthesized separately and combined using chemical techniques.

The polypeptide can also be obtained by isolation from natural sources (eg, by affinity purification using the anti-ESK antibody described in the section below).
The fragment (s) corresponding to the extracellular and / or intracellular regions of ESK can be cleaved from the membrane bound region using limited proteolytic techniques known to those of skill in the art. The amino acid sequence of the fragment thus obtained can be used to design the nucleotide coding sequence for recombinant production of the fragment.

B. Application of ESK The ESK polypeptides of the present invention have applications in (1) therapeutic treatment methods, and (2) drug screening.

B1. Therapeutic Uses and Compositions ESK polypeptides of the present invention are generally used in forebrain-related neurology, including, but not limited to, diseases and disorders associated with ion channel dysfunction. Disorders: dementia such as Alzheimer's disease, depressive and manic depression, anxiety disorders, panic and obsessive-compulsive disorders, eating disorders, attention deficit disorders and hyperactivity disorders, autism, schizophrenia, epilepsy , And in the treatment of neurodegenerative disorders such as Huntington's disease and Parkinson's disease.

ESKs may self-associate to form homomeric ESK channels or may associate with other potassium channel polypeptides to form heteromeric ESK channels. Without wishing to be bound by theory, a polypeptide fragment of ESK, preferably a soluble fragment that binds to an agonist of the ESK channel, binds the agonist of ESK channel by binding the agonist required for ESK channel activity. It can be used to block in effect competing with ESK channels for agonists. An intracellular fragment of ESK (preferably a soluble fragment) can be used to block the interaction of intracellular effector molecules with the intracellular domain of the ESK channel, thereby allowing this E
It interferes with the cellular response induced by the interaction of this intracellular effector molecule with SK channels.

ESK compositions are tested in appropriate in vitro and in vivo animal models of disease to confirm potency, tissue metabolism and estimate dosage according to methods well known in the art.

ESK compositions may be administered by any of a number of routes and methods designed to provide constant and predictable concentrations of the compound at target organs or tissues. The polypeptide composition can be administered alone or in combination with other agents (eg, stabilizing compounds) and / or in combination with other pharmaceutical agents (eg, drugs or hormones). Can be administered.

The ESK composition may be administered by oral means, intravenous means, intramuscular means, transdermal means, subcutaneous means,
It can be administered by a number of routes including, but not limited to, topical, sublingual, or rectal means. The ESK composition can also be administered via liposomes. Such routes of administration and suitable formulations are generally known to those of ordinary skill in the art.

For example, the polypeptide may be administered locally to the epithelial lining of the skin or body cavity, for infection in such areas. Examples of treatable body cavities include the vagina, rectum, and urethra. Conveniently, the polypeptide is formulated in a suppository form for administration to these areas.

The polypeptide may be given via intravenous or intraperitoneal injection. Similarly, the polypeptide can be injected into other localized areas of the body. The polypeptide composition can also be administered via nasal insufflation. Enteral administration is also possible. For such administration, the polypeptide should be formulated in a suitable capsule or elixir for oral administration or in a suppository for rectal administration.

The exemplary modes of administration described above appear to require that the polypeptide be formulated in a suitable carrier, including ointments, gels, suppositories. A suitable formulation is
It is well known to those skilled in the art.

The dosage of this polypeptide will vary, depending on the potency and therapeutic index of the particular polypeptide selected. These parameters can be readily determined by the skilled practitioner. As an example, if the polypeptide inhibits neuronal degradation at a given concentration in vitro, the practitioner will determine that the final desired therapeutic concentration is in this range, calculated based on the expected biodistribution. Know that. Suitable target concentrations range from ng / kg to low mg / kg (for IV administration (
For example, it is in the range of 50 ng / kg body weight to 1 mg / kg body weight.

Therapeutic compositions for use in methods of treatment may include the polypeptide in sterile injectable solution, the polypeptide in an oral delivery vehicle, or the polypeptide in spray form, all of which are well known. It is prepared by the method of. Such a composition comprises a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.

B2. Screening Methods The invention also includes a modulatory effect on the activity of synthetic drugs, antibodies, peptides, or other molecules (ESK channels (eg, one or more ESK polypeptides of the invention. , Such as ESK channel agonists or antagonists)). Such an assay provides a functional ESK channel comprising an ESK polypeptide encoded by a polynucleotide of the invention, contacting the ESK channel with one or more molecules, Determining the regulatory effect of the molecule, and E
Selecting candidate molecules from these molecules that can modulate SK channel activity,
including. Such modulatory agents are useful in treating disease states associated with activation or reduction of ESK channel activity, including but not limited to those described in Section B1 above.

ESK, its ligand binding fragments, catalytic fragments or immunogenic fragments, or oligopeptides thereof, can be used to screen therapeutic compounds in any of a variety of drug screening techniques. The proteins used in such assays may be membrane bound, free in solution, bound to a solid support, present on the cell surface, or located intracellularly. . The formation of binding complexes between the ESK or ESK channel and the agent being tested can be measured. Compounds that inhibit the binding between ESK and its agonist can also be measured.

In one embodiment, the screening system comprises recombinantly expressed ESK. The screened compounds are then tested for their ability to block (inhibit) or enhance (activate) the potassium current activity of ESK channels. In functional screening assays, mammalian cell lines lacking ESK or Xenopus oocytes are used to express ESK.
ESKs can be expressed individually or together with other potassium channel subunit polypeptides. Compounds are screened for their relative efficacy as channel regulators (activators or inhibitors) by comparing relative channel occupancy with the extent of ligand-induced activity or inhibition of potassium ion conductance.

The invention also includes, in a related aspect, an ESK channel modulating agent identified by a screening method that uses ESK, such as the methods described above.

Another technique for drug screening that may be used to provide a high throughput screen for compounds with appropriate binding affinity for ESK channels is described in PCT Application WO, published September 13, 1984. 84/03564 in G
by Eysen in detail. In summary, a large number of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compound is reacted with ESK (eg, either a soluble extracellular fragment of ESK or intact ESK solubilized in detergents or lipid vesicles) and washed. Bound ESK is then detected by methods well known in the art. Substantially purified ESK can also be coated directly onto plates for use in the drug screening techniques described above. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

Antibodies to ESK can also be used in the screening assays according to methods well known in the art, as described in Section IV below. For example, a "sandwich" assay can be performed. In this assay, anti-ESK antibody is immobilized on a solid surface such as a microtiter plate, and solubilized ESK or ESK channel is added. Using such an assay, this ESK
Compounds that bind to the channel can be captured. Alternatively, such an assay
It can be used to measure the ability of a compound to interfere with the binding of a ligand (eg, agonist) to this ESK channel.

IV. Anti-ESK Antibodies In yet another aspect of the invention, purified ESK is used to produce anti-ESK antibodies. This antibody has diagnostic and therapeutic applications related to ESK activity, distribution and expression.

Antibodies to ESK can be generated by methods well known in the art. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, Fab fragments and fragments produced by Fab expression libraries. Antibodies, ie those that block ligand binding, are particularly preferred for therapeutic use.

ESK for antibody induction does not require biological activity; however, the polypeptide fragment or oligopeptide must be antigenic. The peptide used to induce a specific antibody may have an amino acid sequence consisting of at least 10 amino acids, preferably at least 20 amino acids.
Preferably, they should mimic a portion of the amino acid sequence of their native protein and may include the entire amino acid sequence of a small, naturally occurring molecule.
A short stretch of ESK polypeptide can be fused to another protein such as keyhole limpet hemocyanin, and antibodies raised against this chimeric molecule. Procedures well known in the art can be used to generate antibodies to ESK.

For the production of antibodies, various hosts, including goats, rabbits, rats, mice, etc., are injected with ESK or any moiety, fragment or oligopeptide that retains immunogenic properties. , Can be immunized. Depending on the host species,
Various adjuvants may be used to increase the immunological response. Such adjuvants include Freund's adjuvant, mineral gels (eg, aluminum hydroxide), and surface active agents (eg, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. ), But is not limited thereto. BCG
(Bacillus Calmette-Guerin) and Corynebacterium parv
um is potentially a useful human adjuvant.

Monoclonal antibodies against ESK can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These were originally Koehler and Milstein (1975; Nature 256: 4).
95-497), the human B cell hybridoma technology described by Kosbor et al. (1983) Immunol Today 4:72.
Cote et al. (1983) Proc Natl Acad Sci 80:20.
26-2030) and EBV-hybridoma technology (Cole et al. (1984)
) Mol. Cell Biol. 62: 109-120), but is not limited thereto.

Techniques developed for the generation of "chimeric antibodies" (splicing the mouse antibody gene to a human antibody gene to obtain a molecule with appropriate antigen specificity and biological activity) are also used. Can be done (Morrison et al. (1984) Pro
c Natl Acad Sci 81: 6851-6855; Neuberg.
er et al. (1984) Nature 312: 604-608; Takada et al.
1985) Nature 314: 452-454). Alternatively, the techniques described for the production of single chain antibodies (US Pat. No. 4,946,778) can be adapted to produce single chain antibodies specific for ESK.

Antibodies have also been described by Orlandi et al. (1989; Proc Natl Acad.
Sci 86: 3833-3837), and Winter G and Mil.
A recombinant immunoglobulin library or panel of highly specific binding reagents, either by inducing in vivo production in a lymphocyte population, as disclosed in Stein C (1991; Nature 349: 293-299). It can be produced by screening.

Antibody fragments which contain specific binding sites for ESK may also be generated. For example, such fragments include F (ab ′) 2 fragments (which may be produced by pepsin digestion of the antibody molecule) and Fab fragments (which may be disulfide bridges of the F (ab ′) 2 fragment). Can be produced by reduction), but is not limited thereto. Alternatively, the Fab expression library is a monoclonal Fab with the desired specificity.
The b fragment can be constructed to allow rapid and easy identification (Hus
e WD et al. (1989) Science 256: 1275-1281).

A. Diagnostic Applications Various protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificity are well known in the art. Such immunoassays typically involve the formation of a complex between ESK and its specific antibody, and the measurement of complex formation. A two-site, monoclonal-based immunoassay utilizing a monoclonal antibody reactive to two non-interfering epitopes on ESK is preferred, but a competitive binding assay may also be used. These assays are based on Maddox DE et al. (1983, J Exp Med 158: 1211).
It is described in.

Antibodies that specifically bind ESK are useful in the diagnosis of conditions or diseases characterized by the expression of ESK. Alternatively, such antibodies can be used in assays to monitor patients being treated with ESK, its agonists, or its antagonists. Diagnostic assays for ESK proteins include methods of using the antibodies and labels to detect ESK or fragments thereof in cell extracts, tissue extracts, or biological fluids (eg, serum). . The proteins and antibodies of the invention can be used with or without modification. Frequently, the proteins and antibodies will be labeled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules are known in the art.

Various protocols for measuring ESK using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA
), Radioimmunoassay (RIA), and fluorescence activated cell sorting (F
ACS). As noted above, a two-site, monoclonal-based immunoassay utilizing a monoclonal antibody reactive to two non-interfering epitopes on ESK is preferred, but a competitive binding assay can be used. These assays are described, among others, in Maddox et al. (Supra). Such protocols provide the basis for diagnosing ESK expression at altered or abnormal levels. Normal or standard values for ESK expression are those well known in the art, where a cell extract taken from a normal subject (preferably human) is treated with an antibody against ESK under conditions suitable for complex formation. Established by combining. The amount of canonical complex formation can be quantified by various methods, preferably photometrically. The standard value obtained from the normal sample can then be compared to the value obtained from a sample from a subject potentially affected by the disease. Deviation between standard and subject values is evidence of the presence of the disease state.

This antibody assay may be used to determine the level of ESK present in a particular tissue (eg, biopsy tumor tissue or neuronal tissue), whether ESK is overexpressed or underexpressed in that tissue. Or as an indicator of whether or not
It is useful as an indicator of how SK levels respond to drug treatment.

B. Therapeutic Uses In ESK-related conditions (eg, neurological disorders, including but not limited to the disorders described in Section III.B1), therapeutic value is, for example, , E
It can be achieved by administering an antibody specific for ESK, either to block the binding of agonists to the SK channel or to block the ion pore.

The antibody used is preferably a humanized monoclonal antibody, or human Mab produced by known globulin gene library methods. The antibody is typically administered as a sterile solution by IV injection, although other parenteral routes may be appropriate. Typically, the antibody is administered in an amount of about 1-15 mg / kg body weight of the subject. Treatment continues with dosing, for example, every 1 to 7 days, until a therapeutic improvement is observed.

The following examples are illustrated, but are not intended to limit the invention in any way.

Materials and Methods Unless otherwise indicated, restriction enzymes and DNA modifying enzymes are New Engla.
nd Biolabs (Beverly, MA) or Boehringer
Obtained from Mannheim (Indianapolis, IN). Enha
The Necked Chemo-Luminescence (ECL) system is
ersham Corp. (Arlington, Heights, IL). Nitrocellulose paper is available from Schleicher and Schue
11 (Keene, NH). "PBLUESCRIPT II S
"K" was obtained from Stratagene (La Jolla, CA). SD
Materials for S-polyacrylamide gel electrophoresis (SDS-PAGE) are B
Obtained from io-Rad Laboratories (Hercules, CA). Other chemicals are Sigma (St. Louis, MO) or Unit
Purchased from ed States Biochemical (Cleveland, OH).

Example 1 Identification of ESK Nucleic Acid Sequence Shepard & Rae (19) for the isolation of ESK cDNA molecules.
97; Nucleic Acids Res. 25 (15): 3138-318
The procedure of 5) was used with some modifications. Briefly, a human brain cDNA library (Edge BioSystems, Gaithersburg, M.
10-20 μg of cDNA from D) was added to 50-80 ng of biotinylated oligonucleotides, 50 ng of each clamp oligo, and 1 μl of 1N NaOH.
Mix in a total volume of 10 μl. This oligonucleotide sequence has the human EST sequence U6
It was derived from 9184 (SEQ ID NO: 7). After incubating the mixture at room temperature for 15-20 minutes, 40 μl of neutralizing solution (0.12M Tris (pH 7), 2 ×
SSPE, 0.1% Tween 20) was added and further incubated at 37-42 ° C. After 2-3 hours, 20 μl (200 μl) was added to the above reaction mixture.
g) Magnetic beads (Dynabeads) were added and the mixture was incubated for an additional 30 minutes at the above temperature.

To recover the captured cDNA molecules, the supernatant was removed and the magnetic beads were washed 5 times with 0.5 × SSPE, 0.1% Tween20. The beads were then further washed once or twice with TE. Finally, the captured cDNA was eluted with 10 μl of 0.5 × TE at 70 ° C. for 5 minutes. Then
This eluted plasmid cDNA was transformed into E. E. coli cells were transformed and transformants were plated on one or more 15 cm dishes (thousands of colonies per dish). Bacterial colonies were stained with Hybond N filter (Amer
sham, Arlington Heights, IL) in duplicate.

The filters were screened using labeled hybridization probes based on the above EST sequences. The filter was placed in a prehybridization solution (5 × SSPE, 5 × Denhardt's solution, 0.1% SDS)
Prehybridized at 45-50 ° C. for 1 hour without probe, then hybridized with probe overnight. Then filter this filter 2x SSPE
, Wash twice in 0.1% SDS at room temperature for 20 minutes each, and 2x SSPE
, Washed twice at 45-50 ° C for 20 minutes each in 0.1% SDS. The signal was detected by exposure of this filter to X-ray film for several hours.

Positive colonies were subjected to secondary and tertiary screening. Three
Positive colonies from the next screen were cultured. Plasmid DNA, Qia
gen miniprep kit (Qiagen, Santa Clarita, CA
) Was used to isolate from this positive culture and sequenced so that human E
The nucleic acid sequence of the SK1 potassium channel subunit polypeptide was identified.

(Example 2) (Northern analysis) A multiple tissue Northern blot was used as Clon.
We purchased from tech. High Efficiency Hybridiza
motion system (HS-114), Molecular Rease
Purchased from rch Center (Cincinnati, Ohio). Briefly, the blot was first soaked in prehybridization solution (1% SDS and 0.1 M NaCl) for 30 minutes at room temperature, then 100 μg / ml for several hours at 68 ° C. in the absence of probe. HS-1 containing salmon sperm DNA
Incubated in 14 solutions. A cDNA probe was then added and the blot was hybridized overnight at 68 ° C. The blot was then washed under the following conditions: 2 x SSC, 0.05% SDS at room temperature; and 2 x 0.1 x SSPE, 0.1% SDS at 50 ° C. After washing, the blot was exposed to X-ray film. Experiments performed as described above showed that among the tissues tested, ESK1 transcript expression was observed only in the brain (mainly the forebrain).

Example 3 Electrophysiological Measurements I. Whole Cell Patch Clamp Measurements Potassium currents can be measured by the patch clamp method in whole cell settings (H.
amill et al. (1981)). Electrode resistances in the range 2-6 MΩ are suitable. Records are stored in PCLMP6 software (Axon) for data acquisition and analysis.
Axopatch 1C or Axopatch 200A amplifier (Axon Instrument, F) interfaced to Instruments
host city, CA).

The potassium current is recorded (in mM) using an external bath solution consisting of:
140 sodium chloride, 5 potassium chloride, 10 HEPES, 2 calcium chloride, 1 magnesium chloride, and 12 glucose (adjusted to pH 7.4 and 305 mOsM with sodium hydroxide). The internal pipette solution consisted of (in mM): 15 sodium chloride, 125 potassium methanesulfonate,
HEPES 10, EGTA 11, calcium chloride 1, magnesium chloride 2 and glucose 59 (potassium hydroxide pH 7.4 and 295 mOsM
Adjust to). Cells are placed in a flow-through chamber for test application (
0.5-1 ml / min). The current is 30 msec. Every 15 seconds. It is triggered by changing the voltage from a holding potential of -90 mV to 0 mV as a step pulse of duration. The data is sampled at 5 KHz and filtered at 1 KHz. Leakage and capacitance currents are subtracted after measuring the current evoked by the hyperpolarizing pulse.

Example 4 Antispasmodic Activity: DBA / 2 Mouse Spasm Model DBA / 2 mice (18-21 days old; approximately 7-10 g) were treated with Jackson L.
Obtained from laboratories, Bar Harbor, Maine and housed for a minimum of 3 days until acclimatized to laboratory conditions. On the day of the test, mice were injected i.v. with vehicle or test compound (total volume: 5 μl) into their lateral ventricles according to standard methods (Jackson and Scheideler, 1996). c. v. Injection and 30 minutes later exposure to sound stimuli. After injection, the mice were individually housed in the observation chamber and for 30 minutes thereafter, shook (sha).
ing) behavior (sustained whole body tremor) or any other abnormal behavior. High intensity sound stimulation (100-110d for 30 seconds at 14Hz)
Expose to B sine tone. Mice are observed for the presence of clonic and tonic seizures with complete hindlimb extension during 30 seconds of exposure to this sound.

Although the present invention has been described with respect to particular methods and embodiments, it will be appreciated by those skilled in the art that various modifications and changes can be made without departing from the invention.

[Brief description of drawings]

1A-E are E identified herein as human ESK1 (hESK1).
1 shows the nucleic acid sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of the SK polypeptide.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 25/18 A61P 25/24 4C084 25/22 25/28 4C086 25/24 C07H 21/00 4H045 25/28 C07K 14/47 C07H 21/00 16/18 C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12Q 1/68 A 1/21 G01N 33/53 D 5 / 10 M C12Q 1/68 33/566 G01N 33/53 C12P 21/08 C12N 15/00 ZNAA 33/566 A61K 37/02 // C12P 21/08 C12N 5/00 A (81) Designated country EP (AT, BE , CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), AL, AM, AT, AU, AZ, BA, BB, BG, BR, B Y, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Zao, Byron Bee. United States California 94303, Palo Alto, Colorado Avenue 1036 F term (reference) 4B024 AA01 AA11 BA80 CA04 DA06 EA04 GA11 GA19 HA01 HA12 HA15 HA17 4B063 QA01 QA18 QA19 QQ42 QR36 QR56 QR62 QS25 QS34 4B064 AG01 CE65 DA01 CE01 CA27 DA02 CE08 CA19 CA01 CA02 CA08 CA01 CA02 CA02 CA19 CA02 CA08 CA01 CA27 CA02 CA19 CA19 CA02 CA02 CA19 AA26X AA80X AA88X AA90X AA93Y AB01 AC14 BD14 CA24 CA44 CA46 4C057 MM04 4C084 AA06 AA13 BA01 BA22 NA14 ZA02 ZA05 ZA12 ZA16 ZA18 4C086 AA03 EA16 FA20 FA20 FA20A20 EA16 NA20 ZA16 FAC20 4H045 A4

Claims (17)

[Claims] 1. A substantially purified ESK polypeptide comprising a sequence having at least 60% sequence identity to SEQ ID NO: 5. 2. The polypeptide of claim 1, comprising a sequence having at least 70% sequence identity to SEQ ID NO: 2. 3. The polypeptide of claim 1 having the sequence of SEQ ID NO: 2. 4. The polypeptide of claim 1, comprising a sequence having at least 70% sequence identity to SEQ ID NO: 4. 5. The polypeptide of claim 1 having the sequence of SEQ ID NO: 4. 6. An isolated polynucleotide, which comprises:   (A) a sequence encoding the polypeptide according to claim 1, or   (B) Sequence complementary to the sequence of (a) A polynucleotide comprising: 7. An isolated polynucleotide which hybridizes under high stringency conditions to a polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and their complements. A polynucleotide comprising: 8. The polynucleotide of claim 7, having the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. 9. A recombinant expression vector comprising: (a) the polynucleotide of claim 6 and (b) the polynucleotide operably linked to the polynucleotide in a selected host. A vector comprising a regulatory sequence effective for the expression of nucleotides. 10. The vector of claim 9, wherein the polynucleotide comprises a sequence having at least 80% identity to SEQ ID NO: 1 or SEQ ID NO: 3. 11. The vector of claim 10, wherein the polynucleotide has the sequence of SEQ ID NO: 1 or SEQ ID NO: 3. 12. A recombinant eukaryotic cell, which is transfected with the vector of claim 9 and has a functional heterologous ESK expressed by the vector carried on the cell surface. . 13. The cell of claim 12, wherein the ESK has a sequence that is at least 70% identical to SEQ ID NO: 2 or SEQ ID NO: 4. 14. A purified antibody that specifically binds to the polypeptide of claim 1. 15. A method for detecting a polynucleotide encoding ESK in a biological sample, the method comprising: (a) identified as SEQ ID NO: 1 with respect to the nucleic acid material of the biological sample. Hybridizing a polynucleotide fragment derived from the sequence, thereby forming a hybridization complex, wherein the fragment has a length of at least 12 nucleotides; and (b) the hybridization complex. Detecting the presence of the hybridization complex with the presence of a polynucleotide encoding ESK in the biological sample. 16. A method of identifying a candidate compound capable of modulating ESK channel activity, comprising: (a) a test compound comprising a polypeptide containing an amino acid sequence that is at least 60% identical to SEQ ID NO: 5. ESK channel including subunit and the ES
Contacting under conditions where the activity of the K channel can be measured, (b) measuring the effect of the test compound on the activity of the ESK channel, and (c) the effect of the test compound on the activity of the ESK channel Selecting the test compound as a candidate compound if it is above a selected threshold level. 17. A method for detecting ESK in a biological sample, which comprises: (a) contacting the antibody of claim 14 with the biological sample, whereby an antibody-antigen complex. Forming a body; and (b) detecting the antibody-antigen complex, wherein the presence of the antibody-antigen complex correlates with the presence of ESK in the biological sample, Method.