Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL ESK POTASSIUM CHANNEL
P9LYPEPTIDE AND POLYNUCLEOTIDE COMPOSITIONS
dig].d of the Invention
This invention relates to novel human potassium channel polypeptide and
polynucleotide compositions, to the production of these compositions, and to
the use of the
compositions in the diagnosis, prevention, and treatment of disease states.
to Backeround of the Invention
Potassium channels are a heterogeneous group of ion channels that allow
selective
permeation of potassium ions across the plasma membrane, but differ in details
of activation
mechanism, voltage range of activity, and kinetic properties. (Hille, B.
(1992) Ionic Channels
of Excitable Membranes, 2nd Ed. Sinauer, Sunderland, MA; Latorre, R. and
Miller, C.
~s (1983) J. Memb. Biol. 7:11-30). They contribute to numerous physiological
functions, for
example, action potential repolarization, cardiac pacemaking, neuron bursting,
muscle
contraction, hormone secretion, vascular tone regulation, renal ion
reabsorption, learning and
memory, and cell growth and differentiation.
A majority of the potassium channel genes isolated and characterized to date
encode
2o polypeptides containing six probable transmembrane segments and a single
pore-forming P
domain (i.e., 1P/6TM subunits). Channels comprising a distinct family of
1P/6TM K+
channels, represented by the Drosophila 'ether-a-go-go' (eag) K+ channel,
contain a putative
cyclic nucleotide binding domain (cNBD) in the carboxyl terminus. Although the
eag
superfamily is somewhat related to the shaker K+ channel superfamily, the
structural
2s differences between the two are quite considerable, with less than 20%
identity in the
hydrophobic core region. The putative cNBD in eag members shares substantial
similarity to
the cNBD present in cyclic nucleotide-gated cation channels (Wanmke et al.
(1991), Science
252:1560-1564).
Within the eag superfamily, three channel subfamilies are currently known,
3o represented by eag, erg (eag-related gene), and elk (eag-like K+ channel).
In general, two
members of the same subfamily from different species share ~65-70% amino acid
sequence
identity in the region spanning S 1 through the cNBD segment. In contrast, two
different
subfamily members within the same species share only about 40-50% amino acid
sequence
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identity across the same region (Warmke and Ganetzky {1994} P.N.A.S. 91: 3438-
3442).
Channels within the eag superfamily from different animal species can often
express
channel currents with quite different properties. For example, when expressed
in Xenopus
oocytes, Drosophila eag channels are permeable to both K+ and Ca++ ions and
are
modulated by cAMP, while mouse eag channels are not permeable to Ca++ and are
not
modulated by cAMP (Bruggemann et al. (1993) Nature 365:445-448; Ludwig et al.
(1994),
EMBO J.13:4451-4458; Trudea et al.(1995), Science 269: 92-95). The three known
mammalian members ofthe erg subfamily, human ergl (HERG), rat erg2, and rat
erg3, are
activated by membrane depolarization, but their inactivation is faster than
their activation.
~o Unlike ergl and erg2, which are active above the firing threshold, erg3 is
active below firing
threshold, and is thus expected to affect the baseline activity of excitable
cells. The ergl
channel is abundantly expressed in the heart and brain, while erg2 and erg3
are absent from
the heart, but have a wide expression pattern in the nervous system. All three
are expressed in
some sympathetic ganglia (Si et aL, (1997) J. Neurosci. 17(24):9423-9432).
~s Mutations of channels within the eag superfamily have found to cause
profound
deficits in animal physiology. For example, mutations in Drosophila eag cause
excessive
membrane excitability in motor neurons which results in leg shaking behavior
in mutant flies.
Mutations in Drosophila erg can lead to paralysis in affected flies. Mutations
in HERG have
been linked to a fatal cardiac disease, the long QT syndrome type 2 (LQT2;
Curran et al.
20 (1995) Cell 80:795-803; Sanguientti et al. (1995), Cell 81:299-301).
Potassium channels are associated with a variety of disease states. In some
diseases
and disorders, abnormal ion channels are believed to be causative factors,
while other
diseases appear to arise from inappropriate regulation of otherwise normal ion
channels.
Diseases believed to have a particular association with potassium channels
include
2s neurological, cardiovascular, musculoskeletal, and proliferative disorders
such as cancers.
The discovery of novel channel polypeptides which represent a new subfamily of
the
eag K+ channel superfamily, and the polynucleotides which encode them,
satisfies a need in
the art by providing new compositions which are useful in treatment of various
diseases
associated with ion channel dysfunction.
Summary of the Invention
The invention is based on the discovery of a human K+ channel, ESKI (eag-
similar
K+ channel), which is representative of a new ESK subfamily of the eag K+
channel
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superfamily. The invention includes an isolated ESK potassium channel
polypeptide
comprising an amino acid sequence having at least 60 percent, preferably at
least 65 to 70
percent, more preferably at least 80 percent, and most preferably at least 90
percent sequence
identity to the region con esponding to residues 212-668 of the polypeptide
identified herein
s as SEQ ID N0:2, said region identified herein as SEQ ID NO:S. The invention
also includes
(i) fragments of ESK, which are capable of interaction with other proteins,
peptides, or
chemicals, such interaction which alters the functional properties or
cellular/subcellular
localization of ESK, and (ii) a pharmaceutical composition containing ESK or
an ESK
fragment. The invention also includes potassium channels comprising one or
more ESK
t o polypeptides.
In a specific embodiment the invention includes an isolated ESKI polypeptide
comprising an amino acid sequence having at least 60 percent sequence identity
with SEQ ID
N0:2. In other embodiments, the polypeptide comprises a sequence at least 70%,
80%, 90%,
or 95% identical to SEQ ID N0:2. In more specific embodiments, the polypeptide
has the
t s sequence SEQ ID N0:2 or SEQ ID N0:4.
In another aspect the invention includes an isolated polynucleotide having a
sequence
which encodes ESK as described above, or a sequence complementary to the ESK
coding
sequence, and a composition comprising the polynucleotide. The polynucleotide
may be
mRNA, cRNA, DNA, cDNA, genomic DNA, peptide nucleic acid, as well as an
antisense
2o analog thereof. The polynucleotide may encode an ESK polypeptide containing
a sequence
having at least 60% amino acid sequence identity to SEQ ID NO:S. In another
embodiment,
the polynucleotide encodes an ESK1 polypeptide having at least 70% amino acid
sequence
identity to SEQ ID N0:2. The polynucleotide may contain, for example, a coding
sequence
which hybridizes under high-stringency conditions with the polynucleotide
sequence
2s identified as SEQ ID NO:1 or the complement thereof. In specific
embodiments, the
polynucleotide has the sequence identified as SEQ ID NO:1 or SEQ ID N0:3. The
invention
also contemplates fragments of the polynucleotide, preferably at least 12,
more preferably at
least 20 or 30 nucleotides in length.
In another embodiment, the invention includes a nucleic acid molecule of at
least 12
3o nucleotides in length which specifically hybridizes under stringent
conditions to a
polynucleotide sequence encoding any of the polypeptides described above. The
nucleic acid
molecule may be mRNA, DNA, cDNA, genomic DNA, peptide nucleic acid, as well as
an
antisense analog thereof. The invention also includes a polynucleotide
sequence comprising
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the complement of a polynucleotide sequence encoding any of the polypeptides
described
above.
Also disclosed is a recombinant expression vector containing a polynucleotide
encoding ESK or a fragment described above, and, operably linked to the
polynucleotide,
s regulatory elements effective for expression of the protein in a selected
host. Preferred
coding sequences are given above. In a related aspect, the invention includes
a host cell
containing the vector.
The invention further includes a method for producing ESK by recombinant
techniques, by culturing recombinant prokaryotic or eukaryotic host cells
containing nucleic
to acid sequence encoding ESK under conditions promoting expression of the
protein, and
subsequent recovery of the protein from the host cell or the cell culture
medium.
In still another aspect, the invention includes an antibody specific against
ESK. The
antibody has diagnostic and therapeutic applications, particularly in treating
neurological and
neurodegenerative diseases. Treatment methods which employ antisense or coding
sequence
is polynucleotides for inhibiting or enhancing levels of ESK are also
contemplated, as are
treatment methods which employ antibodies specific against ESK. The invention
also
includes methods to alter the expression level of ESK by gene therapy
techniques to achieve
therapeutic benefits in patients.
Diagnostic methods for detecting levels of ESK in specific tissue samples, and
for
2o detecting levels of expression of ESK in tissues, also form part of the
invention. In one
embodiment, a method of detecting a polynucleotide which encodes ESK in a
biological
sample, involves the steps of (a) hybridizing the complement of a
polynucleotide which
encodes ESK to nucleic acid material of a biological sample, thereby forming a
hybridization
complex, and (b) detecting the hybridization complex, wherein the presence of
the complex
2s correlates with the presence of a polynucleotide encoding ESK in the
biological sample.
Methods for detecting mutations in the coding region of ESK are also
contemplated.
Screening methods which employ ESK for identifying a candidate compound
capable
of binding to and modulating the activity of ESK also form part of the
invention. An
exemplary method includes (a) contacting a test compound with ESK, (b)
measuring the
so effect of the test compound on the activity of ESK, and (c) selecting the
test compound as a
candidate compound if its effect on the activity of ESK is above a selected
threshold level.
The activity measured may be, for example, establishment or modulation of
potassium
conductance. In one embodiment, the test compound is a component of a
combinatorial
4
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library. In another embodiment, the test compound is an antibody specific
against the ESK
protein.
The invention also includes, in a related aspect, a compound identified by the
screening methods described above, including purified agonists and
antagonists. The
invention further includes a purified antibody which specifically binds to a
polypeptide
described above.
These and other objects and features of the invention will become more fully
apparent
when the following detailed description is read in conjunction with the
accompanying
drawings.
to
Brief Description of the Figures
Figs. lA-lE show a nucleic acid coding sequence (SEQ ID NO:1) and deduced
amino
acid sequence (SEQ ID N0:2) of an ESK polypeptide identified herein as human
ESKI
(hESKI);
is
Brief Descrin~ion of the Sequences
SEQ ID NO:1 is a nucleic acid sequence encoding hESKI;
SEQ ID N0:2 is the deduced amino acid translation of SEQ ID NO:1;
SEQ ID N0:3 is a nucleic acid sequence encoding a putative hESKl splice
variant;
2o SEQ ID N0:4 is the deduced amino acid translation of the putative hESKI
splice variant;
SEQ ID NO:S is the amino acid sequence of residues 212 to 668 of SEQ ID N0:2;
SEQ ID N0:6 is the amino acid sequence of hERG (GenBank PID g487738); and
SEQ ID N0:7 is the sequence of human EST U69184 (GenBank NID g2739408;
Accession
No. U69184).
Detailed Description of the Invention
I. D it' s
Unless otherwise indicated, all terms used herein have the same meaning as
they
would to one skilled in the art of the present invention. Practitioners are
particularly directed
3o to Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Second
Edition), Cold
Spring Harbor Press, Plainview, N.Y. and Ausubel FM et al. (1989) Current
Protocols in
Molecular Biology, John Wiley & Sons, New York, N.Y., for definitions and
terms of the art.
It is to be understood that this invention is not limited to the particular
methodology,
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protocols, and reagents described, as these may vary.
The term "polypeptide" as used herein refers to a compound made up of a single
chain
of amino acid residues linked by peptide bonds. The term "protein" as used
herein may be
synonymous with the term "polypeptide" or may refer, in addition, to a complex
of two or
more polypeptides.
A "channel" or "channel protein" as used herein refers to a multisubunit
protein
comprising two or more P-domain-containing polypeptide subunits, and may be
formed of
multimers of the same polypeptide (a "homomeric" channel) or of different
polypeptides (a
"heteromeric" channel). Channel proteins may also contain "accessory subunits"
which
t o modulate the activity of the channel.
A "polypeptide belonging to the eag superfamily" is a channel subunit which
contains
a potential P-domain, six predicted transmembrane domains (S1-S6), a cyclic
nucleotide
binding domain (cNBD), and has about 40% or greater sequence identity, in the
region
spanning S 1 through the cNBD segment, to a corresponding aligned region of
another
is polypeptide member of the eag K+ channel superfamily, such as eag or human
ergl (hERG) .
An "eag-similar K+ polypeptide" or "ESK polypeptide" refers to a polypeptide
belonging to the eag superfamily and further containing a sequence having at
least 60
percent, preferably at least 65 to 70 percent, more preferably at least 80
percent, and most
preferably at least 90 percent sequence identity to a region spanning the S 1
through cNBD
2o segment corresponding to residues 212-668 of the polypeptide identified
herein as SEQ ID
N0:2, said region identified herein as SEQ ID NO:S.
"ESKI" refers to an ESK polypeptide comprising a sequence having at least 70
percent, preferably at least 80 percent, more preferably at least 90 percent,
and most
preferably at least 95 percent sequence identity to SEQ ID N0:2.
2s As used herein, reference to "ESK" or "ESKI" is meant to include the full-
length
polypeptide and fi~agments thereof unless the context indicates otherwise.
The term "ESK channel" refers to a multimeric potassium channel comprising at
least
one ESK polypeptide.
The term "mature ESK" refers to the ESK polypeptide as it exists in the cell
after
3o post-translational processing; for example, after removal of a signal
sequence.
The term "modified", when referring to a polypeptide of the invention, means a
polypeptide which is modified either by natural processes, such as processing
or other post-
translational modifications, or by chemical modification techniques which are
well known in
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the art. Among the numerous known modifications which may be present include,
but are
not limited to, acetylation, acylation, amidation, ADP-ribosylation,
glycosylation, GPI anchor
formation, covalent attachment of a lipid or lipid derivative, methylation,
myristlyation,
pegylation, prenylation, phosphorylation, ubiqutination, or any similar
process.
s The term "biologically active" refers to an ESK having structural,
regulatory or
biochemical functions of the naturally occurring ESK including, but not
limited to, the ability
to support potassium ion conductance when self associated into a homomeric
channel, or
when associated with other channel polypeptides into a heteromeric channel.
Likewise,
"immunologically active" defines the capability of a natural, recombinant or
synthetic ESK,
io or any fragment thereof, to induce a specific immune response in
appropriate animals or cells
and to bind with specif c antibodies.
The term "fragment," when refernng to ESK, means a polypeptide which has an
amino acid sequence which is the same as part of but not all of the amino acid
sequence of
ESK, which retains at least one of the functions or activities of ESK, or
which is capable of
is interacting with ESK, other proteins, peptides, or other molecules, to
alter a function or
activity or the cellular/subcellular localization of an ESK channel. Fragments
contemplated
include, but are not limited to, an ESK fragment which retains the ability to
bind a ligand of
an ESK channel, an ESK fragment which blocks the binding of a ligand to an ESK
channel,
or an ESK fragment which retains immunological activity of ESK. The fragment
preferably
2o includes at least 20, more preferably at least 50, contiguous amino acid
residues of ESK.
The term "portion", when referring to a polypeptide of the invention, means a
polypeptide which has an amino acid sequence which is the same as part of the
amino acid
sequence of the present invention or a variant thereof, which does not
necessarily retain any
biological function or activity.
2s A "conservative substitution" refers to the substitution of an amino acid
in one class
by an amino acid in the same class, where a class is defined by common
physicochemical
amino acid sidechain properties and high substitution frequencies in
homologous proteins
found in nature (as determined, e.g., by a standard Dayhoff frequency exchange
matrix or
BLOSUM matrix). Six general classes of amino acid sidechains, categorized as
described
3o above, include: Class I (Cys); Class II (Ser, Thr, Pro, AIa, Gly); Class
III (Asn, Asp, Gln,
Glu); Class IV (His, Arg, Lys); Class V {Ile, Leu, Val, Met); and Class VI
(Phe, Tyr, Trp).
For example, substitution of an Asp for another class III residue such as Asn,
Gln, or Glu, is a
conservative substitution.
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A "non-conservative substitution" refers to the substitution of an amino acid
in one
class with an amino acid from another class; for example, substitution of an
Ala, a class II
residue, with a class III residue such as Asp, Asn, Glu, or Gln.
"Optimal alignment" is defined as an alignment giving the highest percent
identity
score. Such alignment can be performed using a variety of commercially
available sequence
analysis programs, such as the local alignment program LALIGN using a letup of
1, default
parameters and the default PAM. A preferred alignment is the one performed
using the
CLUSTAL-W program in MacVector, operated with default parameters, including an
open
gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM similarity
matrix.
"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 sequences are optimally aligned. Thus, 80% amino acid sequence identity
means that
80% of the amino acids in two or more optimally aligned polypeptide sequences
are identical.
If a gap needs to be inserted into a first sequence to optimally align it with
a second sequence,
is the percent identity is calculated using only the residues that are paired
with a corresponding
amino acid residue (i.e., the calculation does not consider residues in the
second sequences
that are in the "gap" of the first sequence).
A first polypeptide region is said to "correspond" to a second polypeptide
region when
the regions are essentially co-extensive when the sequences containing the
regions are
2o aligned using a sequence alignment program, as above. Corresponding
polypeptide regions
typically contain a similar, if not identical, number of residues. It will be
understood,
however, that corresponding regions may contain insertions or deletions of
residues with
respect to one another, as well as some differences in their sequences.
"Con:esponding" polynucleotide or polypeptide fragments typically contain a
similar,
2s if not identical, number of residues. It will be understood, however, that
corresponding
fragments may contain insertions or deletions of residues with respect to one
another, as well
as some differences in their sequences.
The term "sequence identity" means nucleic acid or amino acid sequence
identity in
two or more aligned sequences, aligned as defined above.
30 "Sequence similarity" between two polypeptides is determined by comparing
the
amino acid sequence and its conserved amino acid substitutes of one
polypeptide to the
sequence of a second polypeptide. Thus, 80% protein sequence similarity means
that 80% of
the amino acid residues in two or more aligned protein sequences are conserved
amino acid
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residues, i.e. are conservative substitutions.
"Hybridization" includes any process by which a strand of a 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 to the
test sequence, or
s vice-versa
"Hybridization conditions" are based on the melting temperature (Tm) of the
nucleic
acid binding complex or probe and are typically classified by degree of
"stringency" of the
conditions under which hybridization is measured. For example, "maximum
stringency"
typically occurs at about Tm-5°C (5° below the Tm of the probe);
"high stringency" at about
io 5-10° below the Tm; "intermediate stringency" at about 10-20°
below the Tm of the probe;
and "low stringency" at about 20-25° below the Tm. Functionally,
maximum stringency
conditions may be used to identify nucleic acid sequences having strict
identity or near-strict
identity with the hybridization probe; while high stringency conditions are
used to identify
nucleic acid sequences having about 80% or more sequence identity with the
probe.
~s An example of "high stringency" conditions includes hybridization at about
65°C in
about Sx SSPE and washing at about 65°C in about O.lx SSPE (where lx
SSPE = 0.15
sodium chloride, 0.010 M sodium phosphate, and 0.001 M disodium EDTA).
The term "gene" as used herein means the segment of DNA involved in producing
a
polypeptide chain; it may include regions preceding and following the coding
region, e.g. 5'
2o untranslated (5' UTR) or "leader" sequences and 3' UTR or "trailer"
sequences, as well as
intervening sequences (introns) between individual coding segments (exons).
An "isolated polynucleotide having a sequence which encodes ESK" is a
polynucleotide which contains the coding sequence of ESK (i) in isolation,
(ii) in
combination with additional coding sequences, such as fusion protein or signal
peptide, in
2s which the ESK coding sequence is the dominant coding sequence, (iii) in
combination with
non-coding sequences, such as introns and control elements, such as promoter
and terminator
elements or 5' and/or 3' untranslated regions, effective for expression of the
coding sequence
in a suitable host, and/or (iv) in a vector or host environment in which the
ESK coding
sequence is a heterologous gene.
so The terms "heterologous DNA" and "heterologous RNA" refer to nucleotides
that are
not endogenous to the cell or part of the genome in which they are present;
generally such
nucleotides have been added to the cell, by transfection, microinjection,
electroporation, or
the like. Such nucleotides generally include at least one coding sequence, but
this coding
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sequence need not be expressed.
The term "isolated" means that the material is removed from its original
environment
(e.g., the natural environment if it is naturally occurring). For example, a
naturally-occurring
polynucleotide or polypeptide present in a living animal is not isolated, but
the same
polynucleotide or polypeptide, separated from some or all of the coexisting
materials in the
natural system, is isolated. Such polynucleotides could be part of a vector
and/or such
polynucleotides or polypeptides could be part of a composition, and still be
isolated in that
such vector or composition is not part of its natural environment.
The term "fragment," when referring to an ESK coding sequence, means a
i o polynucleotide which has a nucleic acid sequence which is the same as part
of but not all of
the nucleic acid sequence of the ESK coding sequence. The fragment preferably
includes at
least 12 contiguous bases of ESK coding sequence.
The term "expression vector" refers to vectors that have the ability to
incorporate and
express heterologous DNA fragments in a foreign cell. Many prokaryotic and
eukaryotic
1s expression vectors are commercially available. Selection of appropriate
expression vectors
is within the knowledge of those having skill in the art.
The term "substantially purified" refers to molecules, either polynucleotides
or
polypeptides, that are removed from their natural environment, isolated or
separated, and are
at least 60% free, preferably 75% free, and most preferably 90% free from
other components
2o with which they are naturally associated.
A "variant" polynucleotide sequence may encode a "variant" amino acid sequence
which is altered by one or more amino acids from the reference polypeptide
sequence. The
variant polynucleotide sequence may encode a variant amino acid sequence which
contains
"conservative" substitutions; wherein the substituted amino acid has
structural or chemical
2s properties similar to the amino acid which it replaces. In addition, or
alternatively, the
variant polynucleotide sequence may encode a variant amino acid sequence which
contains
"non-conservative" substitutions, wherein the substituted amino acid has
dissimilar structural
or chemical properties to the amino acid which it replaces. Variant
polynucleotides may also
encode variant amino acid sequences which contain amino acid insertions or
deletions, or
3o both. Furthermore, a variant polynucleotide may encode the same polypeptide
as the
reference polynucleotide sequence but, due to the degeneracy of the genetic
code, has a
polynucleotide sequence which is altered by one or more bases from the
reference
polynucleotide sequence.
to
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An "allelic variant" is an alternate foam of a polynucleotide sequence which
may have
a substitution, deletion or addition of one or more nucleotides, which does
not substantially
alter the function of the encoded polypeptide.
"Alternative splicing" is a process whereby multiple polypeptide isoforms are
s generated from a single gene, and involves the splicing together of
nonconsecutive exons
during the processing of some, but not all, transcripts of the gene. Thus a
particular exon
may be connected to any one of several alternative exons to form messenger
RNAs. The
alternatively-spliced mRNAs produce polypeptides ("splice variants") in which
some parts
are common while other parts are different.
"Splice variants" of ESK, when referred to in the context of an mRNA
transcript, are
mRNAs produced by alternative splicing of coding regions, i.e., exons, from
the ESK gene.
"Splice variants" of ESK, when referred to in the context of the protein
itself, are ESK
translation products which are encoded by alternatively-spliced ESK mRNA
transcripts.
A "mutant" amino acid or polynucleotide sequence is a variant amino acid
sequence,
is or a variant polynucleotide sequence which encodes a variant amino acid
sequence, which
has significantly altered biological activity from that of the naturally
occurring protein.
A "deletion" is defined as a change in either nucleotide or amino acid
sequence in
which one or more nucleotides or amino acid residues, respectively, are
absent.
An "insertion" or "addition" is that change in a nucleotide or amino acid
sequence
2o which has resulted in the addition of one or more nucleotides or amino acid
residues,
respectively, as compared to the naturally occurring sequence.
A "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 the change in activity of the
polypeptide
2s of the invention. Modulation may relate to an increase or a decrease in
biological activity,
binding characteristics, or any other biological, functional, or immunological
property of the
molecule.
The term "agonist" as used herein, refers to a molecule which, when bound to
the
channel of the present invention, modulates the activity of the channel by
inducing,
so increasing, or prolonging the duration of the biological activity mediated
by the channel.
Agonists may themselves be polypeptides, nucleic acids, carbohydrates, lipids,
or derivatives
thereof, or any other ligand which binds to and modulates the activity of the
channel.
The term "antagonist" as used herein, refers to a molecule which, when bound
to the
11
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channel of the present invention, modulates the activity of the channel by
blocking,
decreasing, or shortening the duration of the biological activity mediated by
the channel.
Antagonists may themselves be polypeptides, nucleic acids, carbohydrates,
lipids, or
derivatives thereof, or any other ligand which binds to and modulates the
activity of the
channel.
The term "humanized antibody" refers to antibody molecule in which one or more
amino acids have been replaced in the non-antigen binding regions in order to
more closely
resemble a human antibody, while still retaining the original binding activity
of the antibody.
"Treating a disease" refers to administering a therapeutic substance effective
to reduce
to the symptoms of the disease and/or lessen the severity of the disease.
II. Polvnucleotides Encoding ESK
The invention provides an isolated ESK polypeptide and an isolated
polynucleotide
encoding the polypeptide. As defined more fully in Section III below, ESK (i)
represents a
is new subfamily of the eag potassium channel superfamily, and (ii) contains
an amino acid
sequence having at least 60 percent sequence identity to a region identified
as SEQ ID NO:S.
An exemplary ESK polypeptide, human ESKI (hESKI), has a sequence identified as
SEQ m
N0:2.
As shown in Fig. 1, SEQ ID NO:1 is a 3829 base nucleic acid sequence which
2o contains an open reading frame encoding a 1080 amino acid polypeptide
identified herein as
an hESKl polypeptide having the sequence SEQ ID N0:2. A putative hESKl splice
variant,
containing a 33 nucleotide insertion between nucleotides 2819 and 2820 of SEQ
ID NO:1
resulting in the 3862 base sequence identified herein as SEQ ID N0:3, yields a
translation
product identical to SEQ ID N0:2 except for an 11 amino acid insertion between
amino acids
2s 855 and 856 resulting in the 1091 amino acid polypeptide SEQ ID N0:4.
Polynuceotides encoding hESKI were discovered in a cDNA library derived from
human brain. Coding sequences were identified, cloned and sequenced
substantially as
described in Example 1. Briefly, a biotin-labeled human probe based on a human
EST
sequence (GenBank Accession No. U69184; SEQ ID N0:7) was used to capture
target
3o cDNA molecules from a brain cDNA library by solution hybridization. After
secondary and
tertiary screening with radiolabeled oligonucleotide or cDNA probes, positive
colonies were
cultured, and plasmid cDNA isolated and sequenced, resulting in the
construction and
identification of a nucleic acid sequence identified as SEQ ID NO:1.
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Northern analysis performed as described in Example 2 showed that, among the
tissues tested, expression of hESKI transcript was observed only in brain,
predominantly
forebrain.
A. ~,olvnucleotide compositions
The polynucleotides of the invention include sequences which encode ESK and
sequences complementary to the coding sequence, and novel fragments of the
polynucleotide.
The polynucleotides may be in the form of RNA or in the form of DNA, and
include mRNA,
cRNA, synthetic RNA and DNA and analogs thereof, cDNA, peptide nucleic acid,
and
to genomic DNA. The polynucleotides may be double-stranded or single-stranded,
and if
single-stranded may be the coding strand or the non-coding (anti-sense,
complementary)
strand.
In a general embodiment, the polynucleotide hybridizes under stringent
conditions,
preferably high-stringency conditions, to the sequence identified as SEQ ID
NO:1, SEQ ID
is N0:3, or the complements thereof. Exemplary hybridization conditions are
described in
Section IIB below. In another embodiment, the polynucleotide encodes an ESK
polypeptide
containing a sequence having at least 60% amino acid sequence identity to SEQ
ID NO:S. In
another embodiment, the polynucleotide encodes an ESKl polypeptide having at
least 70%
amino acid sequence identity to SEQ ID N0:2 or SEQ ID N0:4. In other
embodiments, the
2o polynucleotide of the invention has at least 70%, preferably at least 80%
or 90% sequence
identity with the sequence identified as SEQ ID NO:1, SEQ ID N0:3, or the
complements
thereof. In more specific embodiments, the polynucleotide has the sequence
identified as
SEQ ID NO:1 or SEQ ID N0:3.
The polynucleotides may include the coding sequence of ESK (i) in isolation,
(ii) in
zs combination with additional coding sequences, such as fusion protein or
signal peptide, in
which the ESK coding sequence is the dominant coding sequence, (iii) in
combination with
non-coding sequences, such as introns and control elements, such as promoter
and terminator
elements or 5' and/or 3' untranslated regions, effective for expression of the
coding sequence
in a suitable host, and/or (iv) in a vector or host environment in which the
ESK coding
3o sequence is a heterologous gene.
The polynucleotide may encode a polypeptide fragment of ESK, for example, an
extracellular fragment or an intracellular fragment which has been cleaved
from a
transmembrane domain of ESK.
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The polynucleotides of the present invention may also have the protein coding
sequence fused in-frame to a marker sequence which allows for purification of
ESK. The
marker sequence may be, for example, a hexahistidine tag to provide for
purification of the
mature polypeptide fused to the marker in the case of a bacterial host, or,
the marker
sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7
cells, is
used. The HA tag corresponds to an epitope derived from the influenza
hemagglutinin
protein (Wilson, L, et al. (1984) Cell 37:767).
Also contemplated are novel uses of polynucleotides, also referred to herein
as
oligonucleotides, typically having at least 12 bases, preferably at least 20
or 30 bases,
to corresponding to a region of the coding-sequence polynucleotide or the
complement thereof.
The polynucleotides may be used as probes, primers, antisense agents, and the
like, according
to known methods.
B, pren,~ration of pol~mucleotides
is The polynucleotides may be obtained by screening cDNA libraries using
otigonucleotide probes which can hybridize to or PCR-amplify polynucleotides
which encode
the ESK and fragments disclosed above. cDNA libraries prepared from a variety
of tissues
are commercially available and procedures for screening and isolating cDNA
clones are well-
known to those of skill in the art. Such techniques are described in, for
example, Sambrook
2o et al. (1989) Molecular Cloning: A Laboratory Manual (2nd Edition), Cold
Spring Harbor
Press, Plainview, N.Y. and Ausubel FM et al. (1989) Current Protocols in
Molecular
Biology, John Wiley & Sons, New York, N.Y.
Hybridization conditions are based on the melting temperature (Tm) of the
nucleic
acid binding complex or probe and are typically classified by degree of
"stringency" of the
2s conditions under which hybridization is measured. For example, "maximum
stringency"
typically occurs at about Tm-5°C (5° below the Tm of the probe);
"high stringency" at about
5-10° below the Tm; "intermediate stringency" at about 10-20°
below the Tm of the probe;
and "low stringency" at about 20-25° below the Tm. Functionally,
maximum stringency
conditions may be used to identify nucleic acid sequences having strict
identity or near-strict
3o identity with the hybridization probe; while high stringency conditions are
used to identify
nucleic acid sequences having about 80% or more sequence identity with the
probe. An
example ofhigh stringency conditions includes hybridization at about
65°C in about Sx SSPE
and washing conditions of about 65°C in about O.lx SSPE (where lx SSPE
= 0.15 sodium
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chloride, 0.010 M sodium phosphate, and 0.001 M disodium EDTA).
The polynucleotides may be extended to obtain upstream and downstream
sequences
such as promoters, regulatory elements, and 5' and 3' untranslated regions
(UTRs). Extension
of the available transcript sequence may be performed by numerous methods
known to those
of skill in the art, such as PCR or primer extension (Sambrook et al., supra),
or by the RACE
method using, for example, the Marathon RACE kit (Clontech, Cat. # K1802-1).
Alternatively, the technique of "restriction-site" PCR (Gobinda et al. (1993)
PCR
Methods Applic. 2:318-22), which uses universal primers to retrieve flanking
sequence
adjacent a known locus, may be employed. First, genomic DNA is amplified in
the presence
t o of primer to a linker sequence and a primer specific to the known region.
The amplified
sequences are subjected to a second round of PCR with the same linker primer
and another
specific primer internal to the first one. Products of each round of PCR are
transcribed with
an appropriate RNA polymerase and sequenced using reverse transcriptase.
Inverse PCR can be used to amplify or extend sequences using divergent primers
is based on a known region (Triglia T et al. (1988) Nucleic Acids Res
16:8186). The primers
may be designed using OLIGO(R) 4.06 Primer Analysis Software (1992; National
Biosciences Inc, Plymouth, Minn.), or another appropriate program, to be 22-30
nucleotides
in length, to have a GC content of 50% or more, and to anneal to the target
sequence at
temperatures about 68-72°C. The method uses several restriction enzymes
to generate a
2o suitable fragment in the known region of a gene. The fragment is then
circularized by
intramolecular ligation and used as a PCR template.
Capture PCR (Lagerstrom M et aL (1991) PCR Methods Applic 1:111-19) is a
method for PCR amplification of DNA fragments adjacent to a known sequence in
human
and yeast artificial chromosome DNA. Capture PCR also requires multiple
restriction
2s enzyme digestions and ligations to place an engineered double-stranded
sequence into a
flanking part of the DNA molecule before PCR.
Another method which may be used to retrieve flanking sequences is that of
Parker,
JD et al. (1991; Nucleic Acids Res 19:3055-60). Additionally, one can use PCR,
nested
primers and PromoterFinder'~'M libraries to "walk in" genomic DNA (Clontech,
Palo Alto,
3o CA). This process avoids the need to screen libraries and is useful in
finding intron/exon
junctions. Preferred libraries for screening for full length cDNAs are ones
that have been
size-selected to include larger cDNAs. Also, random primed libraries are
preferred in that
they will contain more sequences which contain the 5' and upstream regions of
genes. A
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randomly primed library may be particularly useful if an oligo d(T) library
does not yield a
full-length cDNA. Genomic libraries are useful for extension into the f
nontranslated
regulatory region.
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, then joined to form continuous
sequences up to
several hundred bases.
C. A_nnlications of noly~~ucleotides
to The polynucleotide coding sequences and novel oligonucleotides of the
invention
have a variety of uses in ( 1 ) synthesis of ESK, (2) diagnostics, (3) gene
mapping, and (4)
therapeutics.
C 1. Synthesis of ESK
In accordance with the present invention, polynucleotide sequences which
encode
~s ESK, splice variants, fragments of the polypeptide, fusion proteins, or
functional equivalents
thereof, collectively referred to herein as "ESK", may be used in recombinant
DNA
molecules that direct the expression of ESK in appropriate host cells. Due to
the inherent
degeneracy of the genetic code, other nucleic acid sequences which encode
substantially the
same or a functionally equivalent amino acid sequence may be used to clone and
express
2o ESK.
As will be understood by those of skill in the art, it may be advantageous to
produce
ESK-encoding nucleotide sequences possessing non-naturally occurring codons.
Codons
preferred by a particular prokaryotic or eukaryotic host (Murray, E. et al.
(1989) Nuc Acids
Res 17:477-508) can be selected, for example, to increase the rate of ESK
polypeptide
2s expression or to produce recombinant RNA transcripts having desirable
properties, such as a
longer half life, than transcripts produced from naturally occurnng sequence.
The polynucleotide sequences of the present invention can be engineered in
order to
alter an ESK coding sequence for a variety of reasons, including but not
limited to, alterations
which modify the cloning, processing and/or expression of the gene product.
For example,
3o alterations may be introduced using techniques which are well known in the
art, e.g., site-
directed mutagenesis, to insert new restriction sites, to alter glycosylation
patterns, to change
codon preference, to produce splice variants, etc.
The present invention also includes recombinant constructs comprising one or
more
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of the sequences as broadly described above. The constructs comprise a vector,
such as a
plasmid or viral vector, into which a sequence of the invention has been
inserted, in a forward
or reverse orientation. In a preferred aspect of this embodiment, the
construct further
comprises regulatory sequences, including, for example, a promoter, operably
linked to the
s sequence. Large numbers of suitable vectors and promoters are known to those
of skill in the
art, and are commercially available. Appropriate 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 which are genetically
engineered with
vectors of the invention, and the production of proteins and polypeptides of
the invention by
to recombinant techniques. Host cells are genetically engineered {i.e.,
transduced, transformed
or transfected) with the vectors of this invention which may be, for example,
a cloning vector
or an expression vector. The vector may be, for example, in the form of a
plasmid, a viral
particle, a phage, etc. The engineered host cells can be cultured in
conventional nutrient
media modified as appropriate for activating promoters, selecting
transformants or
is amplifying the ESK gene. The culture conditions, such as temperature, pH
and the like, are
those previously used with the host cell selected for expression, and will be
apparent to those
skilled in the art.
The polynucleotides of the present invention may be included in any one of a
variety
of expression vectors for expressing a polypeptide. Such vectors include
chromosomal,
2o nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;
bacterial
plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from
combinations of
plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox
virus, and
pseudorabies. However, any other vector may be used as long as it is
replicable and viable in
the host. The appropriate DNA sequence may be inserted into the vector by a
variety of
2s procedures. In general, the DNA sequence is inserted into an appropriate
restriction
endonuclease sites) by procedures known in the art. Such procedures and
related sub-
cloning procedures are deemed to be within the scope of those skilled in the
art.
The DNA sequence in the expression vector is operatively linked to an
appropriate
transcription control sequence (promoter) to direct mRNA synthesis. Examples
of such
3o promoters include: LTR or SV40 promoter, the E. coli lac or trp promoter,
the phage lambda
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
1~
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appropriate sequences for amplifying expression. In addition, the expression
vectors
preferably contain one or more selectable marker genes to provide a phenotypic
trait for
selection of transformed host cells such as dihydrofolate reductase or
neomycin resistance for
eukaryotic cell culture, or such as tetracycline or ampicillin resistance in
E. coli.
The vector containing the appropriate DNA sequence as described above, as well
as
an appropriate promoter or control sequence, may be employed to transform an
appropriate
host to permit the host to express the protein. Examples of appropriate
expression hosts
include: bacterial cells, such as E. coli, Streptomyces, and Salmonella
typhimurium; fungal
cells, such as yeast; insect cells such as Drosophila and Spodoptera Sf9;
mammalian cells
to such as CHO, COS, BHK, HEK 293 or Bowes melanoma; adenoviruses; plant
cells, etc. It is
understood that not all cells or cell lines will be capable of producing fully
functional ESK;
for example, bacterial expression is contemplated for the production of
fragments of ESK
which may not retain all functions of ESK. The selection of an appropriate
host is deemed to
be within the scope of those skilled in the art from the teachings herein. The
invention is not
t s limited by the host cells employed.
In bacterial systems, a number of expression vectors may be selected depending
upon
the use intended for ESK. For example, when large quantities of ESK or
fragments thereof
are needed for the induction of antibodies, vectors which direct high level
expression of
fusion proteins that are readily purified may be desirable. Such vectors
include, but are not
20 limited to, multifunctional E. coli cloning and expression vectors such as
Bluescript~
(Stratagene), in which the ESK coding sequence may be ligated into the vector
in-frame with
sequences for the amino-terminal Met and the subsequent 7 residues of beta-
galactosidase so
that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster (1989) J.
Biol. Chem
264:5503-5509); pET vectors (Novagen, Madison WI); and the like.
2s 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
reviews, see Ausubel et al. (supra) and Grant et al. (1987; Methods in
Enzymology 153:516-
544).
In cases where plant expression vectors are used, the expression of a sequence
3o encoding ESK 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) may
be used alone or in combination with the omega leader sequence from TMV
(Takamatsu et
al. (1987) EMBO J 6:307-311). Alternatively, plant promoters such as the small
subunit of
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RLBISCO (Coruzzi et al (1984) EMBO J 3:1671-1680; Broglie et al. (1984)
Science
224:838-843); or heat shock promoters (Winter J and Sinibaldi RM (1991)
Results. Probl.
Cell Differ. 17:85-105) may be used. These constructs can be introduced into
plant cells by
direct DNA transformation or pathogen-mediated transfection. For reviews of
such
s techniques, see Hobbs S or Murry LE (1992) in McGraw Hill Yearbook of
Science and
Technology, McGraw Hill, New York, N.Y., pp 191-196; or Weissbach and
Weissbach
(1988) Methods for Plant Molecular Biology, Academic Press, New York, N.Y., pp
421-463.
ESK may also be expressed in an insect system. In one such system, Autographa
californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express
foreign genes
to in Spodoptera frugiperda Sf9 cells or in Trichoplusia larvae. The ESK
coding sequence is
cloned into a nonessential region of the virus, such as the polyhedrin gene,
and placed under
control of the polyhedrin promoter. Successful insertion of ESK coding
sequence will render
the polyhedrin gene inactive and produce recombinant virus lacking coat
protein coat. The
recombinant viruses are then used to infect S. frugiperda cells or
Trichoplusia larvae in
is which ESK is expressed (Smith et al. (1983) J Virol 46:584; Engelhard EK et
al. (1994) Proc
Nat Acad Sci 91:3224-3227).
In mammalian host cells, a number of viral-based expression systems may be
utilized.
In cases where an adenovirus is used as an expression vector, an ESK coding
sequence may
be ligated into an adenovirus transcription/translation complex consisting of
the late promoter
2o and tripartite leader sequence. Insertion in a nonessential E1 or E3 region
of the viral
genome will result in a viable virus capable of expressing ESK in infected
host cells (Logan
and Shenk (1984) Proc Natl Acad Sci 81:3655-3659). In addition, transcription
enhancers,
such as the rous sarcoma virus (RSV) enhancer, may be used to increase
expression in
mammalian host cells.
2s Specific initiation signals may also be required for efficient translation
of an ESK
coding sequence. These signals include the ATG initiation codon and adjacent
sequences. In
cases where ESK coding sequence, its initiation codon and upstream sequences
are inserted
into the appropriate expression vector, no additional translational control
signals may be
needed. However, in cases where only coding sequence, or a portion thereof, is
inserted,
so exogenous transcriptional control signals including the ATG initiation
codon must be
provided. Furthermore, the initiation codon must be in the correct reading
frame to ensure
transcription of the entire insert. Exogenous transcriptional elements and
initiation codons
can be of various origins, both natural and synthetic. The efficiency of
expression may be
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enhanced by the inclusion of enhancers appropriate to the cell system in use
(Scharf D et al.
(1994) Results Probl Cell Differ 20:125-62; Bittner et al. (1987) Methods in
Enzymol
153:516-544).
In a further embodiment, the present invention relates to host cells
containing the
s above-described constructs. The host cell can be a higher eukaryotic cell,
such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host
cell can be a
prokaryotic cell, such as a bacterial cell. Introduction of the construct into
the host cell can
be effected by calcium phosphate transfection, DEAF-Dextran mediated
transfection, or
electmporation (Davis, L., Dibner, M., and Battey, I. (1986) Basic Methods in
Molecular
to Biology). Cell-free translation systems can also be employed to produce
polypeptides using
RNAs derived from the DNA constructs of the present invention. ESK cRNA may be
microinjected into cells, such as Xenopus laevis oocytes, for production of
ESK for
electrophysiological measurements or other assays.
A host cell strain may be chosen for its ability to modulate the expression of
the
is inserted sequences or to pmcess the expressed protein in the desired
fashion. Such
modifications of the protein include, but are not limited to, acetylation,
carboxylation,
glycosylation, phosphorylation, lipidation and acylation. Post-translational
processing which
cleaves a "prepro" form of the protein may also be important for correct
insertion, folding
and/or function. Different host cells such as CHO, HeLa, BHK, MDCK, 293, WI38,
etc. have
zo specific cellular machinery and characteristic mechanisms for such post-
translational
activities and may be chosen to ensure the correct modification and processing
of the
introduced, foreign protein. For practicing certain aspects of the invention,
such as
electrophysiological measurements described below, it is appreciated that it
may be desirable
that the host cell lack endogenous functionally expressed potassium channels
having current
2s characteristics similar to those exhibited or modulated by the ESK channel
described herein.
For long-term, high-yield production of recombinant proteins, stable
expression is
preferred. For example, cell lines which stably express ESK may be transformed
using
expression vectors which contain viral origins of replication or endogenous
expression
elements and a selectable marker gene. Following the introduction of the
vector, cells may
3o be allowed to grow for 1-2 days in an enriched media before they are
switched to selective
media. The purpose of the selectable marker is to confer resistance to
selection, and its
presence allows growth and recovery of cells which successfully express the
introduced
sequences. Resistant clumps of stably transformed cells can be proliferated
using tissue
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culture techniques appropriate to the cell type.
Host cells transformed with a nucleotide sequence encoding ESK may 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 understood by those of skill in the art, expression vectors
containing
polynucleotides encoding ESK can be designed with signal sequences which
direct secretion
of ESK through a prokaryotic or eukaryotic cell membrane.
ESK may also be expressed as a recombinant protein with one or more additional
to polypeptide domains added to facilitate protein purification. Such
purification facilitating
domains include, but are not limited to, metal chelating peptides such as
histidine-tryptophan
modules that allow purification on immobilized metals, protein A domains that
allow
purification on immobilized immunoglobulin, and the domain utilized in the
FLAGS
extension/affinity purification system (Immunex Corp, Seattle, Wash.). The
inclusion of a
is protease-cleavable polypepdde linker sequence between the purification
domain and ESK is
useful to facilitate purification. One such expression vector provides for
expression of a
fusion protein compromising ESK (e.g., a soluble ESK fi~agment) fused to a
polyhistidine
region separated by an enterokinase cleavage site. The histidine residues
facilitate
purification on IMIAC (immobilized metal ion affinity chromatography, as
described in
2o Porath et al. (1992) Protein Expression and Purification 3:263-281) while
the enterokinase
cleavage site provides a means for isolating ESK from the fusion protein. pGEX
vectors
(Promega, Madison, Wis.) may also be used to express foreign polypeptides as
fusion
proteins with glutathione S-transferase (GST). In general, such fusion
proteins are soluble
and can easily be purified from lysed cells by adsorption to ligand-agarose
beads (e.g.,
2s glutathione-agarose in the case of GST-fusions) followed by elution in the
presence of free
ligand.
Following transformation of a suitable host strain and growth of the host
strain to an
appropriate cell density, the selected promoter is induced by appropriate
means (e.g.,
temperature shift or chemical induction) and cells are cultured for an
additional period. Cells
3o are typically harvested by centrifugation, disrupted by physical or
chemical means, and the
resulting crude extract retained for further purification. Microbial cells
employed in
expression of proteins can be disrupted by any convenient method, including
freeze-thaw
cycling, sonication, mechanical disruption, or use of cell lysing agents, or
other methods,
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which are well know to those skilled in the art.
ESK can be recovered and purified from recombinant cell cultures by any of a
number
of methods well known in the art, including ammonium sulfate or ethanol
precipitation, acid
extraction, anion or cation exchange chromatography, phosphocellulose
chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite
chromatography, and lectin chromatography. Protein refolding steps can be
used, as
necessary, in completing configuration of the mature protein. Finally, high
performance
liquid chromatography (HPLC) can be employed for final purification steps.
C2. Dia og,~ stic a~nlications
to The polynucleotides of the present invention may be used for a variety of
diagnostic
purposes. The polynucleotides may be used to detect and quantitate expression
of ESK in
patient's cells, e.g. biopsied tissues, by detecting the presence of mltNA
coding for ESK.
This assay typically involves obtaining total mltNA from the tissue and
contacting the
mRNA with a nucleic acid probe. The probe is a nucleic acid molecule of at
least 12
~s nucleotides, preferably at least 20 or at least 30 nucleotides, capable of
specifically
hybridizing with a sequence included within the sequence of a nucleic acid
molecule
encoding ESK under hybridizing conditions, detecting the presence of mRNA
hybridized to
the probe, and thereby detecting the expression of ESK. This assay can be used
to distinguish
between absence, presence, and excess expression of ESK and to monitor levels
of ESK
zo expression during therapeutic intervention.
The invention also contemplates the use of the polynucleotides as a diagnostic
for
diseases resulting from inherited defective ESK genes. These genes can be
detected by
comparing the sequences of the defective (i.e., mutant) ESK gene with that of
a normal one.
Association of a mutant ESK gene with abnormal ESK activity (for example,
abnormal
2s channel activity) may be verified. In addition, mutant ESK genes can be
inserted into a
suitable vector for expression in a functional assay system as yet another
means to verify or
identify mutations. Once mutant genes have been identified, one can then
screen populations
of interest for carriers of the mutant gene.
The invention also includes a method of determining if a subject is at risk
for a
3o disorder associated with, e.g., abnormal ESK channel activity. The method
involves
detecting at least one of a) aberrant modification or mutation of a
polynucleotide sequence
encoding ESK, b) misregulation and c) aberrant post-translational modif cation
of ESK. In
one embodiment, detecting the genetic lesion includes determining the presence
of at least
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one of a deletion of one or more nucleotides from an ESK gene, an addition of
one or more
nucleotides to an ESK gene, a substitution of one or more nucleotides of the
gene, a gross
chromosomal arrangement of the gene, an alteration in the level of mRNA
transcript of the
gene; the presence of an abnormal splicing pattern of an mRNA transcript of
the gene; a non-
s wild type level of ESK protein expression. In a preferred embodiment, a ESK
polynucleotide of the present invention is combined with the nucleic acid of a
cell and
hybridization of the polynucleotide to the nucleic acid is determined. Failure
of the
polynucleotide to hybridize or a reduction of hybridization signal are
indicative of a mutation
in the ESK gene.
io Individuals carrying mutations in the gene of the present invention may be
detected at
the DNA level by a variety of techniques. Nucleic acids used for diagnosis may
be obtained
from a patient's cells, including but not limited to such as from blood,
urine, saliva, placenta,
tissue biopsy and autopsy material. Genomic DNA may be used directly for
detection or may
be amplified enzymatically by using PCR (Saiki, et al. (1986) Nature 324:163-
166) prior to
~s analysis. RNA or cDNA may also be used for the same purpose. As an example,
PCR
primers complementary to the nucleic acid of the present invention can be used
to identify
and analyze mutations in the gene of the present invention. Deletions and
insertions can be
detected by a change in size of the amplified product in comparison to the
normal genotype.
Point mutations can be identified by hybridizing amplified DNA to radiolabeled
RNA
20 of the invention or alternatively, radiolabeled antisense DNA sequences of
the invention.
Sequence changes at specific locations may also be revealed by nuclease
protection assays,
such RNase and S 1 protection or the chemical cleavage method (e.g. Cotton, et
al. (1985)
Proc. Natl. Acad. Sci. USA 85:4397-4401), or by differences in melting
temperatures.
"Molecular beacons" (Kostrikis L.G. et al. (1998) Science 279:1228-1229),
hairpin-shaped,
2s single-stranded synthetic oligonucleotides containing probe sequences which
are
complementary to the nucleic acid of the present invention, may also be used
to detect point
mutations or other sequence changes as well as monitor expression levels of
ESK. Such
diagnostics would be particularly useful for, e.g., prenatal testing.
Another method for detecting mutations uses two DNA probes which are designed
to
3o hybridize to adjacent regions of a target, with abutting bases, where the
region of known or
suspected mutations) is at or near the abutting bases. The two probes may be
joined at the
abutting bases, e.g., in the presence of a ligase enzyme, but only if both
probes are correctly
base paired in the region of probe junction. The presence or absence of
mutations is then
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detectable by the presence or absence of ligated probe.
Also suitable for detecting mutations in the ESK coding sequence are
oligonucleotide
array methods based on sequencing by hybridization (SBH), as described, for
example, in
U.S. Patent No. 5,547,839. In a typical method, the DNA target analyte is
hybridized with an
s array of oligonucleotides formed on a microchip. The sequence of the target
can then be
"read" from the pattern of target binding to the array
C3. Gene map i~ng,
The sequences of the present invention are also valuable for chromosome
identification. The sequence is specifically targeted to and can hybridize
with a particular
~o location on an individual human chromosome. Moreover, there is a current
need for
identifying particular sites on the chromosome. Few chromosome marking
reagents based on
actual sequence data (repeat polymorphisms) are presently available for
marking
chromosomal location. The mapping of DNAs to chromosomes according to the
present
invention is an important first step in correlating those sequences with genes
associated with
~ s disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers
(preferably 12-30 bp) from the ESK cDNA. Computer analysis of the 3'
untranslated region is
used to rapidly select primers that do not span more than one exon in the
genomic DNA,
which would complicate the amplification process. These primers are then used
for PCR
2o screening of somatic cell hybrids containing individual human chromosomes.
Only those
hybrids containing the human gene corresponding to the primer will yield an
amplified
fragment.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a
particular
DNA to a particular chromosome. Using the present invention with the same
oligonucleotide
2s primers, sublocalization can be achieved with panels of fragments from
specific
chromosomes or pools of large genomic clones in an analogous manner. Other
mapping
strategies that can similarly be used to map to its chromosome include in situ
hybridization,
prescreening with labeled flow-sorted chromosomes and preselection by
hybridization to
construct chromosome specific-cDNA libraries.
3o Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase
chromosomal spread can be used to provide a precise chromosomal location in
one step. This
technique can be used with cDNA as short as 50 or 60 bases. For a review of
this technique,
see Verma et al. (1988) Human Chromosomes: a Manual of Basic Techniques,
Pergamon
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Press, New York.
Once a sequence has been mapped to a precise chromosomal location, the
physical
position of the sequence on the chromosome can be correlated with genetic map
data. Such
data are found, for example, in the OMIM database (Center for Medical
Genetics, Johns
Hopkins University, Baltimore, MD and National Center for Biotechnology
Information,
National Library of Medicine, Bethesda, MD). The OMIM gene map presents the
cytogenetic
map location of disease genes and other expressed genes. The OMIM database
provides
information on diseases associated with the chromosomal location. Such
associations include
the results of linkage analysis mapped to this interval, and the correlation
of translocations
to and other chromosomal aberrations in this area with the advent of polygenic
diseases, such as
cancer.
C4. Therapeutic ap~ications
Polynucleotides which encode ESK, or complements of the polynucleotides, may
also
be used for therapeutic purposes. Expression of ESK may be modulated through
antisense
is technology, which controls gene expression through complementary
polynucleotides, i.e.
antisense DNA or RNA, to the control, 5' or regulatory regions of the gene
encoding ESK.
For example, the 5' coding portion of the polynucleotide sequence which codes
for the
protein of the present invention is used to design an antisense
oligonucleotide of from about
to 40 base pairs in length. Oligonucleotides derived from the transcription
start site, e.g.
2o between positions -10 and +10 from the start site, are preferred. An
antisense DNA
oligonucleotide is designed to be complementary to a region of the gene
involved in
transcription (Lee et al. (1979) Nucl. Acids Res. 6:3073; Cooney et al. (1988)
Science
241:456; and Dervan et al. (1991) Science 251: 1360), thereby preventing
transcription and
the production of ESK. An antisense RNA oligonucleotide hybridizes to the mRNA
in vivo
2s and blocks translation of the mRNA molecule into ESK protein (Okano (1991)
J.
Neurochem. 56:560). The antisense constructs can be delivered to cells by
procedures known
in the art such that the antisense RNA or DNA may be expressed in vivo.
The therapeutic polynucleotides of the invention may be employed in
combination
with a suitable pharmaceutical carrier. Such compositions comprise a
therapeutically
3o effective amount of the compound, and a pharmaceutically acceptable carrier
or excipient.
Such a carrier includes but is not limited to saline, buffered saline,
dextrose, water, glycerol,
ethanol, and combinations thereof. The formulation should suit the mode of
administration.
The polypeptides, and agonist and antagonist compounds which are polypeptides,
2s
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may also be employed in accordance with the present invention by expression of
such
polypeptides in vivo, which is often referred to as "gene therapy." Cells from
a patient may
be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex
vivo, with the
engineered cells then being provided to a patient to be treated with the
polypeptide. Such
methods are well-known in the art. For example, cells may be engineered by
procedures
known in the art by use of a retroviral particle containing RNA encoding a
polypeptide of the
present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide in
vivo by
procedures known in the art. As known in the art, a producer cell for
producing a retroviral
to particle containing RNA encoding the polypeptide of the present invention
may be
administered to a patient for engineering cells in vivo and expression of the
polypeptide in
vivo. These and other methods for administering a polypeptide of the present
invention by
such method should be apparent to those skilled in the art from the teachings
of the present
invention. For example, the expression vehicle for engineering cells may be,
for example, an
is adenovirus which may be used to engineer cells in vivo after combination
with a suitable
delivery vehicle (Yeh P., et al. (1997) FASEB J 11:615-623)
Retroviruses from which the retroviral plasmid vectors mentioned above may be
derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen
necrosis
virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian
leukosis virus,
2o gibbon ape leukemia virus, adenovirus, Myeloproliferative Sarcoma Virus,
and mammary
tumor virus.
The retroviral plasmid vector is employed to transduce packaging cell lines to
form
producer cell lines. Examples of packaging cells which may be transfected
include, but are
not limited to, the PESO1, PA317, psi-2, psi-AM, PA12, T19-14X, VT-19-17-H2,
psi-CRE,
2s psi-CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller
(1990; Human
Gene Therapy, Vol. 1, pg. 5-14). The vector may transduce the packaging cells
through any
means known in the art. Such means include, but are not limited to,
electroporation, the use
of liposomes, and CaP04 precipitation. In one alternative, the retroviral
plasmid vector may
be encapsulated into a liposome, or coupled to a lipid, and then administered
to a host.
3o The producer cell line generates infectious retroviral vector particles
which include
the nucleic acid sequences) encoding the polypeptides. Such retroviral vector
particles then
may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
The transduced
eukaryotic cells will express the nucleic acid sequences) encoding the
polypeptide.
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Eukaryotic cells which may be transduced include, but are not limited to,
embryonic stem
cells, embryonic carcinoma cells, as well as hematopoietic stem cells,
hepatocytes,
fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial
epithelial cells.
The genes introduced into cells may be placed under the control of inducible
promoters, such as the radiation-inducible Egr-1 promoter, (Maceri, H.J., et
al. (1996) Cancer
Res 56(19):4311), to stimulate ESK production or antisense inhibition in
response to
radiation, e.g., radiation therapy for treating tumors.
III. ESK and ESK ,hannels
to The invention is based in part on the structwal similarity between ESK and
other
known subunits of the eag potassium channel superfamily, such as human ergl
(hERG). An
exemplary ESK polypeptide sequence SEQ ID N0:2 contains 1080 residues, six
potential
transmembrane domains spanning approximately residues 212 to 239 (S 1 ), 259
to 277 (S2),
297 to 320 (S3), 329 to 349 {S4), 356 to 378 (SS), and 477 to 501 (S6), and
potential N-
ts linked glycosylation sites at residues Asn418, Asn425, Asn433, Asn467, and
Asn496. The
hESKI sequence SEQ ID N0:2 also contains a potential pore-forming P-domain
spanning
approximately residues 449 to 468 (P 1 ) and a putative cyclic nucleotide
binding domain
(cNBD) spanning about residues 601-668. These structural motifs are
characteristic of eag
channel subunits.
2o The hESK amino acid sequence SEQ ID N0:2 and the hERG sequence SEQ
ID N0:6 were aligned using the CLUSTAL-W pairwise alignment program of
MacVectorTM
software (ver. 6.01; Oxford Molecular Ltd, Oxford, UK) using the default
pairwise
parameters. The proteins have an overall amino acid sequence identity of about
37%, which
increases to about 47% identity (216/457 identical residues) in the region
spanning the S 1
2s through the cNBD segment, corresponding to residues 212-668 of SEQ ID N0:2,
the region
identified herein as SEQ ID NO:S. The level of sequence identity in the S1-
cNBD region
indicates that ESK represents a new subfamily of the eag channel superfamily.
In general,
two members of the same subfamily from different species share ~65-70% amino
acid
sequence identity in the region spanning S 1 through the cNBD segment. In
contrast, two
3o different subfamily members within the same species share only about 40-50%
amino acid
sequence identity across the same region (Warmke and Ganetzky (1994) P.N.A.S.
91: 3438-
3442).
The substantially purified ESK of the invention includes a polypeptide
containing an
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amino acid sequence having at least 60 percent, preferably at least 65 to 70
percent, more
preferably at least 80 percent, and most preferably at least 90 or 95 percent
sequence identity
to SEQ ID NO:S. In other embodiments, the invention includes a polypeptide
having at least
70 percent, at least 80 percent, at least 90 percent, or at least 95 percent
sequence identity to
s SEQ ID N0:2 or SEQ ID N0:4, or having the sequence SEQ ID N0:2 or SEQ ID
N0:4. 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, also as defined above. Also contemplated are fragments derived
from ESK,
which are capable of interacting with other polypeptides, proteins, or other
molecules, such
t o interaction which alters the functional properties or the
cellular/subcellular localization of the
ESK channel. The invention also includes a substantially purified potassium
channel which
contain at least one ESK subunit.
The polypeptide sequence variations may include those that are considered
conserved
and non-conserved substitutions, as defined above. Thus, for example, a
polypeptide with a
is sequence having at least 80% sequence identity with the polypeptide
identified as SEQ ID
N0:2 (1080 amino acids) contains up to 216 amino acid substitutions when
optimally
aligned as defined above. In a more specific embodiment, the polypeptide has a
sequence
substantially identical (97-100% identical) to SEQ ID N0:2 or SEQ ID N0:4. ESK
may be
(i) a polypeptide in which one or more of the amino acid residues in a
sequence listed above
2o are substituted with a conserved or non-conserved amino acid residue
(preferably a conserved
amino acid residue), or (ii) a polypeptide in which one or more of the amino
acid residues
includes a substituent group, or (iii) a polypeptide in which the ESK is fused
with another
compound, such as a compound to increase the half life of the polypeptide (for
example,
polyethylene glycol (PEG)), or (iv) a polypeptide in which additional amino
acids are fused
2s to ESK, or (v) an isolated fragment of the polypeptide. Such fragments,
variants and
derivatives are deemed to be within the scope of those skilled in the art from
the teachings
herein. In particular, splice variants of the polypeptide are also
contemplated.
A. Preparation of ESK
3o Recombinant methods for producing and isolating ESK and fragments are
described
above.
In addition to recombinant production, fragments and portions of ESK may be
produced by direct peptide synthesis using solid-phase techniques (cf Stewart
et al. (1969)
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Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield J
(1963) J Am
Chem Soc 85:2149-2154). In vitro peptide synthesis may be performed using
manual
techniques or by automation. Automated synthesis may be achieved, for example,
using
Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City,
Calif.) in
s accordance with the instructions provided by the manufacturer. Portions of
ESK may be
chemically synthesized separately and combined using chemical methods.
The polypeptide may also be obtained by isolation from natural sources, e.g.,
by
affinity purification using the anti- ESK antibody described in the section
below. A
fragment or fragments corresponding to extracellular regions and/or the
intracellular regions
0 of ESK may be cleaved from the membrane-bound regions using limited
proteolysis
techniques known to those of skill in the art. The amino acid sequence of a
fragment so
obtained may be used to design nucleotide coding sequence for recombinant
production of
the fragment.
is B. ~~Dlications of ESK
The ESK polypeptide of the invention has uses in (1) therapeutic treatment
methods
and (2) drug screening.
B 1. Theraveutic uses and compositions
The ESK polypeptide of the invention is generally useful in treating diseases
and
2o disorders associated with ion channel dysfunction, such as, for example,
forebrain-related
neurological disorders including, but not limited to, dementias such as
Alzheimer's disease,
depressive and manic-depressive disorders, anxiety, panic, and obsessive-
compulsive
disorders, eating disorders, attention-deficit and hyperactivity disorders,
autism,
schizophrenia, epilepsy, and neurodegenerative disorders such as Huntington's
and
2s Parkinson's diseases.
ESK may self associate to form a homomeric ESK channel, or may associate with
other potassium channel polypeptides to form a heteromeric ESK channel. While
not
intending to be bound by theory, a polypeptide fragment of ESK, preferably a
soluble
fragment which binds an agonist of the ESK channel, may be employed to inhibit
activity of
3o the ESK channel by binding an agonist which is necessary for ESK channel
activity, in effect
competing with ESK channel for agonist. An intracellular fragment of ESK,
preferably a
soluble fragment, may be used to block the interaction of intracellular
effector molecules with
an intracellular domain of the ESK channel, thus preventing the cellular
response induced by
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the interaction of the intracellular effector molecule with the ESK channel.
ESK compositions are tested in appropriate in vitro and in vivo animal models
of
disease, to confirm efficacy, tissue metabolism, and to estimate dosages,
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 a consistent and predictable concentration of compound at
the target
organ or tissue. The polypeptide compositions may be administered alone or in
combination
with other agents, such as stabilizing compounds, and/or in combination with
other
pharmaceutical agents such as drugs or hormones.
to ESK compositions may be administered by a number of routes including, but
not
limited to oral, intravenous, intramuscular, transdermal, subcutaneous,
topical, sublingual, or
rectal means. ESK compositions may also be administered via liposomes. Such
administration routes and appropriate formulations are generally known to
those of skill in
the art.
is For example, the polypeptide may be given topically to the skin or
epithelial linings
of body cavities, for infections in such regions. Examples of treatable body
cavities include
the vagina, the rectum and the urethra. Conveniently, the polypeptide would be
formulated
into suppository form for administration to these areas.
The poiypeptide can be given via intravenous or intraperitoneal injection.
Similarly,
2o the polypeptide may be injected to other localized regions of the body. The
polypeptide may
also be administered via nasal insufflation. Enteral administration is also
possible. For such
administration, the polypeptide should be formulated into an appropriate
capsule or elixir for
oral administration, or into a suppository for rectal administration.
The foregoing exemplary administration modes will likely require that the
2s polypeptides be formulated into an appropriate carrier, including
ointments, gels,
suppositories. Appropriate formulations are well known to persons skilled in
the art.
Dosage of the polypeptide will vary, depending upon the potency and
therapeutic
index of the particular polypeptide selected. These parameters are easily
determinable by the
skilled practitioner. As an example, if the polypeptide inhibits neuronal cell
degradation in
3o vitro at a given concentration, the practitioner will know that the final
desired therapeutic
concentration will be this range, calculated on the basis of expected
biodistribution. An
appropriate target concentration is in the ng/kg to low mg/kg range, e.g., SO
ngJkg to 1 mg/kg
body weight, for IV administration.
SUBSTTTUTE SHEET (RULE Z6)
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A therapeutic composition for use in the treatment method can include the
polypeptide in a sterile injectabie solution, the polypeptide in an oral
delivery vehicle, or the
polypeptide in a nebulized form, all prepared according to well known methods.
Such
compositions comprise a therapeutically effective amount of the compound, and
a
pharmaceutically acceptable carrier or excipient. Such a carrier includes but
is not limited to
saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations
thereof.
B2. Screening methods
The present invention also includes an assay for identifying molecules, such
as
synthetic drugs, antibodies, peptides, or other molecules, which have a
modulating effect on
to the activity of an ESK channel, e.g. agonists or antagonists of an ESK
channel comprising
one or more ESK poiypeptides of the present invention. Such an assay comprises
the steps of
providing a functional ESK channel comprising ESK polypeptides encoded by the
polynucleotides of the present invention, contacting the ESK channel with one
or more
molecules to detemline the modulating effect of the molecules on the activity
of the channel,
is and selecting from the molecules a candidate molecule capable of modulating
ESK channel
activity. Such modulating agents are useful in the treatment of disease
conditions associated
with activation or reduction of ESK channel activity, including but not
limited to those
described in section B 1 above.
ESK, its ligand-binding, catalytic, or immunogenic fragments, or oligopeptides
2o thereof, can be used for screening therapeutic compounds in any of a
variety of drug
screening techniques. The protein employed in such a test may be membrane-
bound, free in
solution, affixed to a solid support, borne on a cell surface, or located
intracellularly. The
formation of binding complexes between ESK, or an ESK channel, and the agent
being tested
may be measured. Compounds which inhibit binding between ESK and its agonists
may also
2s be measured.
In one embodiment, the screening system includes recombinantly expressed ESK,
and
the compounds screened are tested for their ability to block (inhibit) or
enhance (activate) the
potassium current activity of ESK channel. In a functional screening assay,
mammalian cell
tines or Xenopus oocytes which lack ESK are used to express ESK. ESK may be
expressed
3o individually or together with other potassium channel subunit polypeptides.
Compounds are
screened for their relative effectiveness as channel modulators, e.g.,
activators or inhibitors,
by comparing the relative channel occupancy to the extent of ligand-induced
activation or
inhibition of potassium ion conductance.
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The invention also includes, in a related aspect, an ESK channel modulating
agent
identified by screening methods employing ESK such as those described above.
Another technique for drug screening which may be used provides for high
throughput screening of compounds having suitable binding affinity to the ESK
channel is
described in detail by Geysen in PCT Application WO 84/03564, published on
Sep. 13, 1984.
In summary, large numbers of different small peptide test compounds are
synthesized on a
solid substrate, such as plastic pins or some other surface. The peptide test
compounds are
reacted with ESK (as either a soluble extracellular fragment of ESK, or intact
ESK
solubilized in detergents or in lipid vesicles), and washed. Bound ESK is then
detected by
to methods well known in the art. Substantially purified ESK can also be
coated directly onto
plates for use in the aforementioned drug screening techniques. Alternatively,
non-
neutralizing antibodies can be used to capture the peptide and immobilize it
on a solid
support.
Antibodies to ESK, as described in Section IV. below, may also be used in
screening
~s assays according to methods well known in the art. For example, a
"sandwich" assay may be
performed, in which an anti- ESK antibody is affixed to a solid surface such
as a microtiter
plate and solubilized ESK or ESK channel is added. Such an assay can be used
to capture
compounds which bind to the ESK channel. Alternatively, such an assay may be
used to
measure the ability of compounds to interfere with the binding of a ligand,
such as an agonist,
2o to the ESK channel.
IV. Anti-ESK antibodies
In still another aspect of the invention, purified ESK is used to produce anti-
ESK
antibodies which have diagnostic and therapeutic uses related to the activity,
distribution, and
2s expression of ESK.
Antibodies to ESK may be generated by methods well known in the art. Such
antibodies may include, but are not limited to, polyclonal, monoclonal,
chimeric, humanized,
single chain, Fab fragments and fragments produced by an Fab expression
library.
Antibodies, i.e., those which block ligand binding, are especially preferred
for therapeutic
3o use.
ESK for antibody induction does not require biological activity; however, the
polypeptide fragment or oligopeptide must be antigenic. Peptides used to
induce specific
antibodies may have an amino acid sequence consisting of at least ten amino
acids, preferably
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at least 20 amino acids. Preferably they should mimic a portion of the amino
acid sequence of
the natural protein and may contain the entire amino acid sequence of a small,
naturally
occurring molecule. Short stretches of an ESK polypeptide may be fused with
another protein
such as keyhole limpet hemocyanin and antibody produced against the chimeric
molecule.
Procedures well known in the art can be used for the production of antibodies
to ESK.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, etc
may be immunized by injection with ESK or any portion, fragment or
oligopeptide which
retains immunogenic properties. Depending on the host species, various
adjuvants may be
used to increase immunological response. Such adjuvants include but are not
limited to
t o Freund's, mineral gels such as aluminum hydroxide, and surface active
substances such as
lysolecithin, platonic polyols, polyanions, peptides, oil emulsions, keyhole
limpet
hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum
are potentially useful human adjuvants.
Monoclonal antibodies to ESK may be prepared using any technique which
provides
~s for the production of antibody molecules by continuous cell lines in
culture. These include
but are not limited to the hybridoma technique originally described by Koehler
and Milstein
(1975; Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et
al. (1983)
lmmunol Today 4:72; Cote et al. (1983) Proc Natl Acad Sci 80:2026-2030) and
the EBV-
hybridoma technique (Cole, et al. (1984) Mol. Cell Biol. 62:109-120).
2o Techniques developed for the production of "chimeric antibodies", the
splicing of
mouse antibody genes to human antibody genes to obtain a molecule with
appropriate antigen
specificity and biological activity can also be used (Mornson et al. {1984)
Proc Natl Acad Sci
81:6851-6855; Neuberger et al. (1984) Nature 312:604-608; Takeda et al. (1985)
Nature
314:452-454). Alternatively, techniques described for the production of single
chain
2s antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single-chain
antibodies
specific for ESK.
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening recombinant immunoglobulin libraries or panels of
highly specific
binding reagents as disclosed in Orlandi et al. (1989; Proc Natl Acad Sci
86:3833-3837), and
3o Winter G and Milstein C (1991; Nature 349:293-299).
Antibody fragments which contain specific binding sites for ESK may also be
generated. For example, such fragments include, but are not limited to, the
F(ab')2 fragments
which can be produced by pepsin digestion of the antibody molecule and the Fab
fragments
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which can be generated by reducing the disulfide bridges of the F(ab')2
fragments.
Alternatively, Fab expression libraries may be constructed to allow rapid and
easy
identif cation of monoclonal Fab fragments with the desired specificity (Huse
WD et a1
(1989) Science 256:1275-1281).
A. Dia ostic applications
A variety of protocols for competitive binding or immunoradiometric assays
using
either polyclonal or monoclonal antibodies with established specificities are
well known in
the art. Such immunoassays typically involve the formation of complexes
between ESK and
io its specific antibody and the measurement of complex formation. A two-site,
monoclonal-
based immunoassay utilizing monoclonal antibodies reactive to two
noninterfering epitopes
on ESK is preferred, but a competitive binding assay may also be employed.
These assays are
described in Maddox DE et al. (1983, J Exp Med 158:1211).
Antibodies which specifically bind ESK are useful for the diagnosis of
conditions or
is diseases characterized by expression of ESK. Alternatively, such antibodies
may be used in
assays to monitor patients being treated with ESK, its agonists, or its
antagonists. Diagnostic
assays for ESK protein include methods utilizing the antibody and a label to
detect ESK or its
fragments in extracts of cells, tissues, or biological fluids such as sera.
The proteins and
antibodies of the present invention may be used with or without modification.
Frequently, the
2o proteins and antibodies will be labeled by joining them, either covalently
or noncovalently,
with a reporter molecule. A wide variety of reporter molecules are known in
the art.
A variety of 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 fluorescent
activated
2s cell sorting (FACS). As noted above, a two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on ESK is
preferred, but a
competitive binding assay may be employed. These assays are described, among
other places,
in Maddox, et al. (supra). Such protocols provide a basis for diagnosing
altered or abnormal
levels of ESK expression. Normal or standard values for ESK expression are
established by
3o combining cell extracts taken from normal subjects, preferably human, with
antibody to ESK
under conditions suitable for complex formation which are well known in the
art. The amount
of standard complex formation may be quantified by various methods, preferably
by
photometric methods. Then, standard values obtained from normal samples may be
compared
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with values obtained from samples from subjects potentially affected by
disease. Deviation
between standard and subject values establishes the presence of disease state.
The antibody assays are useful to determine the level of ESK present in a
particular
tissue, e.g., biopsied tumor tissue or neuronal tissue, as an indication of
whether ESK is being
overexpressed or underexpressed in the tissue, or as an indication of how ESK
levels are
responding to drug treatment.
B. Therapeutic uses
In conditions associated with ESK, such as neurological disorders including
but not
Io limited to those described in Section IILB1 above, therapeutic value may be
achieved by
administering an antibody specific against ESK, to inhibit, for example,
binding of an agonist
to the ESK channel, or to block the ion pore.
The antibody employed is preferably a humanized monoclonal antibody, or a
human
Mab produced by known globulin-gene library methods. The antibody is
administered
is typically as a sterile solution by IV injection, although other parenteral
routes may be
suitable. Typically, the antibody is administered in an amount between about 1-
15 mg/kg
body weight of the subject. Treatment is continued, e.g., with dosing every 1-
7 days, until a
therapeutic improvement is seen.
The following examples illustrate but in no way are intended to limit the
present
zo invention.
MATERIALS AND METHODS
Unless otherwise indicated, restriction enzymes and DNA modifying enzymes were
obtained from New England Biolabs {Beverly, MA) or Boehringer Mannheim
(Indianapolis,
2s III. The Enhanced Chemo-Luminescence (ECL) system were obtained from
Amersham
Corp. (Arlington, Heights, IL). Nitrocellulose paper was obtained from
Schleicher and
Schuell (Keene, NH). "pBLUESCRIPT II SK'" was obtained from Stratagene (La
Jolla, CA).
Materials for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were obtained
from Bio-
Rad Laboratories (Hercules, CA). Other chemicals were purchased from Sigma
(St. Louis,
3o MO) or United States Biochemical (Cleveland, OH).
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Examgle 1
Identifi~tion of ESK Nucleic Acid Sequences
To isolate ESK cDNA molecules, the procedure of Shepard & Rae (1997; Nucleic
Acids Res. 25(15): 3183-3185) was used with some modifications. Briefly, 10 to
20 ug
cDNA from a human brain cDNA library (Edge BioSystems, Gaithersburg, MD) was
mixed
with 50-80 ng biotinylated oligonucleotide, 50 ng of each clamp oligo, and 1
ul of 1N NaOH
in a total volume of 10 ul. The oligonucleotide sequences were derived from
human EST
sequence U69184 (SEQ ID N0:7). After the mixture was incubated at RT for 15-20
min, 40
ul of neutralization solution (0.12 M Tris, pH 7, 2x SSPE, 0.1 % Tween 20) was
added, and
~o fiuther incubated at 37 - 42°C. Two to three hours later, to the
above reaction mix, 20 ul (200
ug) magnetic beads (Dynabeads) was added, and the mixture was further
incubated at the
above temperature for 30 min.
To recover captured cDNA molecules, the supernatant was removed and the
magnetic
beads were washed 5 times with O.Sx SSPE, 0.1% Tween 20. The beads were then
fiuther
~s washed with TE once or twice. Finally, the captured cDNA was eluted with 10
ul of O.Sx TE
at 70°C for 5 min. The eluted plasmid cDNA was then transform into E.
coli cells and
transformants were plated on one or more 1 S-cm dishes (a few thousand
colonies per dish).
Bacterial colonies were lifted onto Hybond N filters (Amersham, Arlington
Heights, II,) in
duplicate.
zo Filters were screened using a labeled hybridization probe based on the
above EST
sequence. The filters were prehybridized without probe in the prehybridization
solution (Sx
SSPE, SxDenhardt's solution, 0.1% SDS) at 45-50 °C for 1 hour, and then
hybridized with
probe overnight. The filters were then washed twice for 20 minutes each at
room temperature
in 2x SSPE, 0.1% SDS, and twice for 20 minutes each at 45-50 °C in 2x
SSPE, 0.1% SDS.
2s Signals were detected by a few hours of exposure of the filters to X-ray
film.
Positive colonies were subjected to secondary and tertiary screenings.
Positive
colonies from tertiary screening were cultured. Plasmid DNA was isolated from
the positive
cultures using a Qiagen miniprep kit (Qiagen, Santa Clarita, CA) and
sequenced, resulting in
the identification of nucleic acid sequences of a human ESKl potassium channel
subunit
3o polypeptide.
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Example 2
Northern Analvsis
Multiple tissue Northern blots were purchased from Clontech. High Efficiency
Hybridization System (HS-114) was purchased from Molecular Research Center
(Cincinnati,
s Ohio). Briefly, the blot was first soaked in prehybridization solution (1%
SDS and O.1M
NaCI) for 30 min at room temperature, and then was incubated in HS-114
solution with 100
pg/ml salmon sperm DNA in the absence of probe for a few hours 68 °C.
The cDNA probe
was then added and the blot was let to hybridize at 68 °C overnight.
The blot was then
washed under the following conditions: twice in 2x SSC, 0.05% SDS, at room
temperature;
o and twice in O.l 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,
expression of ESK1 transcript was observed only in brain, predominantly
forebrain.
Exg~
is Elect~oghysiological Measurements
I. Whole Cell Patch Clamp Measurements
Potassium currents can be measured by the patch clamp method in the whole cell
configuration (Hamill, et al., 1981). Electrode resistances ranging from 2-6
MS2 are
appropriate. Recordings can be made with either an Axopatch 1 C or Axopatch
200A ampli-
2o fier (Axon Instruments, Foster City, CA) interfaced to PCLMP6 software
(Axon Instruments)
for data acquisition and analysis.
Potassium currents are recorded utilizing an external bath solution consisting
of (in
mM): 140 sodium chloride, 5 potassium chloride, 10 HEPES, 2 calcium chloride,
1
magnesium chloride, and I2 glucose, adjusted to pH 7.4 with sodium hydroxide
and 305
2s mOsM. The internal pipette solution consists of (in mM): 15 sodium
chloride,125 potassium
methanesulphonate, 10 HEPES, 11 EGTA, 1 calcium chloride, 2 magnesium chloride
and 59
glucose, adjusted to pH 7.4 with potassium hydroxide and 295 mOsM. For test
application,
cells are placed in a flow through chamber (0.5-1 ml/min). Currents are
elicited by changing
the voltage from a holding potential of -90 mV to 0 mV, as a step pulse of 30
msec. duration
3o every 15 sec. Data are sampled at 5 KHz and filtered at 1 KHz. Leak and
capacitance
currents are subtracted after measuring currents elicited by hyperpolarizing
pulses.
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Exams a 4
Anticonvulsant Activity: DBA/2 Mouse Seizure Model
DBA/2 mice (18-21 days old; approx. 7-10 g) are obtained from Jackson
Laboratories, Bar Harbor, Maine, and are housed for a minimum of three days to
acclimate
them to laboratory conditions. On the day of the test, mice are injected
i.c.v. into the lateral
ventricle with vehicle or test compound (total volume: 5 pl) according to
standard methods
(Jackson and Scheideler, 1996) 30 minutes prior to exposure to sound stimulus.
After
injection, the mice are individually housed in observation chambers and are
observed over the
following 30 min. for evidence of shaking behavior (persistent whole body
shakes) or any
~o other abnormal behaviors. The animals are exposed to a high intensity sound
stimulus (100-
110 dB sinusoidal tone at 14 Hz for 30 s). Mice are observed for the presence
of clonic and
tonic seizures with full hindlimb extension during the 30 s exposure to the
sound.
While the invention has been described with reference to specific methods and
embodiments, it is appreciated that various modifications and changes may be
made without
i s departing from the invention.
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