Note: Descriptions are shown in the official language in which they were submitted.
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C7F2- A NOVEL POTASSIUM CHANNEL ~3-SUBUNIT
FIELD OF THE INVENTION
The present invention relates to a newly identified potassium channel ~i-
subunit. The invention also relates to polynucleotides encoding the ~i-
subunit. The
invention further relates to methods using ~i-subunit polypeptides and
polynucleotides, as a target for diagnosis and treatment in channel-mediated
disorders. The invention further relates to drug-screening methods using the
polypeptides and polynucleotides to identify agonists and antagonists,
applicable to
diagnosis and treatment. The invention further encompasses agonists, and
antagonists based on the a-subunit polypeptides and polynucleotides. The
invention
further relates to procedures for producing the ~i-subunit polypeptides and
polynucleotides.
BACKGROUND OF THE INVENTION
The flow of potassium through the plasma membrane affects diverse
biological processes including action-potential firing and control of cell
volume.
Potassium channels are ubiquitous integral membrane proteins serving numerous
functions in excitable and nonexcitable cells (McManus, O. B., J. Bioenerg.
Biomembr. 23:537-560 (1991)). Many different classes of potassium channels
have
evolved and have been separated into classes on the basis of their biophysical
properties, physiological regulation, and pharmacology (Hille, B., Ionic
Channels of
Excitable Membranes, Sunderland, M. A. Sinauer (1992); Rudy, B., Neuroscience
25:729-749 (1988)). Major types include voltage-dependent, calcium-activated,
and
ATP-sensitive channels. Some subtypes exist within the classifications.
However,
certain functional features are shared among many types of potassium channels
(Kukuljan et al., Am. J. Physiol. 268 (Cell Physiol. 3~:C535-C556 (1995)).
Potassium channel-forming proteins can be grouped into three families that
differ in the number of transmembrane segments. The largest family contains
six
membrane-spanning segments. Inward rectifiers comprise the second family with
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subunits having two transmembrane segments. The third family contains only one
transmembrane segment. These channels have been studied using recombinant DNA
techniques. The information has been reviewed in Kukuljan et al., cited above.
High conductance calcium-activated potassium channels are a group of
proteins with a number of unique features. The channels are activated by
intracellular calcium, as well as membrane depolarization. The channels
display a
high single-channel conductance and are highly selective for potassium. They
are
sensitive to specific toxins, such as charybdotoxin that binds to a receptor
site located
on the external vestibule of the channel and prevents potassium flow by
physical
occlusion of the pore.
Knaus et al. (J. Biol. Chem. 269:3921-3924 (1994)) reported on the subunit
composition of the high conductance calcium-activated potassium channel from
smooth muscle. This potassium channel is reported to be composed of two
subunits,
a and Vii, of 62 and 31 kilodaltons, respectively. Amino acid sequence
analysis
showed a high sequence homology with two cloned high conductance potassium
channels from Drosophila. An antipeptide antibody directed against the amino
acid
sequence of one of the a-subunit fragments could also immunoprecipitate, under
nondenaturing conditions, the ~3-subunit, demonstrating specific noncovalent
association of both subunits. The results indicated that the a-subunit of this
specific
high conductance potassium channel is a member of a specific family of
potassium
channels and forms a noncovalent complex with a ~-subunit. The reference
reported
a specific and tight interaction between the two polypeptides. The following
model
was proposed. The a-subunit is the central ion channel-forming element and
contains the receptor for the various blocking toxins. A tetramer a-subunit is
noncovalently associated with four ~i-subunits. The ~i-subunits are in close
proximity
(less than 12 t~) to the pore-forming and receptor carrying subunit. This high
conductance potassium channel ~i-subunit shares characteristics with the ~i-
subunit of
rat brain sodium channels and the Y-subunit of skeletal muscle L-type calcium
channels and may be analogous in structure and/or function. It is speculated
that this
subunit is a conserved constituent of many voltage- and calcium-dependent
potassium channels.
2
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Knaus et al. (J. Biol. Chem. 269:17274-1727$ (1994)) disclosed the primary
sequence and immunological characterization of the ~3-subunit of the high
conductance calcium-activated potassium channel from smooth muscle. The amino
acid sequence was used to design oligonucleotide probes with which cDNAs
encoding the protein were isolated. The protein was reported to contain two
hydrophobic (putative transmembrane) domains bearing little sequence homology
to
subunits of other known ion channels. Reports had suggested that the ~i-
subunit
plays a role in modulating the properties of the pore-forming subunit. For
example,
co-expression of sodium or calcium channel a- and ~i-subunits had been
10 demonstrated to modulate the currents expressed from the a-subunits alone.
The
reference also reported small regions of homology with other ~i-subunits. It
is
reported, for example, that the ~i2- subunit of the rabbit cardiac calcium
channel
contains a stretch of eight amino acids that are 100% homologous to a region
of the
(3-subunit of the channel under study.
15 McManus et al. (Neuron 14:b45-650 (1995)) examined the functional
contribution of the ~i-subunit properties of high conductance potassium
channels
expressed heterologously in Xenopus oocytes. The reference reported that co-
expression of the bovine smooth muscle high conductance potassium channel ~-
subunit has dramatic effects on the properties of expressed mouse brain a-
subunits.
20 The reference noted that expression of an a-subunit alone is sufficient to
generate
potassium channels that are gated by voltage and intracellular calcium.
Nevertheless,
channels from oocytes injected with cDNAs encoding both a- and ~3-subunits
were
much more sensitive to activation voltage and calcium than channels composed
of
the a-subunit alone. Expression levels, single channel conductance, and ion
25 selectivity appeared unaffected. Further, channels from oocytes expressing
both
subunits were sensitive to a potent agonist of native high conductance
potassium
channels, whereas channels composed of the a-subunit alone were insensitive.
Thus,
in addition to its effects on channel gating, the (3-subunit conferred
sensitivity to
DHS-I, a potent agonist of native high conductance potassium channels.
30 Accordingly, whereas expression of the ~i-subunit alone did not result in a
functional
potassium channel, a coexpression with the a-subunit formed channels with
3
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biophysical and pharmacological properties distinct from channels formed by
the a-
subunit alone. These properties more closely resemble those of native high
conductance potassium channels. The report concluded that based on the effect
on
sensitivity of the channel to voltage and calcium conferred by the ~i-subunit,
that the
5 ~i-subunit may form part of the transduction machinery of the channel. This
reference also showed that these properties could be conferred by chimeric
multimers
in which a ~-subunit from one tissue was able to modulate the a-subunit from
another tissue. The possibility was raised that regulated expression of ~i-
subunits, as
in tissue-specific or developmental-specific regulation, could constitute a
mechanism
for generating functional diversity among mammalian high conductance potassium
channels.
Meera et al. (FEBS. Lett. 382:84-88 (1996)) disclosed the importance of
calcium concentration for the functional coupling between a- and ~3-subunits
of high
conductance potassium channels. The reference pointed out that these channels
are
15 unique in that they are modulated not only by voltage, but also by calcium
in the
micromolar range. They referred to the ~i-subunit as "the regulatory subunit
for the
pore-forming a-subunit." The reference demonstrated that intracellular calcium
concentration controls the functional coupling between a- and (3-subunits of
the
complex in a concentration range relevant to cellular excitation. The ~i-
subunit used
20 for the experiments was derived from human smooth muscle. The experiments
were
performed by injecting cRNA into Xenopus oocytes. Channel currents and number
of channels were recorded. The results were reported as demonstrating that a
minimum calcium concentration was required to switch a- and (3-subunits to a
functional activated mode. It was proposed that a rise in local calcium
concentration
25 would induce a conformational change in one or both of the subunits,
triggering the
functional coupling and causing the a-subunit to respond much more efficiently
to
calcium and voltage. Prior to this work, it was thought that the channels were
calcium- and voltage-activated and would never open in the virtual absence of
calcium. However, the report demonstrated that the channel a-subunit will open
at a
30 low calcium concentration and, in fact, becomes independent of calcium at
concentrations lower than 100 nM, operating according to a purely voltage-
regulated
4
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mode. Similarly, the results provided evidence for a calcium dependent
mechanism
that switches the a-subunit from a calcium-independent to a calcium-dependent
mode and from a (3-subunit-null interaction to a (3-subunit-activated mode.
The ~i-subunits of voltage-gated potassium channels have been recently
reviewed (Barry et al., Ann. Rev. Physiol. 58:363-394 (1996)).
Oberst et al. (Oncogene 14:1109-1116 (1997)) recently identified a nucleic
acid sequence in quail cDNA in which the corresponding gene encodes a 200
amino
acid protein with 46-48% amino acid sequence identity to regulatory ~i-
subunits of
the bovine, human, and canine high conductance calcium-activated potassium
channel. Studies of gene expression in v-myc-transformed quail embryo
fibroblasts
led to the isolation of a clone hybridizing in the normal, but not in the
transformed
fibroblasts. Subsequent analysis revealed that the sequence was expressed in
all
normal avian fibroblasts tested, but was undetectable in a variety of cell
lines
transformed by a variety of oncogenes or chemical carcinogens. It was
suggested
that the protein encoded by this sequence is a regulatory subunit of a calcium-
activated potassium channel potentially involved in the regulation of cell
proliferation.
Rhodes et al. (J. Neurosci. 17:8246-8258 (1997)) examined the association
and colocalization of two mammalian a-subunits with several potassium channel
a-
subunits in adult rat brain. The experiment showed that the two subunits
associate
with virtually all of the a-subunits examined. It was suggested that the
differential
expression and association of cytoplasmic ~3-subunits with pore-forming a-
subunits
could significantly contribute to the complexity and heterogeneity of voltage-
gated
potassium channels in excitable cells. The results provided a biochemical and
neuroanatomical basis for the differential contribution of a and (3 subunits
to
electrophysiologically diverse neuronal potassium currents.
Ion channels are a major target for drug action and development.
Accordingly, it is valuable to the field of pharmaceutical development to
identify and
characterize previously unknown ion channel components. The present invention
advances the state of the art by providing a previously unidentified human
potassium
channel ~3-subunit.
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SUMMARY OF THE INVENTION
It is a general object of the invention to modulate ion channels.
Therefore, it is an object of the invention to identify novel ion channel
components.
It is a specific object of the invention to provide novel ion channel p-
subunit
polypeptides, herein referred to as C7F2 polypeptides, that are useful as
reagents or
targets in assays applicable to treatment and diagnosis of ion-channel-
mediated
disorders.
It is a further object of the invention to provide polynucleotides
corresponding to the novel ~3-subunit polypeptides that are useful as targets
and
reagents in assays applicable to treatment and diagnosis of ion-channel-
mediated
disorders and useful for producing novel ion channel polypeptides by
recombinant
methods.
A specific object of the invention is to identify compounds that act as
agonists or antagonists and modulate the function or expression of the ~3-
subunit.
A further specific object of the invention is to provide the compounds that
modulate the expression or function of the p-subunit for treatment and
diagnosis of
ion-channel-related disorders.
The novel ~i-subunit polypeptides and polynucleotides of the invention are
useful for the treatment of ~3-subunit-associated or related disorders,
including, for
example, central nervous system (CNS) disorders, cardiovascular system
disorders,
and musculoskeletal system disorders. ~i-subunit-associated or related
disorders also
include disorders of tissues in which the novel ~i-subunit C7F2 is expressed,
e.g.,
heart, placental, lung, kidney, prostate, testicular, ovarian, spleen, small
and large
intestine, colon, or thymus tissues, as well as in brain tissues, including
cerebellum,
cerebral cortex, medulla, spinal cord, occipital lobe, frontal lobe, temporal
lobe,
putanem, amygdala, caudate, corpus colosum, hippocampus, substantia nigra,
subthalamus and thalamus.
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The invention is thus based on the identification of a novel potassium channel
(3-subunit.
This ~3-subunit is useful for modulating ion channels in view of its
interaction
with the pore-forming a-subunit. Accordingly, by using the ~i-subunit to
modulate
a-subunit activity, ion channel modulation is provided.
The ~i-subunit is also useful per se as a target or reagent for treatment and
diagnosis.
The invention thus provides isolated ~-subunit polypeptides including a
polypeptide having the amino acid sequence shown in SEQ ID NO 1, or the amino
I 0 acid sequence encoded by the cDNA deposited as ATCC No. on
("the deposited cDNA").
The invention also provides isolated ~i-subunit nucleic acid molecules having
the sequence shown in SEQ ID NO 2 or in the deposited cDNA.
The invention also provides variant polypeptides having an amino acid
15 sequence that is substantially homologous to the amino acid sequence shown
in SEQ
ID NO 1 or encoded by the deposited cDNA.
The invention also provides variant nucleic acid sequences that are
substantially homologous to the nucleotide sequence shown in SEQ ID NO 2 or in
the deposited cDNA.
20 The invention also provides fragments of the polypeptide shown in SEQ ID
NO 1 and nucleotide shown in SEQ ID NO 2, as well as substantially homologous
fragments of the polypeptide or nucleic acid.
The invention also provides vectors and host cells for expression of the (3-
subunit nucleic acid molecules and polypeptides and particularly recombinant
vectors
25 and host cells.
The invention also provides methods of making the vectors and host cells and
methods for using them to produce the ~i-subunit nucleic acid molecules and
polypeptides.
The invention also provides antibodies that selectively bind the (3-subunit
30 polypeptides and fragments.
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The invention also provides methods of screening for compounds that
modulate the expression or activity of the ~i-subunit polypeptides. Modulation
can
be at the level of the polypeptide ~i-subunit or at the level of controlling
the
expression of nucleic acid expressing the ~i-subunit polypeptide.
The invention also provides a process for modulating (3-subunit expression or
activity using the screened compounds, including to treat conditions related
to
expression or activity of the (3-subunit polypeptides.
The invention also provides diagnostic assays for determining the presence,
level, or activity of the ~i-subunit polypeptides or nucleic acid molecules in
a
biological sample.
The invention also provides diagnostic assays for determining the presence of
a mutation in the ~i-subunit polypeptides or nucleic acid molecules.
DESCRIPTION OF THE DRAWINGS
Figure 1 shows the C7F2 ~i-subunit nucleotide sequence (SEQ ID NO 2) and
the deduced amino acid sequence (SEQ ID NO 1 ). The amino acid sequence is
numbered with respect to nucleotides (below). It is predicted that amino acids
1-19
constitute the amino terminal intracellular domain, 20-40 constitute the first
transmembrane domain, 41-167 constitute the extracellular loop, 168-192
constitute
the second transmembrane domain, and 193-210 constitute the carboxyl terminal
intracellular domain.
Figures 2A and B show a sequence comparison of the C7F2 ~3-subunit
amino acid sequence with (A) the sequence of a human calcium-activated
potassium
channel ~i-subunit (SEQ ID NO 3) (Meera, P. et al., FEBSLetters 382:84-88
(1996))
and (B) a quail calcium-activated potassium channel ~3-subunit (SEQ ID NO 4)
(Oberst, C. et al., Oncogene 14:1109-1116 (1997)). Sequences were aligned
using
the Clustal W ( 1.74) multiple sequence alignment program using default
parameters.
Figure 3 shows an analysis of the C7F2 ~i-subunit amino acid sequence:
a(3turn and coil regions; hydrophilicity; amphipathic regions; flexible
regions;
antigenic index; and surface probability.
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Figure 4 shows a C7F2 R-subunit hydrophobicity plot. Regions of high
hydrophobicity corresponding to transmembrane segments occur from amino acids
20-40 and 168-192.
Figure 5 shows an analysis of the C7F2 a-subunit open reading frame for
5 amino acids corresponding to specific functional sites. A glycosylation site
is found
corresponding to the site at amino acid 56, which would be in the
extracellular loop.
A second glycosylation site is found at the site of amino acid 93, also in the
extracellular loop. A cyclic AMP or cyclic GMP dependent protein kinase
phosphorylation site is found at the site of amino acid 210, which is in the
carboxy
10 terminal intracellular segment. A protein kinase C phosphorylation site
corresponds
to the site at amino acid 19, which is just outside the beginning of the first
transmembrane domain. This corresponds to the protein kinase A phosphorylation
site in one of the ~i-subunits previously referenced (Knaus, H.G. et al., J.
Biol. Chem.
269:17274-17278 (1994)). A site corresponding to a casein kinase II
15 phosphorylation site is found at the site of amino acid 14, also in the
amino terminal
intracellular segment close to the beginning of the first transmembrane
segment. A
second casein kinase II phosphorylation site is found at the site of amino
acid 167,
which is in the extracellular loop just adjacent to the beginning of the
second
transmembrane segment.
20 Figure 6 shows the time constants of activation and deactivation of the
mouse maxi-K channel (mSlo) when expressed in HEK293 cells alone or when co-
expressed with the C7F2 (3-subunit. Note the increase in activation and
deactivation time constants when mSlo is co-expressed with C7F2.
Figure 7 shows half maximal channel activation of the mouse maxi-K
25 channel (mSlo) in the presence of 3 ~M Ca++when mSlo is expressed alone or
co-
expressed with the C7F2 p-subunit. Note the consistent and highly significant
20
mV hyperpolarizing (leftward) shift of half maximal activation of mSlo when co-
expressed with C7F2.
Figure 8 shows half maximal channel activation of the human maxi-K
30 channel (hSlo) in the presence of 3 pM Ca'+when hSlo is expressed alone or
co-
9
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expressed with the C7F2 ~i-subunit. Note the 20-50 mV depolarizing (rightward)
shift of half maximal channel activation of hSlo when co-expressed with C7F2.
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides
The invention is based on the discovery of a novel potassium channel (3-
subunit. An expressed sequence tag (EST) was identified in a monkey striatum
library. This EST had homology to a quail putative potassium channel ~i-
subunit
(Oberst et al., cited above) and a human calcium-activated potassium channel
(3-
1 S subunit (Meera et al., cited above). A human EST was identified with
similarity to
the 3' end of the monkey EST. This human EST was sequenced and found to be
nearly identical to the 3' end of the monkey clone. 'This EST was used in a
Northern
blot analysis for expression in various human tissues.
The gene is expressed preferentially in brain with highest expression in the
cortical regions but with expression in other regions and in the spinal cord.
In the
brain the following tissues showed a positive signal upon Northern blotting:
cerebellum, cerebral cortex, medulla, spinal cord, occipital lobe, frontal
lobe,
temporal lobe, putanem, amygdala, caudate, corpus colosum, hippocampus,
substantia nigra, subthalamus and thalamus. However, expression is also found
in
heart, kidney, placenta, lung, prostate, testes, ovary and small and large
intestine.
Using the sequence as a probe, a full-length human clone from fetal brain was
identified and sequenced and designated C7F2.
The invention thus relates to a novel potassium channel ~i-subunit having the
deduced amino acid sequence shown in Figure 1 (SEQ ID NO 1 ) or having the
amino
acid sequence encoded by the deposited cDNA, ATCC No.
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The deposit will be maintained under the terms of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms. The deposit is
provided
as a convenience to those of skill in the art and is not an admission that a
deposit is
required under 35 U.S.C. ~112. The deposited sequence, as well as the
polypeptide
encoded by the sequence, is incorporated herein by reference and controls in
the
event of any conflict, such as a sequencing error, with description in this
application.
The "C7F2 ~i-subunit polypeptide" or "C7F2 ~3-subunit protein" refers to the
polypeptide in SEQ ID NO 1 or encoded by the deposited cDNA. The term "~3-
10 subunit protein" or "p-subunit polypeptide", however, further includes the
variants
described herein, as well as fragments derived from the full length C7F2 ~i-
subunit
polypeptide and variants.
T'he'present invention thus provides an isolated or purified C7F2 potassium
channel ~i-subunit polypeptide and variants and fragments thereof.
15 The C7F2 p-subunit polypeptide is a 210 residue protein exhibiting 5
structural domains. The amino terminal intracellular domain is identified to
be
within residues 1 to about residue 19 in SEQ ID NO 1. The first transmembrane
domain is identified to be within residues from about 20 to about 40 in SEQ ID
NO
1. The extracellular loop is identified to be within residues from about 41 to
167 in
20 SEQ ID NO 1. The second transmembrane domain is identified to be within
residues
from about 168 to about 192 in SEQ ID NO 1. The carboxy terminal intracellular
domain is identified to be within residues from about 193 to 210.
As used herein, a polypeptide is said to be "isolated" or "purified" when it
is
substantially free of cellular material when it is isolated from recombinant
and non-
25 recombinant cells, or free of chemical precursors or other chemicals when
it is
chemically synthesized. A polypeptide, however, can be joined to another
polypeptide with which it is not normally associated in a cell and still be
considered
"isolated" or "purified."
The ~-subunit polypeptides can be purified to homogeneity. It is understood,
30 however, that preparations in which the polypeptide is not purified to
homogeneity
are useful and considered to contain an isolated form of the polypeptide. The
critical
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feature is that the preparation allows for the desired function of the
polypeptide, even
in the presence of considerable amounts of other components. Thus, the
invention
encompasses various degrees of purity.
In one embodiment, the language "substantially free of cellular material"
includes preparations of the (3-subunit polypeptide having less than about 30%
(by
dry weight) other proteins (i.e., contaminating protein), less than about 20%
other
proteins, less than about 10% other proteins, or less than about 5% other
proteins.
When the R-subunit polypeptide is recombinantly produced, it can also be
substantially free of culture medium, i.e., culture medium represents less
than about
20%, less than about 10%, or less than about 5% of the volume of the protein
preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of the ~i-subunit polypeptide in which it is separated
from
chemical precursors or other chemicals that are involved in its synthesis. In
one
15 embodiment, the language "substantially free of chemical precursors or
other
chemicals" includes preparations of the polypeptide having less than about 30%
(by
dry weight) chemical precursors or other chemicals, less than about 20%
chemical
precursors or other chemicals, less than about 10% chemical precursors or
other
chemicals, or less than about 5% chemical precursors or other chemicals.
In one embodiment, the ~i-subunit polypeptide comprises the amino acid
sequence shown in SEQ ID NO 1. However, the invention also encompasses
sequence variants. Variants include a substantially homologous protein encoded
by
the same genetic locus in an organism, i.e., an allelic variant. Variants also
encompass proteins derived from other genetic loci in an organism, but having
25 substantial homology to the C7F2 ~i-subunit protein of SEQ ID NO 1.
Variants also
include proteins substantially homologous to the C7F2 ~i-subunit protein but
derived
from another organism, i.e., an ortholog. Variants also include proteins that
are
substantially homologous to the C7F2 ~i-subunit protein that are produced by
chemical synthesis. Variants also include proteins that are substantially
homologous
to the C7F2 ~i-subunit protein that are produced by recombinant methods. It is
12
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understood, however, that variants exclude any amino acid sequences disclosed
prior
to the invention.
As used herein, two proteins (or a region of the proteins) are substantially
homologous when the amino acid sequences are at least about 55-60%, typically
at
least about 70-75%, more typically at least about 80-85%, and most typically
at least
about 90-95% or more homologous. A substantially homologous amino acid
sequence, according to the present invention, will be encoded by a nucleic
acid
sequence hybridizing to the nucleic acid sequence, or portion thereof, of the
sequence
shown in SEQ ID NO 2 under stringent conditions as more fully described below.
To determine the percent homology of two amino acid sequences, or of two
nucleic acids, the sequences are aligned for optimal comparison purposes
(e.g., gaps
can be introduced in the sequence of one protein or nucleic acid for optimal
alignment with the other protein or nucleic acid). The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide positions are
then
compared. When a position in one sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the other sequence,
then the
molecules are homologous at that position. As used herein, amino acid or
nucleic
acid "homology" is equivalent to amino acid or nucleic acid "identity". The
percent
homology between the two sequences is a function of the number of identical
positions shared by the sequences (i.e., per cent homology equals the number
of
identical positions/total number of positions times 100).
The invention also encompasses polypeptides having a lower degree of
identity but having sufficient similarity so as to perform one or more of the
same
functions performed by the C7F2 ~3-subunit polypeptide. Similarity is
determined by
conserved amino acid substitution. Such substitutions are those that
substitute a
given amino acid in a polypeptide by another amino acid of like
characteristics.
Conservative substitutions are likely to be phenotypically silent. Typically
seen as
conservative substitutions are the replacements, one for another, among the
aliphatic
amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser
and Thr,
exchange of the acidic residues Asp and Glu, substitution between the amide
residues
Asn and Gln, exchange of the basic residues Lys and Arg and replacements among
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the aromatic residues Phe, Tyr. Guidance concerning which amino acid changes
are
likely to be phenotypically silent are found in Bowie et al., Science 247:1306-
1310
( 1990).
TABLE 1. Conservative Amino Acid Substitutions.
Aromatic Phenylalanine
Tryptophan
Tyrosine
Hydrophobic Leucine
Isoleucine
Valine
Polar G lutamine
Asparagine
Basic Arginine
Lysine
Histidine
Acidic Aspartic Acid
Glutamic Acid
Small Alanine
Serine
Threonine
Methionine
Glycine
Both identity and similarity can be readily calculated (Computational
Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988;
Biocomputing.~ Informatics and Genome Projects, Smith, D.W., ed., Academic
Press, New York, 1993; Computer Analysis of Sequence Data, Part I , Griffin,
A.M.,
and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in
Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis
Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991
).
14
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Preferred computer program methods to determine identify and similarity
between
two sequences include, but are not limited to, GCG program package (Devereux,
J.,
et al., Nucleic Acids Res. 12(1):387 (1984)), BLASTP, BLASTN, FASTA (Atschul,
S.F. et al., J. Molec. Biol. 215:403 (1990)).
A variant polypeptide can differ in amino acid sequence by one or more
substitutions, deletions, insertions, inversions, fusions, and truncations or
a
combination of any of these.
Variant polypeptides can be fully functional or can lack function in one or
more activities. Thus, in the present case, variations can affect the
function, for
example, of one or more of the regions con esponding to ligand binding,
transmembrane association, phosphorylation, and a-subunit interaction.
Fully functional variants typically contain only conservative variation or
variation in non-critical residues or in non-critical regions. Functional
variants can
also contain substitution of similar amino acids which result in no change or
an
insignificant change in function. Alternatively, such substitutions may
positively or
negatively affect function to some degree.
Non-functional variants typically contain one or more non-conservative
amino acid substitutions, deletions, insertions, inversions, or truncation or
a
substitution, insertion, inversion, or deletion in a critical residue or
critical region.
As indicated, variants can be naturally-occurring or can be made by
recombinant means or chemical synthesis to provide useful and novel
characteristics
for the (3-subunit polypeptide. This includes preventing immunogenicity from
pharmaceutical formulations by preventing protein aggregation, for example if
soluble peptides corresponding to the extracellular loop are used.
Useful variations include alteration of ligand binding characteristics. For
example, one embodiment involves a variation at the binding site that results
in
increased or decreased extent or rate of ligand binding. A further useful
variation at
the same site can result in a higher or lower affinity for ligand. Useful
variations also
include changes that provide affinity for another ligand. Another useful
variation
provides for reduced or increased affinity for the a-subunit or for binding by
a
different a-subunit than the one with which the p-subunit is normally
associated.
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Another useful variation provides for reduced or increased rate or extent of
activation
of the a-subunit. Another useful variation provides a fusion protein in which
one or
more segments is operatively fused to one or more segments from another ~i-
subunit.
Another useful variation provides for an increase or decrease in
phosphorylation or
glycosylation.
Amino acids that are essential for function can be identified by methods
known in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis
{Cunningham et al., Science 244:1081-1085 (1989)). The latter procedure
introduces
single alanine mutations at every residue in the molecule. The resulting
mutant
molecules are then tested for biological activity such as ligand binding, a-
subunit
association or activation, or channel currents. Sites that are critical for
ligand binding
and a-subunit modulation can also be determined by structural analysis such as
crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith
et al., J.
Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).
The invention also includes polypeptide fragments of the C7F2 (3-subunit
protein. Fragments can be derived from the amino acid sequence shown in SEQ ID
NO 1. However, the invention also encompasses fragments of the variants of the
~i-
subunit protein as described herein.
The fragments to which the invention pertains, however, are not to be
construed as encompassing fragments that may be disclosed prior to the present
invention.
Fragments can retain one or more of the biological activities of the protein,
for example the ability to bind to an a-subunit or ligand. Biologically active
fragments can comprise a domain or motif, e.g., an extracellular domain, one
or more
transmembrane domains, a-subunit binding domain, or intracellular domains or
functional parts thereof. Such peptides can be, for example, 7, 10, 15, 20,
30, 35, 36,
37, 38, 39, 40, 50, 100 or more amino acids in length.
Possible fragments include, but are not limited to: 1) peptides comprising
from about amino acid 1 to about amino acid 19 of SEQ ID NO 1; 2) peptides
comprising from about amino acid 20 to about amino acid 40 of SEQ ID NO 1; 3)
peptides comprising from about amino acid 41 to about amino acid 167 of SEQ ID
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NO 1; 4) peptides comprising from about amino acid 168 to about amino acid
192;
and 5) peptides comprising from about amino acid 193 to amino acid 210, or
combinations of these fragments such as two, three, or four domains. Other
fragments include fragments containing the various functional sites described
herein
such as phosphorylation sites such as around amino acids 210, 19, 14, and 167,
and
glycosylation sites around amino acids 56 and 93. Fragments, for example, can
extend in one or both directions from the functional site to encompass S, 10,
15, 20,
30, 40, 50, or up to 100 amino acids. Further, fragments can include
subfragments of
the specific domains mentioned above, which subfragments retain the function
of the
domain from which they are derived. Fragments also include amino acid
sequences
greater than 71 amino acids. Fragments also include antigenic fragments and
specifically those shown to have a high antigenic index in Figure 3. Further
specific
fragments include amino acids 1 to 29, 306 to 326, and fragments including but
larger than amino acids 1-29, 30-65, 67-252, 254-305, 306-326, 330-338, 342-
347,
353-361, and 366-382.
Accordingly, possible fragments include fragments defining the site of
association between the ~i and a subunits, fragments defining a ligand binding
site,
fragments defining a glycosylation site, fragments defining membrane
association,
and fragments defining phosphorylation sites. By this is intended a discrete
fragment
that provides the relevant function or allows the relevant function to be
identified. In
a preferred embodiment, the fragment contains the sites) of a and ~i subunit
association.
The invention also provides fragments with immunogenic properties. These
contain an epitope-bearing portion of the C7F2 (3-subunit protein and
variants. These
epitope-bearing peptides are useful to raise antibodies that bind specifically
to a ~3-
subunit polypeptide or region or fragment. These peptides can contain at least
7, at
least 14, or between at least about 1 S to about 30 amino acids. Peptides
having a
high antigenic index are shown in Figure 3.
Non-limiting examples of antigenic polypeptides that can be used to generate
antibodies include peptides derived from the extracellular domain.
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The epitope-bearing ~i-subunit and polypeptides may be produced by any
conventional means (Houghten, R.A., Proc. Natl. Acad. Sci. USA 82:5131-5135
(1985)). Simultaneous multiple peptide synthesis is described in U.S. Patent
No.
4,631,211.
Fragments can be discrete (not fused to other amino acids or polypeptides) or
can be within a larger polypeptide. Further, several fragments can be
comprised
within a single larger polypeptide. In one embodiment a fragment designed for
expression in a host can have heterologous pre- and pro-polypeptide regions
fused to
the amino terminus of the ~3-subunit fragment and an additional region fused
to the
I 0 carboxyl terminus of the fragment.
The invention thus provides chimeric or fusion proteins. These comprise a (3-
subunit protein operatively linked to a heterologous protein having an amino
acid
sequence not substantially homologous to the ~i-subunit protein. "Operatively
linked" indicates that the ~i-subunit protein and the heterologous protein are
fused in-
1 S frame. The heterologous protein can be fused to the N-terminus or C-
terminus of the
~i-subunit protein.
In one embodiment the fusion protein does not affect ~i-subunit function per
se. For example, the fusion protein can be a GST-fusion protein in which the
(3-
subunit sequences are fused to the C-terminus of the GST sequences. Other
types of
20 fusion proteins include, but are not limited to, enzymatic fusion proteins,
for example
beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions and
Ig
fusions. Such fusion proteins, particularly poly-His fusions, can facilitate
the
purification of recombinant p-subunit protein. In certain host cells (e.g.,
mammalian
host cells), expression and/or secretion of a protein can be increased by
using a
25 heterologous signal sequence. Therefore, in another embodiment, the fusion
protein
contains a heterologous signal sequence at its N-terminus.
EP-A-O 464 533 discloses fusion proteins comprising various portions of
immunoglobin constant regions. The Fc is useful in therapy and diagnosis and
thus
results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
In
30 drug discovery, for example, human proteins have been fused with Fc
portions for
the purpose of high-throughput screening assays to identify antagonists.
Bennett et
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al., Journal of Molecular Recognition 8:52-58 ( 1995) and Johanson et al., The
Journal ofBiological Chemistry 270,16:9459-9471 (1995). Thus, this invention
also
encompasses soluble fusion proteins containing a ~i-subunit polypeptide and
various
portions of the constant regions of heavy or light chains of immunoglobulins
of
various subclass (IgG, IgM, IgA, IgE). Preferred as immunoglobulin is the
constant
part of the heavy chain of human IgG, particularly IgGl, where fusion takes
place at
the hinge region. For some uses it is desirable to remove the Fc after the
fusion
protein has been used for its intended purpose, for example when the fusion
protein is
to be used as antigen for immunizations. In a particular embodiment, the Fc
part can
be removed in a simple way by a cleavage sequence which is also incorporated
and
can be cleaved with factor Xa.
A chimeric or fusion protein can be produced by standard recombinant DNA
techniques. For example, DNA fragments coding for the different protein
sequences
are ligated together in-frame in accordance with conventional techniques. In
another
embodiment, the fusion gene can be synthesized by conventional techniques
including automated DNA synthesizers. Alternatively, PCR amplification of gene
fragments can be carried out using anchor primers which give rise to
complementary
overhangs between two consecutive gene fragments which can subsequently be
annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et
al.,
Current Protocols in Molecular Biology, 1992). Moreover, many expression
vectors
are commercially available that already encode a fusion moiety (e.g., a GST
protein).
A ~i-subunit protein-encoding nucleic acid can be cloned into such an
expression
vector such that the fusion moiety is linked in-frame to the ~i-subunit
protein.
Another form of fusion protein is one that directly affects ~i-subunit
functions. Accordingly, a ~i-subunit polypeptide encompassed by the present
invention in which one or more of the ~i-subunit segments has been replaced by
homologous segments from another ~i-subunit. Various permutations are
possible.
The various segments include the intracellular amino and carboxy terminal
domains,
the two transmembrane domains, and the extracellular loop domain. More
specifically, the functional domains include the domain containing the ligand
binding
site, the domains containing the phosphorylation sites, and the domain
containing the
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site that functions to bind a-subunit or modulate a-subunit activation. Any of
these
domains or subregions thereof containing a specific site can be replaced with
the
corresponding domain or subregion from another ~i-subunit protein, or other
subunit
protein that modulates a-subunit activation. Accordingly, one or more of the
specific
domains or functional subregions can be combined with those from another
subunit
that modulates an a-subunit. Thus, chimeric ~i-subunits can be formed in which
one
or more of the native domains or subregions has been replaced.
The invention also encompasses chimeric channels in which an a-subunit
other than the one with which the a-subunit is naturally found is substituted.
The (3-
subunit can therefore be tested for the ability to modulate other a-subunits.
Using
assays directed towards these a-subunits as end points, allows the assessment
of the
(3-subunit function. With this type of construct, an a-subunit can be made
responsive
to a ligand by which it is not normally activated. Thus, by substitution of
the ~3-
subunit, a ligand binding to that p-subunit can be used to modulate the
activity of the
a-subunit.
The isolated [3-subunit protein can be purified from cells that naturally
express it, such as from brain, heart, kidney, prostate, placenta, lung,
testes, ovary
and intestine, purified from cells that have been altered to express it
(recombinant), or
synthesized using known protein synthesis methods.
In one embodiment, the protein is produced by recombinant DNA techniques.
For example, a nucleic acid molecule encoding the ~i-subunit polypeptide is
cloned
into an expression vector, the expression vector introduced into a host cell
and the
protein expressed in the host cell. The protein can then be isolated from the
cell by
an appropriate purification scheme using standard protein purification
techniques.
Polypeptides often contain amino acids other than the 20 amino acids
commonly referred to as the 20 naturally-occurring amino acids. Further, many
amino acids, including the terminal amino acids, may be modified by natural
processes, such as processing and other post-translational modifications, or
by
chemical modification techniques well known in the art. Common modifications
that
occur naturally in polypeptides are described in basic texts, detailed
monographs, and
the research literature, and they are well known to those of skill in the art.
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Accordingly, the polypeptides also encompass derivatives or analogs in
which a substituted amino acid residue is not one encoded by the genetic code,
in
which a substituent group is included, in which the mature polypeptide is
fused with
another compound, such as a compound to increase the half life of the
polypeptide
(for example, polyethylene glycol), or in which the additional amino acids are
fused
to the mature polypeptide, such as a leader or secretory sequence or a
sequence for
purification of the mature polypeptide or a pro-protein sequence.
Known modifications include, but are not limited to, acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin, covalent
attachment of a
heme moiety, covalent attachment of a nucleotide or nucleotide derivative,
covalent
attachment of a lipid or lipid derivative, covalent attachment of
phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation, demethylation, formation
of
covalent crosslinks, formation of cystine, formation of pyroglutamate,
formylation,
gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation,
iodination, methylation, myristoylation, oxidation, proteolytic processing,
phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-
RNA
mediated addition of amino acids to proteins such as arginylation, and
ubiquitination.
Such modifications are well known to those of skill in the art and have been
described in great detail in the scientific literature. Several particularly
common
modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation
of
glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are
described in most basic texts, such as Proteins - Structure and Molecular
Properties,
2nd Ed., T.E. Creighton, W. H. Freeman and Company, New York (1993). Many
detailed reviews are available on this subject, such as by Wold, F.,
Posttranslational
Covalent Modification ofProteins, B.C. Johnson, Ed., Academic Press, New York
1-
12 (1983); Seifter et al., Meth. Enzymol. 182: 626-646 (1990) and Rattan et
al., Ann.
N. Y. Acad. Sci. 663:48-62 (1992).
As is also well known, polypeptides are not always entirely linear. For
instance, polypeptides may be branched as a result of ubiquitination, and they
may be
circular, with or without branching, generally as a result of post-translation
events,
including natural processing event and events brought about by human
manipulation
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which do not occur naturally. Circular, branched and branched circular
polypeptides
may be synthesized by non-translational natural processes and by synthetic
methods.
Modifications can occur anywhere in a polypeptide, including the peptide
backbone, the amino acid side-chains and the amino or carboxyl termini.
Blockage
of the amino or carboxyl group in a polypeptide, or both, by a covalent
modification,
is common in naturally-occurring and synthetic polypeptides. For instance, the
amino terminal residue of polypeptides made in E coli, prior to proteolytic
processing, almost invariably will be N-formylmethionine.
The modifications can be a function of how the protein is made. For
recombinant polypeptides, for example, the modifications will be determined by
the
host cell posttranslational modification capacity and the modification signals
in the
polypeptide amino acid sequence. Accordingly, when glycosylation is desired, a
polypeptide should be expressed in a glycosylating host, generally a
eukaryotic cell.
Insect cells often carry out the same posttranslational glycosylations as
mammalian
cells and, for this reason, insect cell expression systems have been developed
to
efficiently express mammalian proteins having native patterns of
glycosylation.
Similar considerations apply to other modifications.
The same type of modification may be present in the same or varying degree
at several sites in a given polypeptide. Also, a given polypeptide may contain
more
than one type of modification.
Polypeptide Uses
The ~3-subunit polypeptides, as well as the a-subunit nucleic acid molecules,
modulators of these polypeptides, and antibodies (also referred to herein as
"active
compounds") of the invention are useful in the modulation, diagnosis, and
treatment
of (3-subunit-associated or related disorders, also referred to as C7F2-
associated or
related disorders. Such disorders include, for example, central nervous system
(CNS)
disorders, cardiovascular system disorders, and musculoskeletal system
disorders.
CNS disorders include, but are not limited to, cognitive and neurodegenerative
disorders such as Alzheimer's disease and dementias related to Alzheimer's
disease
(such as Pick's disease), senile dementia, Huntington's disease, amyotrophic
lateral
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sclerosis, Parkinson's disease and other Lewy diffuse body diseases, Gilles de
la
Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis,
progressive
supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease autonomic
function
disorders such as hypertension and sleep disorders, and neuropsychiatric
disorders,
such as depression, schizophrenia, schizoaffective disorder, korsakofPs
psychosis,
learning or memory disorders, e.g., amnesia or age-related memory loss,
attention
deficit disorder, dysthymic disorder, major depressive disorder, mania,
obsessive-
compulsive disorder, psychoactive substance use disorders, anxiety, phobias,
panic
disorder, as well as bipolar affective disorder, e.g., severe bipolar
affective (mood)
disorder (BP-I), bipolar affective (mood) disorder with hypomania and major
depression (BP-II), neurological disorders, e.g., migraine, and obesity.
Further CNS-
related disorders include, for example, those listed in the American
Psychiatric
Association's Diagnostic and Statistical manual of Mental Disorders (DSM), the
most current version of which is incorporated herein by reference in its
entirety.
15 ~i-subunit-associated or related disorders can detrimentally affect
conveyance
of sensory impulses from the periphery to the brain (e.g., pain disorders)
and/or
conductance of motor impulses from the brain to the periphery; integration of
reflexes; interpretation of sensory impulses (e.g., pain); or emotional,
intellectual
(e.g., learning and memory), or motor processes.
Cardiovascular system disorders include, but are not limited to,
arteriosclerosis,
ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall
remodeling, ventricular remodeling, rapid ventricular pacing, coronary
microembolism, tachycardia, bradycardia, pressure overload, aortic bending,
coronary artery ligation, vascular heart disease, atrial fibrilation, long-QT
syndrome,
congestive heart failure, sinus node
disfunction, angina, heart failure, hypertension, atrial fibrillation, atrial
flutter, dilated
cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, coronary
artery
disease, coronary artery spasm, or arrhythmia. C7F2-mediated or related
disorders
also include disorders of the musculoskeletal system such as paralysis and
muscle
weakness, e.g., ataxia, myotonia, and myokymia.
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(3-subunit-associated or related disorders also include disorders of tissues
in
which C7F2 is expressed, e.g., heart, placental, lung, kidney, prostate,
testicular,
ovarian, spleen, small and large intestine, colon, or thymus tissues, as well
as in brain
tissues, including cerebellum, cerebral cortex, medulla, spinal cord,
occipital lobe,
frontal lobe, temporal lobe, putanem, amygdala, caudate, corpus colosum,
hippocampus, substantia nigra, subthalamus and thalamus.
The ~-subunit polypeptides and nucleotide sequences encoding the
polypeptides find use in modulating a (3-subunit function or activity. By
"modulating" is intended the upregulating or downregulating of a response.
That is,
the (3-subunit polypeptide and nucleic acid compositions of the invention
affect the
targeted activity in either a positive or negative fashion.
The (3-subunit-associated or related activities include, but are not limited
to,
an activity that involves a potassium channel, e.g., a potassium channel in a
neuronal
cell or a muscle cell, associated with receiving, conducting, and transmitting
signals
in, for example, the nervous system. Potassium-channel mediated activities
include
release of neurotransmitters, e.g., dopamine or norepinephrine, from cells,
e.g.,
neuronal cells; modulation of resting potential of membranes, wave forms and
frequencies of action potentials, and thresholds of excitation; and modulation
of
processes such as integration of sub-threshold synaptic responses and the
conductance of back-propagating action potentials in, for example, neuronal
cells or
muscle cells. (3-subunit-associated or related activities also include
activities which
involve a potassium channel in nonneuronal cells, e.g., placental, lung,
kidney,
prostate, testicular, ovarian, spleen, small intestine, colon, or thymus
cells, such as
membrane potential, cell volume, and pH regulation. (3-subunit-associated or
related
activities include activities involved in muscle function such as maintenance
of
muscle membrane potential, regulation of muscle contraction andrelaxation, and
coordination. A preferred (3-subunit activity is modulation or regulation of
the pore-
forming a-subunit of a potassium channel, particularly activation of the a-
subunit.
Accordingly, in one aspect, this invention provides a method for identifying a
compound suitable for treating a (3-subunit-associated or related disorder by
contacting a C7F2 ~-subunit polypeptide, or a cell expressing a C7F2 ~i-
subunit
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polypeptide, with a test compound and determining whether the C7F2 (i-subunit
polypeptide binds to the test compound, thereby identifying a compound
suitable for
treating a (3-subunit-associated or related disorder.
The ~i-subunit polypeptides are useful for producing antibodies specific for
the C7F2 ~3-subunit protein, regions, or ftagments.
The ~i-subunit polypeptides are also useful in drug screening assays, in cell-
based or cell-free systems. Cell-based systems can be native i.e., cells that
normally
express the ~i-subunit protein, as a biopsy or expanded in cell culture. In
one
embodiment, however, cell-based assays involve recombinant host cells
expressing
the ~i-subunit protein.
The polypeptides can be used to identify compounds that modulate (3-subunit
activity. Both C7F2 ~i-subunit protein and appropriate variants and fragments
can be
used in high throughput screens to assay candidate compounds for the ability
to bind
to the ~3-subunit. These compounds can be further screened against a
functional ~3-
subunit to determine the effect of the compound on the (3-subunit activity.
Compounds can be identified that activate (agonist) or inactivate (antagonist)
the ~i-
subunit to a desired degree.
The ~-subunit polypeptides can be used to screen a compound for the ability
to stimulate or inhibit interaction between the ~i-subunit protein and a
target molecule
that normally interacts with the (3-subunit protein. The target can be ligand
or
another channel subunit with which the ~i-subunit protein normally interacts
(for
example, the a-subunit in the potassium channel). The target can be a molecule
that
modifies the ~i-subunit such as by phosphorylation, for example, casein
lcinase II.
The assay includes the steps of combining the ~i-subunit protein with a
candidate
compound under conditions that allow the ~i-subunit protein or fragment to
interact
with the target molecule, and to detect the formation of a complex between the
protein and the target or to detect the biochemical consequence of the
interaction
with the ~i-subunit protein and the target, such as ion currents or any of the
associated
effects of the currents, phosphorylation, change in cell volume, mutagenesis,
or
transformation.
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The invention also encompasses chimeric channels in which a (3-subunit is
associated with a heterologous a-subunit. Thus, the ~i-subunit can be used to
modulate heterologous a-subunits, as a target for drug screening and in
diagnosis and
treatment.
Candidate compounds include, for example, 1 ) small organic and inorganic
molecules (e.g., molecules obtained from combinatorial and natural product
libraries); 2) phosphopeptides (e.g., members of random and partially
degenerate,
directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778
(1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-
idiotypic,
chimeric, and single chain antibodies as well as Fab, F(ab~)2, Fab expression
library
fragments, and epitope-binding fragments of antibodies); and 4) peptides such
as
soluble peptides, including Ig-tailed fusion peptides and members of random
peptide
libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al.,
Nature
354:84-86 ( 1991 )) and combinatorial chemistry-derived molecular libraries
made of
D- and/or L- configuration amino acids.
The invention provides other end points to identify compounds that modulate
(stimulate or inhibit) ~i-subunit activity. The assays typically involve an
assay of
events in channeling that indicate ~i-subunit activity. A preferred assay
involves the
activation of the a-subunit.
Assays allowing the assessment of ~-subunit activity are known to those of
skill in the art and can be found, for example, in McManus et al. (1995),
Knaus et al.
( 1996), Knaus et al. ( 1994), Meera et al. , and Oberst et al. , cited above.
Binding and/or modulating (activating or inhibiting) compounds can also be
screened by using chimeric subunit proteins in which the ligand binding or a-
subunit
binding region is replaced by a heterologous region. For example, an a-subunit
binding region can be used that interacts with a different a-subunit than that
which is
recognized by the native ~i-subunit. Accordingly, a different end-point assay
is
available. Alternatively, one or two transmembrane regions can be replaced
with
transmembrane portions specific to a host cell that is different from the
native host
cell from which the native (3-subunit is derived. This allows for assays to be
performed in other than the original host cell. Alternatively, the ligand
binding
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region can be replaced by a region binding a different ligand, thus, enabling
an assay
for test compounds that interact with the heterologous ligand binding region
but still
cause channeling function, including a-subunit activation.
The ~-subunit polypeptides are also useful in competition binding assays in
methods designed to discover compounds that interact with the ~i-subunit.
Thus, a
compound is exposed to a (3-subunit polypeptide under conditions that allow
the
compound to bind or to otherwise interact with the polypeptide. Competing (3-
subunit polypeptide is also added to the mixture. If the test compound
interacts with
the competing ~-subunit polypeptide, it decreases the amount of complex formed
or
activity from the R-subunit target. This type of assay is particularly useful
in cases in
which compounds are sought that interact with specific regions of the ~i-
subunit.
Thus, the polypeptide that competes with the target ~-subunit region is
designed to
contain peptide sequences corresponding to the region of interest.
A ~i-subunit is also useful for assessing function of a given a-subunit. Thus,
alteration in channel currents, number of receptors, cell transformation, or
any other
biological end point can be assessed using the ~i-subunit of the present
invention in
cell-based or cell-free assays with a given a-subunit. Mutation in the a-
subunit can
be detected by any of the various end points. Moreover, mutations in the ~i-
subunit
that complement (i.e., correct) mutations in the a-subunit can be identified
through
cell-based or cell-free assays. Such assays could even be performed at the
level of
the organism, as with a transgenic animal (see below).
To perform cell free drug screening assays, it is desirable to immobilize
either
the ~3-subunit protein, or fragment, or its target molecule to facilitate
separation of
complexes from uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay.
Techniques for immobilizing proteins on matrices can be used in the drug
screening assays. In one embodiment, a fusion protein can be provided which
adds a
domain that allows the protein to be bound to a matrix. For example,
glutathione-S-
transferase/flh385 fusion proteins can be adsorbed onto glutathione sepharose
beads
(Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates,
which
are then combined with the cell lysates (e.g., 35S-labeled) and the candidate
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compound, and the mixture incubated under conditions conducive to complex
formation (e.g., at physiological conditions for salt and pH). Following
incubation,
the beads are washed to remove any unbound label, and the matrix immobilized
and
radiolabel determined directly, or in the supernatant after the complexes are
dissociated. Alternatively, the complexes can be dissociated from the matrix,
separated by SDS-PAGE, and the level of ~i-subunit-binding protein found in
the
bead fraction quantitated from the gel using standard electrophoretic
techniques. For
example, either the polypeptide or its target molecule can be immobilized
utilizing
conjugation of biotin and streptavidin using techniques well known in the art.
Alternatively, antibodies reactive with the protein but which do not interfere
with
binding of the protein to its target molecule can be derivatized to the wells
of the
plate, and the protein trapped in the wells by antibody conjugation.
Preparations of a
p-subunit-binding protein and a candidate compound are incubated in the ~i-
subunit
protein-presenting wells and the amount of complex trapped in the well can be
quantitated. Methods for detecting such complexes, in addition to those
described
above for the GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the ~i-subunit protein target molecule, or
which are
reactive with ~i-subunit protein and compete with the target molecule; as well
as
enzyme-linked assays which rely on detecting an enzymatic activity associated
with
the target molecule.
Modulators of ~3-subunit protein activity identified according to these drug
screening assays can be used to treat a subject with a disorder mediated by
the ~i-
subunit. These methods of treatment include the steps of administering the
modulators of protein activity in a pharmaceutical composition as described
herein,
to a subject in need of such treatment.
The ~-subunit polypeptides also are useful to provide a target for diagnosing
a disease or predisposition to disease mediated by the ~-subunit protein.
Accordingly, methods are provided for detecting the presence, or levels of,
the ~i-
subunit protein in a cell, tissue, or organism. The method involves contacting
a
biological sample with a compound capable of interacting with the (3-subunit
protein
such that the interaction can be detected.
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One agent for detecting ~-subunit protein is an antibody capable of
selectively binding to ~i-subunit protein. A biological sample includes
tissues, cells
and biological fluids isolated from a subject, as well as tissues, cells and
fluids
present within a subject.
The (3-subunit protein also provides a target for diagnosing active disease,
or
predisposition to disease, in a patient having a variant ~i-subunit protein.
Thus, ~-
subunit protein can be isolated from a biological sample, assayed for the
presence of
a genetic mutation that results in aberrant ~i-subunit protein. This includes
amino
acid substitution, deletion, insertion, rearrangement, (as the result of
aberrant splicing
events), and inappropriate post-translational modification. Analytic methods
include
altered electrophoretic mobility, altered tryptic peptide digest, altered ~i-
subunit
activity in cell-based or cell-free assay, alteration in ligand, a-subunit, or
antibody-
binding pattern, altered isoelectric point, direct amino acid sequencing, and
any other
of the known assay techniques useful for detecting mutations in a protein.
In vitro techniques for detection of ~i-subunit protein include enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations and
immunofluorescence. Alternatively, the protein can be detected in vivo in a
subject
by introducing into the subject a labeled anti-R-subunit antibody. For
example, the
antibody can be labeled with a radioactive marker whose presence and location
in a
subject can be detected by standard imaging techniques. Particularly useful
are
methods which detect the allelic variant of a ~i-subunit protein expressed in
a subject
and methods which detect fragments of a R-subunit protein in a sample.
The ~i-subunit polypeptides are also useful in pharmacogenomic analysis.
Pharmacogenomics deal with clinically significant hereditary variations in the
response to drugs due to altered drug disposition and abnormal action in
affected
persons. See, e.g., Eichelbaum, M. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-
11):983-985 and Linder, M.W. (1997) Clin. Chem. 43(2):254-266. The clinical
outcomes of these variations result in severe toxicity of therapeutic drugs in
certain
individuals or therapeutic failure of drugs in certain individuals as a result
of
individual variation in metabolism. Thus, the genotype of the individual can
determine the way a therapeutic compound acts on the body or the way the body
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metabolizes the compound. Further, the activity of drug metabolizing enzymes
effects both the intensity and duration of drug action. Thus, the
pharmacogenomics
of the individual permit the selection of effective compounds and effective
dosages
of such compounds for prophylactic or therapeutic treatment based on the
individual's genotype. The discovery of genetic polymorphisms in some drug
metabolizing enzymes has explained why some patients do not obtain the
expected
drug effects, show an exaggerated drug effect, or experience serious toxicity
from
standard drug dosages. Polymorphisms can be expressed in the phenotype of the
extensive metabolizes and the phenotype of the poor metabolizes.
Accordingly, genetic polymorphism may lead to allelic protein variants of the
~3-subunit protein in which one or more of the ~i-subunit functions in one
population
is different from those in another population. The polypeptides thus allow a
target to
ascertain a genetic predisposition that can affect treatment modality. Thus,
in a
ligand-based treatment, for example, polymorphism may give rise to sites that
are
more or less active in ligand binding, and channel activation. Accordingly,
ligand
choice or dosage could be modified to maximize the therapeutic effect within a
given
population containing a polymorphism. As an alternative to genotyping,
specific
polymorphic polypeptides could be identified.
The ~i-subunit polypeptides are also useful for monitoring therapeutic effects
during clinical trials and other treatment. Thus, the therapeutic
effectiveness of an
agent that is designed to increase or decrease gene expression, protein levels
or ~i-
subunit activity can be monitored over the course of treatment using the ~i-
subunit
polypeptides as an end-point target.
The p-subunit polypeptides are also useful for treating a ~i-subunit-
associated
disorders. Accordingly, methods for treatment include the use of soluble
subunit or
fragments of the ~i-subunit protein that compete for molecules interacting
with the
extracellular portions of the subunit. These ~i-subunits or fragments can have
a
higher affinity for the molecule so as to provide effective competition.
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Antibodies
The invention also provides antibodies that selectively bind to the subunit
protein and its variants and fragments. An antibody is considered to
selectively bind,
even if it also binds to other proteins that are not substantially homologous
with the
~i-subunit protein. These other proteins share homology with a fragment or
domain
of the (3-subunit protein. This conservation in specific regions gives rise to
antibodies that bind to both proteins by virtue of the homologous sequence. In
this
case, it would be understood that antibody binding to the subunit protein is
still
selective.
Regions showing a high antigenic index are shown in Figure 3. Antibodies
are preferably prepared from these regions or from discrete fragments in these
regions. However, antibodies can be prepared from any region of the peptide as
described herein.
Antibodies can be polyclonal or monoclonal. An intact antibody, or a
fragment thereof (e.g. Fab or F(ab~)2) can be used.
Detection can be facilitated by coupling (i.e., physically linking) the
antibody
to a detectable substance. Examples of detectable substances include various
enzymes, prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent materials, and radioactive materials. Examples of suitable
enzymes
include horseradish peroxidase, alkaline phosphatase, ~3-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable fluorescent
materials
include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a
luminescent material includes luminol; examples of bioluminescent materials
include
luciferase, luciferin, and aequorin, and examples of suitable radioactive
material
include 125I~ ~311~ 35s or 3H.
To generate antibodies, an isolated p-subunit polypeptide is used as an
immunogen to generate antibodies using standard techniques for polyclonal and
monoclonal antibody preparation. Either the full-length protein or antigenic
peptide
fragment can be used. Fragments having a high antigenic index are shown in
Figure
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3. A preferred fragment produces an antibody that diminishes or completely
prevents
association between the a and p subunits. Accordingly, a preferred antibody is
one
that diminishes or completely inhibits association between the two subunits.
An
antigenic fragment will typically comprise at least 7 contiguous amino acid
residues.
The antigenic peptide can comprise, however, at least 12, at least 14 amino
acid
residues, at least 15 amino acid residues, at least 20 amino acid residues, or
at least
30 amino acid residues. In one embodiment, fragments correspond to regions
that
are located on the surface of the protein, e.g., hydrophilic regions.
An appropriate immunogenic preparation can be derived from native,
recombinantly expressed, protein or chemically synthesized peptides.
Antibody Uses
The antibodies can be used to isolate a ~i-subunit protein by standard
techniques, such as affinity chromatography or immunoprecipitation. The
antibodies
can facilitate the purification of the natural ~i-subunit protein from cells
and
recombinantly produced ~i-subunit protein expressed in host cells.
The antibodies are useful to detect the presence of ~i-subunit protein in
cells
or tissues to determine the pattern of expression of the ~i-subunit among
various
tissues in an organism and over the course of normal development.
The antibodies can be used to detect ~3-subunit protein in situ, in vitro, or
in a
cell lysate or supernatant in order to evaluate the abundance and pattern of
expression.
The antibodies can be used to assess abnormal tissue distribution or abnormal
expression during development.
Antibody detection of fragments of the full length ~i-subunit protein can be
used to identify p-subunit turnover.
Further, the antibodies can be used to assess ~-subunit expression in disease
states such as in active stages of the disease or in an individual with a
predisposition
toward disease related to p-subunit function. When a disorder is caused by an
inappropriate tissue distribution, developmental expression, or level of
expression of
the ~-subunit protein, the antibody can be prepared against the normal ~i-
subunit
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protein. If a disorder is characterized by a specific mutation in the ~3-
subunit protein,
antibodies specific for this mutant protein can be used to assay for the
presence of the
specific mutant R-subunit protein.
The antibodies can also be used to assess normal and aberrant subcellular
localization of cells in the various tissues in an organism.
Antibodies can be developed against the whole [3-subunit or portions of the
~i-subunit, for example, the intracellular regions, the extracellular region,
the
transmembrane regions, and specific functional sites such as the site of
Iigand
binding, the site of interaction with the a-subunit, or sites that are
phosphorylated, for
example by casein kinase II.
The diagnostic uses can be applied, not only in genetic testing, but also in
monitoring a treatment modality. Accordingly, where treatment is ultimately
aimed
at correcting (3-subunit expression level or the presence of aberrant ~i-
subunits and
aberrant tissue distribution or developmental expression, antibodies directed
against
the ~i-subunit or relevant fragments can be used to monitor therapeutic
efficacy.
Additionally, antibodies are useful in pharmocogenomic analysis. Thus,
antibodies prepared against polymorphic ~3-subunit proteins can be used to
identify
individuals that require modified treatment modalities.
The antibodies are also useful as diagnostic tools as an immunological
marker for aberrant (3-subunit protein analyzed by electrophoretic mobility,
isoelectric point, tryptic peptide digest, and other physical assays known to
those in
the art.
The antibodies are also useful for tissue typing. Thus, where a specific ~i-
subunit protein has been correlated with expression in a specific tissue,
antibodies
that are specific for this ~i-subunit protein can be used to identify a tissue
type.
The antibodies are also useful in forensic identification. Accordingly, where
an individual has been correlated with a specific genetic polymorphism
resulting in a
specific polymorphic protein, an antibody specific for the polymorphic protein
can be
used as an aid in identification.
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The antibodies are also useful for inhibiting subunit function, for example,
blocking ligand binding or a-subunit binding and/or activation. Subunit
function
involving the extracellular loop is particularly amenable to antibody
inhibition.
These uses can also be applied in a therapeutic context in which treatment
involves inhibiting subunit function. Antibodies can be prepared against
specific
fragments containing sites required for function or against intact (3-subunit
associated
with a cell.
The invention also encompasses kits for using antibodies to detect the
presence of a ~i-subunit protein in a biological sample. The kit can comprise
antibodies such as a labeled or labelable antibody and a compound or agent for
detecting a-subunit protein in a biological sample; means for determining the
amount
of ~3-subunit protein in the sample; and means for comparing the amount of (3-
subunit protein in the sample with a standard. The compound or agent can be
packaged in a suitable container. The kit can further comprise instructions
for using
the kit to detect ~i-subunit protein.
Polynucleotides
The nucleotide sequence in SEQ ID NO 2 was obtained by sequencing the
deposited human full length cDNA. Accordingly, the sequence of the deposited
clone is controlling as to any discrepancies between the two and any reference
to the
sequence of SEQ ID NO 2 includes reference to the sequence of the deposited
cDNA.
The specifically disclosed cDNA comprises the coding region, 5' and 3'
untranslated sequences (SEQ ID NO 2). In one embodiment, the subunit nucleic
acid
comprises only the coding region.
The human C7F2 ~i-subunit cDNA is approximately 1737 nucleotides in
length and encodes a full length protein that is approximately 210 amino acid
residues in length. The nucleic acid is expressed in brain, heart, kidney,
placenta,
lung, prostate, testes, ovary, and small and large intestine. Structural
analysis of the
amino acid sequence of SEQ ID NO 1 is provided in Figure 4, a hydropathy plot.
The figure shows the putative structure of the two transmembrane domains, the
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extracellular loop and the two intracellular domains. As used herein, the term
"transmembrane domain" (or "region" or "segment") refers to a structural amino
acid
motif which includes a hydrophobic helix that spans the plasma membrane.
The invention provides isolated polynucleotides encoding a C7F2 ~i-subunit
protein. The term "C7F2 ~i-subunit polynucleotide" or "C7F2 (3-subunit nucleic
acid" refers to the sequence shown in SEQ ID NO 2 or in the deposited cDNA.
The
term "a-subunit polynucleotide" or "~i-subunit nucleic acid" further includes
variants
and fragments of the C7F2 polynucleotide.
An "isolated" ~3-subunit nucleic acid is one that is separated from other
nucleic acid present in the natural source of the ~i-subunit nucleic acid.
Preferably,
an "isolated" nucleic acid is free of sequences which naturally flank the
nucleic acid
(i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the
genomic DNA
of the organism from which the nucleic acid is derived. However, there can be
some
flanking nucleotide sequences, for example up to about SKB. The important
point is
that the nucleic acid is isolated from flanking sequences such that it can be
subjected
to the specific manipulations described herein such as recombinant expression,
preparation of probes and primers, and other uses specific to the ~i-subunit
nucleic
acid sequences.
Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule,
can be substantially free of other cellular material, or culture medium when
produced
by recombinant techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be fused to
other
coding or regulatory sequences and still be considered isolated.
For example, recombinant DNA molecules contained in a vector are
considered isolated. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or purified
(partially or substantially) DNA molecules in solution. Isolated RNA molecules
include in vivo or in vitro RNA transcripts of the isolated DNA molecules of
the
present invention. Isolated nucleic acid molecules according to the present
invention
further include such molecules produced synthetically.
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The ~i-subunit polynucleotides can encode the mature protein plus additional
amino or carboxy terminal amino acids, or amino acids interior to the mature
polypeptide (when the mature form has more than one polypeptide chain, for
instance). Such sequences may play a role in processing of a protein from
precursor
to a mature form, facilitate protein trafficking, prolong or shorten protein
half life or
facilitate manipulation of a protein for assay or production, among other
things. As
generally is the case in situ, the additional amino acids may be processed
away from
the mature protein by cellular enzymes.
The ~i-subunit polynucleotides include, but are not limited to, the sequence
I 0 encoding the mature polypeptide alone, the sequence encoding the mature
polypeptide and additional coding sequences, such as a leader or secretory
sequence
(e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature
polypeptide, with or without the additional coding sequences, plus additional
non-
coding sequences, for example introns and non-coding 5' and 3' sequences such
as
transcribed but non-translated sequences that play a role in transcription,
mRNA
processing (including splicing and polyadenylation signals), ribosome binding
and
stability of mRNA. In addition, the polynucleotide may be fused to a marker
sequence encoding, for example, a peptide that facilitates purification.
(3-subunit polynucleotides can be in the form of RNA, such as mRNA, or in
the form DNA, including cDNA and genomic DNA obtained by cloning or produced
by chemical synthetic techniques or by a combination thereof. The nucleic
acid,
especially DNA, can be double-stranded or single-stranded. Single-stranded
nucleic
acid can be the coding strand (sense strand) or the non-coding strand (anti-
sense
strand).
One ~3-subunit nucleic acid comprises the nucleotide sequence shown in SEQ
ID NO 2, corresponding to human fetal brain cDNA.
The invention further provides variant subunit polynucleotides, and
fragments thereof, that differ from the nucleotide sequence shown in SEQ ID NO
2
due to degeneracy of the genetic code and thus encode the same protein as that
encoded by the nucleotide sequence shown in SEQ ID NO 2.
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The invention also provides ~3-subunit nucleic acid molecules encoding the
variant polypeptides described herein. Such polynucleotides may be naturally
occurring, such as allelic variants (same locus), homologs (different locus),
and
orthologs (different organism), or may be constructed by recombinant DNA
methods
or by chemical synthesis. Such non-naturally occurring variants may be made by
mutagenesis techniques, including those applied to polynucleotides, cells, or
organisms. Accordingly, as discussed above, the variants can contain
nucleotide
substitutions, deletions, inversions and insertions.
Variation can occur in either or both the coding and non-coding regions. The
variations can produce both conservative and non-conservative amino acid
substitutions.
Orthologs, homologs, and allelic variants can be identified using methods
well known in the art. These variants comprise a nucleotide sequence encoding
a ~i-
subunit that is at least about 55-60%, typically at least about 70-75%, more
typically
at least about 80-85%, and most typically at least about 90-95% or more
homologous
to the nucleotide sequence shown in SEQ ID NO: 2 or a fragment of this
sequence.
Such nucleic acid molecules can readily be identified as being able to
hybridize
under stringent conditions, to the nucleotide sequence shown in SEQ ID NO 2 or
a
fragment of the sequence. It is understood that stringent hybridization does
not
indicate substantial homology where it is due to general homology, such as
poly A
sequences, or sequences common to all or most proteins, all K+ channel ~i-
subunits,
or all channel ~i-subunits. Moreover, it is understood that variants do not
include any
of the nucleic acid sequences that may have been disclosed prior to the
invention.
As used herein, the term "hybridizes under stringent conditions" is intended
to describe conditions for hybridization and washing under which nucleotide
sequences encoding a ~i-subunit at least 55-60% homologous to each other
typically
remain hybridized to each other. The conditions can be such that sequences at
least
about 65%, at least about 70%, or at least about 75% or more homologous to
each
other typically remain hybridized to each other. Such stringent conditions are
known
to those skilled in the art and can be found in Current Protocols in Molecular
Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent
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hybridization conditions are hybridization in 6X sodium chloride/sodium
citrate
(SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1%
SDS at
50-65°C. In one embodiment, an isolated ~i-subunit nucleic acid
molecule that
hybridizes under stringent conditions to the sequence of SEQ ID NO 2
corresponds
to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-
occurring" nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
The invention also provides polynucleotides that comprise a fragment of the
full length ~i-subunit polynucleotides. The fragment can be single or double
stranded
and can comprise DNA or RNA. The fragment can be derived from either the
coding
or the non-coding sequence, e.g., transcriptional regulatory sequence.
In one embodiment, ~i-subunit coding region nucleic acid is at least 216
nucleotides in length and hybridizes under stringent conditions to the nucleic
acid
molecule comprising the nucleotide sequence of SEQ ID NO 2. Fragments also
1 S include those nucleic acid sequences encoding the specific domains
described herein.
Fragments also include nucleic acids encoding the entire coding sequence.
Fragments also include nucleic acids encoding the mature protein. Fragments
also
include nucleic acid sequences encoding two or more domains. Fragments also
include nucleic acid sequences corresponding to the amino acids at the
specific
functional sites described herein. Fragments further include nucleic acid
sequences
encoding a portion of the amino acid sequence described herein but further
including
flanking nucleotide sequences at the 5' andlor 3' regions. Other fragments can
include subfragments of the specific domains or sites described herein.
Fragments
also include nucleic acid sequences corresponding to specific amino acid
sequences
described above or fragments thereof. In these embodiments, the nucleic acid
is at
least 20, 30, 40, 50, 100, 250 or 500 nucleotides in length. Nucleic acid
fragments,
according to the present invention, are not to be construed as encompassing
those
fragments that may have been disclosed prior to the invention.
However, it is understood that a ~i-subunit fragment includes any nucleic acid
sequence that does not include the entire gene.
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(3-subunit nucleic acid fragments include nucleic acid molecules encoding a
polypeptide comprising an amino terminal intracellular domain including amino
acid
residues from 1 to about 19, a polypeptide comprising the first transmembrane
domain (amino acid residues from about 20 to about 40), a polypeptide
comprising
the extracellular loop domain (amino acid residues from about 41 to about
167), a
polypeptide comprising the second transmembrane domain (amino acid residues
from about 168 to about 192) and a polypeptide comprising the carboxy terminal
intracellular domain (amino acid residues from about 193 to 210). Where the
location of the domains have been predicted by computer analysis, one of
ordinary
I 0 skill would appreciate that the amino acid residues constituting these
domains can
vary depending on the criteria used to define the domains.
The invention also provides ~i-subunit nucleic acid fragments that encode
epitope bearing regions of the p-subunit proteins described herein.
The isolated ~i-subunit polynucleotide sequences, and especially fragments,
1 S are useful as DNA probes and primers.
For example, the coding region of a ~i-subunit gene can be isolated using the
known nucleotide sequence to synthesize an oligonucleotide probe. A labeled
probe
can then be used to screen a cDNA library, genomic DNA library, or mRNA to
isolate nucleic acid corresponding to the coding region. Further, primers can
be used
20 in PCR reactions to clone specific regions of ~i-subunit genes.
A probe/primer typically comprises substantially purified oligonucleotide.
The oligonucleotide typically comprises a region of nucleotide sequence, as
described above, that hybridizes under stringent conditions to at least about
20,
typically about 25, more typically about 40, 50 or 75 consecutive nucleotides
of SEQ
25 ID NO 2 sense or anti-sense strand or other ~i-subunit polynucleotides. A
probe
further comprises a label, e.g., radioisotope, fluorescent compound, enzyme,
or
enzyme co-factor.
Polynucleotide Uses
30 The ~i-subunit polynucleotides are useful as a hybridization probe for cDNA
and genomic DNA to isolate a full-length cDNA and genomic clones encoding the
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polypeptide described in SEQ ID NO 1 and to isolate cDNA and genomic clones
that
correspond to variants producing the same polypeptide shown in SEQ ID NO 1 or
the other variants described herein. Variants can be isolated from the same
tissue and
organism from which the polypeptide shown in SEQ ID NO 1 was isolated,
different
tissues from the same organism, or from different organisms. This method is
useful
for isolating genes and cDNA that are developmentally controlled and therefore
may
be expressed in the same tissue at different points in the development of an
organism.
The probe can correspond to any sequence along the entire length of the gene
encoding the p-subunit. Accordingly, it could be derived from 5' noncoding
regions,
the coding region, as specified above, and 3' noncoding regions.
The nucleic acid probe can be, for example, the full-length cDNA of SEQ ID
NO 1, or a fragment thereof, as described above. The probe can be an
oligonucleotide of at least 20, 30, 50, 100, 250 or 500 nucleotides in length
and
sufficient to specifically hybridize under stringent conditions to mRNA or
DNA.
Fragments of the polynucleotides described herein are also useful to
synthesize larger fragments or full-length polynucleotides described herein.
For
example, a fragment can be hybridized to any portion of an mRNA and a larger
or
full-length cDNA can be produced.
The fragments are also useful to synthesize antisense molecules of desired
length and sequence.
The R-subunit polynucleotides are also useful as primers for PCR to amplify
any given region of a ~i-subunit polynucleotide.
The p-subunit polynucleotides are also useful for constructing recombinant
vectors. Such vectors include expression vectors that express a portion of, or
all of,
the ~i-subunit polypeptides. Vectors also include insertion vectors, used to
integrate
into another polynucleotide sequence, such as into the cellular genome, to
alter in situ
expression of ~3-subunit genes and gene products. For example, an endogenous R-
subunit coding sequence can be replaced via homologous recombination with all
or
part of the coding region containing one or more specifically introduced
mutations.
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The p-subunit polynucleotides are also useful as probes for determining the
chromosomal positions of the ~i-subunit polynucleotides by means of in situ
hybridization methods.
The ~3-subunit polynucleotide probes are also useful to determine patterns of
the presence of the gene encoding the ~i-subunits and their variants with
respect to
tissue distribution, for example whether gene duplication has occurred and
whether
the duplication occurs in all or only a subset of tissues. The genes can be
naturally
occurring or can have been introduced into a cell, tissue, or organism
exogenously.
The ~i-subunit polynucleotides are also useful for designing ribozymes
corresponding
to all, or a part, of the mRNA produced from genes encoding the
polynucleotides
described herein.
The ~3-subunit polynucleotides are also useful for constructing host cells
expressing a part, or all, of the ~i-subunit polynucleotides and polypeptides.
The ~3-subunit polynucleotides are also useful for constructing transgenic
animals expressing all, or a part, of the ~i-suburut polynucleotides and
polypeptides.
The ~i-subunit polynucleotides are also useful for making vectors that express
part, or all, of the ~i-subunit polypeptides.
The ~-subunit polynucleotides are also useful as hybridization probes for
determining the level of ~3-subunit nucleic acid expression. Accordingly, the
probes
can be used to detect the presence of, or to determine levels of, ~-subunit
nucleic acid
in cells, tissues, and in organisms. The nucleic acid whose level is
determined can be
DNA or RNA. Accordingly, probes corresponding to the polypeptides described
herein can be used to assess gene copy number in a given cell, tissue, or
organism.
This is particularly relevant in cases in which there has been an
amplification of the
~3-subunit genes.
Alternatively, the probe can be used in an in situ hybridization context to
assess the position of extra copies of the ~-subunit genes, as on
extrachromosomal
elements or as integrated into chromosomes in which the ~-subunit gene is not
normally found, for example as a homogeneously staining region.
These uses are relevant for diagnosis of disorders involving an increase or
decrease in ~i-subunit expression relative to normal results.
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In vitro techniques for detection of mRNA include Northern hybridizations
and in situ hybridizations. In vitro techniques for detecting DNA includes
Southern
hybridizations and in situ hybridization.
Probes can be used as a part of a diagnostic test kit for identifying cells or
tissues that express a ~i-subunit protein, such as by measuring a level of a
subunit-
encoding nucleic acid in a sample of cells from a subject e.g., mRNA or
genomic
DNA, or determining if a ~3-subunit gene has been mutated.
Nucleic acid expression assays are useful for drug screening to identify
compounds that modulate (3-subunit nucleic acid expression or activity.
The invention thus provides a method for identifying a compound that can be
used to treat a disorder associated with nucleic acid expression of the p-
subunit gene.
The method typically includes assaying the ability of the compound to modulate
the
expression of the ~i-subunit nucleic acid and thus identifying a compound that
can be
used to treat a disorder characterized by undesired ~-subunit nucleic acid
expression.
The assays can be performed in cell-based and cell-free systems. Cell-based
assays include cells naturally expressing the ~i-subunit nucleic acid or
recombinant
cells genetically engineered to express specific nucleic acid sequences.
Alternatively, candidate compounds can be assayed in vivo in patients or in
transgenic animals.
The assay for (3-subunit nucleic acid expression can involve direct assay of
nucleic acid levels, such as mRNA levels, or on collateral compounds involved
in the
signal pathway (such as cyclic AMP or phosphatidylinositol turnover). Further,
the
expression of genes that are up- or down-regulated in response to the ~i-
subunit
protein signal pathway can also be assayed. In this embodiment the regulatory
regions of these genes can be operably linked to a reporter gene such as
luciferase.
Thus, modulators of a-subunit gene expression can be identified in a method
wherein a cell is contacted with a candidate compound and the expression of
mRNA
determined. The level of expression of ~3-subunit mRNA in the presence of the
candidate compound is compared to the level of expression of p-subunit mRNA in
the absence of the candidate compound. The candidate compound can then be
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identified as a modulator of nucleic acid expression based on this comparison
and be
used, for example to treat a disorder characterized by aberrant nucleic acid
expression. When expression of mRNA is statistically significantly greater in
the
presence of the candidate compound than in its absence, the candidate compound
is
identified as a stimulator of nucleic acid expression. When nucleic acid
expression is
statistically significantly less in the presence of the candidate compound
than in its
absence, the candidate compound is identified as an inhibitor of nucleic acid
expression.
Accordingly, the invention provides methods of treatment, with the nucleic
acid as a target, using a compound identified through drug screening as a gene
modulator to modulate (3-subunit nucleic acid expression. Modulation includes
both
up-regulation (i.e. activation or agonization) or down-regulation (suppression
or
antagonization) of nucleic acid expression.
Alternatively, a modulator for ~i-subunit nucleic acid expression can be a
small molecule or drug identified using the screening assays described herein
as long
as the drug or small molecule inhibits the ~i-subunit nucleic acid expression.
The ~3-subunit polynucleotides are also useful for monitoring the
effectiveness of modulating compounds on the expression or activity of the (3-
subunit
gene in clinical trials or in a treatment regimen. Thus, the gene expression
pattern
can serve as a barometer for the continuing effectiveness of treatment with
the
compound, particularly with compounds to which a patient can develop
resistance.
The gene expression pattern can also serve as a marker indicative of a
physiological
response of the affected cells to the compound. Accordingly, such monitoring
would
allow either increased administration of the compound or the administration of
alternative compounds to which the patient has not become resistant.
Similarly, if
the level of nucleic acid expression falls below a desirable level,
administration of the
compound could be commensurately decreased.
The ~i-subunit polynucleotides are also useful in diagnostic assays for
qualitative changes in ~i-subunit nucleic acid, and particularly in
qualitative changes
that lead to pathology. The polynucleotides can be used to detect mutations in
~i-
subunit genes and gene expression products such as mRNA. The polynucleotides
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can be used as hybridization probes to detect naturally occurring genetic
mutations in
the (3-subunit gene and thereby determining whether a subject with the
mutation is at
risk for a disorder caused by the mutation. Mutations include deletion,
addition, or
substitution of one or more nucleotides in the gene, chromosomal rearrangement
such as inversion or transposition, modification of genomic DNA such as
aberrant
methylation patterns or changes in gene copy number such as amplification.
Detection of a mutated form of the ~i-subunit gene associated with a
dysfunction
provides a diagnostic tool for an active disease or susceptibility to disease
when the
disease results from overexpression, underexpression, or altered expression of
a ~i-
subunit protein.
Individuals carrying mutations in the (3-subunit gene can be detected at the
nucleic acid level by a variety of techniques. Genomic DNA can be analyzed
directly or can be amplified by using PCR prior to analysis. RNA or cDNA can
be
used in the same way.
In certain embodiments, detection of the mutation involves the use of a
probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Patent Nos.
4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively,
in a
ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-
1080
(1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can
be
particularly useful for detecting point mutations in the gene (see Abravaya et
al.,
Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of
collecting a sample of cells from a patient, isolating nucleic acid (e.g.,
genomic,
mRNA or both) from the cells of the sample, contacting the nucleic acid sample
with
one or more primers which specifically hybridize to a gene under conditions
such
that hybridization and amplification of the gene (if present) occurs, and
detecting the
presence or absence of an amplification product, or detecting the size of the
amplification product and comparing the length to a control sample. Deletions
and
insertions can be detected by a change in size of the amplified product
compared to
the normal genotype. Point mutations can be identified by hybridizing
amplified
DNA to normal RNA or antisense DNA sequences.
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Alternatively, mutations in a ø-subunit gene can be directly identified, for
example, by alterations in restriction enzyme digestion patterns determined by
gel
electrophoresis.
Further, sequence-specific ribozymes (U.S.Patent No. 5,498,531) can be used
to score for the presence of specific mutations by development or loss of a
ribozyme
cleavage site.
Perfectly matched sequences can be distinguished from mismatched
sequences by nuclease cleavage digestion assays or by differences in melting
temperature.
Sequence changes at specific locations can also be assessed by nuclease
protection assays such as RNase and S 1 protection or the chemical cleavage
method.
Furthermore, sequence differences between a mutant subunit gene and a
wild-type gene can be determined by direct DNA sequencing. A variety of
automated sequencing procedures can be utilized when performing the diagnostic
assays (Biotechniques 19:448 (1995)), including sequencing by mass
spectrometry
(see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.
Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol.
38:147-159 (1993)).
Other methods for detecting mutations in the gene include methods in which
protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or
RNA/DNA duplexes (Myers et al., Science 230:1242 (1985); Cotton et al., PNAS
85:4397 (1988); Saleeba et al., Meth. Enrymol. 217:286-295 (1992)),
electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS
86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et
al.,
Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type
fragments in polyacrylamide gels containing a gradient of denaturant is
assayed using
denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)).
Examples of other techniques for detecting point mutations include, selective
oligonucleotide hybridization, selective amplification, and selective primer
extension.
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The (3-subunit polynucleotides are also useful for testing an individual for a
genotype that while not necessarily causing the disease, nevertheless affects
the
treatment modality. Thus, the polynucleotides can be used to study the
relationship
between an individual's genotype and the individual's response to a compound
used
for treatment (pharmacogenomic relationship). In the present case, for
example, a
mutation in the ~i-subunit gene that results in altered affinity for ligand,
for example,
could result in an excessive or decreased drug effect with standard
concentrations of
ligand. Alternatively, for example, a mutation in the subunit gene that
results in an
altered interaction with the a-subunit could result in an increased or
decreased drug
effect with standard concentrations of a drug that affects this functional
interaction.
Accordingly, the ~i-subunit polynucleotides described herein can be used to
assess
the mutation content of the (3-subunit gene in an individual in order to
select an
appropriate compound or dosage regimen for treatment.
Thus polynucleotides displaying genetic variations that affect treatment
provide a diagnostic target that can be used to tailor treatment in an
individual.
Accordingly, the production of recombinant cells and animals containing these
polymorphisms allow effective clinical design of treatment compounds and
dosage
regimens.
The ~i-subunit polynucleotides are also useful for chromosome identification
when the sequence is identified with an individual chromosome and to a
particular
location on the chromosome. First, the DNA sequence is matched to the
chromosome by in situ or other chromosome-specific hybridization. Sequences
can
also be correlated to specific chromosomes by preparing PCR primers that can
be
used for PCR screening of somatic cell hybrids containing individual
chromosomes
from the desired species. Only hybrids containing the chromosome containing
the
gene homologous to the primer will yield an amplified fragment.
Sublocalization
can be achieved using chromosomal fragments. Other strategies include
prescreening with labeled flow-sorted chromosomes and preselection by
hybridization to chromosome-specific libraries. Further mapping strategies
include
fluorescence in situ hybridization which allows hybridization with probes
shorter
than those traditionally used. Reagents for chromosome mapping can be used
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individually to mark a single chromosome or a single site on the chromosome,
or
panels of reagents can be used for marking multiple sites and/or multiple
chromosomes. Reagents corresponding to noncoding regions of the genes actually
are preferred for mapping purposes. Coding sequences are more likely to be
conserved within gene families, thus increasing the chance of cross
hybridizations
during chromosomal mapping.
The (3-subunit polynucleotides can also be used to identify individuals from
small biological samples. This can be done for example using restriction
fragment-
length polymorphism (RFLP) to identify an individual. Thus, the
polynucleotides
described herein are useful as DNA markers for RFLP {See U.S. Patent No.
5,272,057). Furthermore, the ~3-subunit sequence can be used to provide an
alternative technique which determines the actual DNA sequence of selected
fragments in the genome of an individual. Thus, the ~i-subunit sequences
described
herein can be used to prepare two PCR primers from the S' and 3' ends of the
sequences. These primers can then be used to amplify DNA from an individual
for
subsequent sequencing.
Panels of corresponding DNA sequences from individuals prepared in this
manner can provide unique individual identifications, as each individual will
have a
unique set of such DNA sequences. It is estimated that allelic variation in
humans
occurs with a frequency of about once per each S00 bases. Allelic variation
occurs to
some degree in the coding regions of these sequences, and to a greater degree
in the
noncoding regions. The (3-subunit sequences can be used to obtain such
identification sequences from individuals and from tissue. The sequences
represent
unique fragments of the human genome. Each of the sequences described herein
can,
to some degree, be used as a standard against which DNA from an individual can
be
compared for identification purposes.
If a panel of reagents from the sequences is used to generate a unique
identification database for an individual, those same reagents can later be
used to
identify tissue from that individual. Using the unique identification
database,
positive identification of the individual, living or dead, can be made from
extremely
small tissue samples.
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The ~i-subunit polynucleotides can also be used in forensic identification
procedures. PCR technology can be used to amplify DNA sequences taken from
very small biological samples, such as a single hair follicle, body fluids
(eg. blood,
saliva, or semen). The amplified sequence can then be compared to a standard
allowing identification of the origin of the sample.
The (3-subunit polynucleotides can thus be used to provide polynucleotide
reagents, e.g., PCR primers, targeted to specific loci in the human genome,
which can
enhance the reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA sequence that is
unique
to a particular individual). As described above, actual base sequence
information can
be used for identification as an accurate alternative to patterns formed by
restriction
enzyme generated fragments. Sequences targeted to the noncoding region are
particularly useful since greater polymorphism occurs in the noncoding
regions,
making it easier to differentiate individuals using this technique. Fragments
are at
/east 12 bases.
The (3-subunit polynucleotides can further be used to provide polynucleotide
reagents, e.g., labeled or labelable probes which can be used in, for example,
an in
situ hybridization technique, to identify a specific tissue. This is useful in
cases in
which a forensic pathologist is presented with a tissue of unknown origin.
Panels of
~i-subunit probes can be used to identify tissue by species and/or by organ
type.
In a similar fashion, these primers and probes can be used to screen tissue
culture for contamination (i.e. screen for the presence of a mixture of
different types
of cells in a culture).
Alternatively, the p-subunit polynucleotides can be used directly to block
transcription or translation of ~i-subunit gene expression by means of
antisense or
ribozyme constructs. Thus, in a disorder characterized by abnormally high or
undesirable ~i-subunit gene expression, nucleic acids can be directly used for
treatment.
The ~i-subunit polynucleotides are thus useful as antisense constructs to
control ~i-subunit gene expression in cells, tissues, and organisms. A DNA
antisense
polynucleotide is designed to be complementary to a region of the gene
involved in
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transcription, preventing transcription and hence production of ~i-subunit
protein. An
antisense RNA or DNA polynucleotide would hybridize to the mRNA and thus block
translation of mRNA into ~i-subunit protein.
Examples of antisense molecules useful to inhibit nucleic acid expression
include antisense molecules complementary to a fragment of the 5' untranslated
region of SEQ ID NO 2 which also includes the start codon and antisense
molecules
which are complementary to a fragment of the 3' untranslated region of SEQ ID
NO
2.
Alternatively, a class of antisense molecules can be used to inactivate mRNA
in order to decrease expression of ~i-subunit nucleic acid. Accordingly, these
molecules can treat a disorder characterized by abnormal or undesired subunit
nucleic acid expression. This technique involves cleavage by means of
ribozymes
containing nucleotide sequences complementary to one or more regions in the
mRNA that attenuate the ability of the mRNA to be translated. Possible regions
include coding regions and particularly coding regions corresponding to the
functional activities of the ~i-subunit protein.
The ~i-subunit polynucleotides also provide vectors for gene therapy in
patients containing cells that are aberrant in ~i-subunit gene expression.
Thus,
recombinant cells, which include the patient's cells that have been engineered
ex vivo
and returned to the patient, are introduced into an individual where the cells
produce
the desired ~i-subunit protein to treat the individual.
The invention also encompasses kits for detecting the presence of a ~i-subunit
nucleic acid in a biological sample. For example, the kit can comprise
reagents such
as a labeled or labelable nucleic acid or agent capable of detecting ~i-
subunit nucleic
acid in a biological sample; means for determining the amount of ~i-subunit
nucleic
acid in the sample; and means for comparing the amount of (3-subunit nucleic
acid in
the sample with a standard. The compound or agent can be packaged in a
suitable
container. The kit can further comprise instructions for using the kit to
detect ~i-
subunit mRNA or DNA.
Vectors/Host Cells
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The invention also provides vectors containing the ~i-subunit polynucleotides
and to host cells containing the ~i-subunit polynucleotides. As described more
fully
below, vectors can be used for cloning or expression but are preferably used
for
expression of the ~i-subunit. Preferably expression systems include host cells
in
which both the a and j3 subunits are expressed. The term "vector" refers to a
vehicle,
preferably a nucleic acid molecule, that can transport the ~3-subunit
polynucleotides.
When the vector is a nucleic acid molecule, the ~i-subunit polynucleotides are
covalently linked to the vector nucleic acid. With this aspect of the
invention, the
vector includes a plasmid, single or double stranded phage, a single or double
stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC,
PAC,
YAC, OR MAC.
A vector can be maintained in the host cell as an extrachromosomal element
where it replicates and produces additional copies of the R-subunit
polynucleotides.
Alternatively, the vector may integrate into the host cell genome and produce
additional copies of the (3-subunit polynucleotides when the host cell
replicates.
The invention provides vectors for the maintenance (cloning vectors) or
vectors for expression (expression vectors) of the ~i-subunit polynucleotides.
The
vectors can function in procaryotic or eukaryotic cells or in both (shuttle
vectors).
Expression vectors contain cis-acting regulatory regions that are operably
linked in the vector to the ~i-subunit polynucleotides such that transcription
of the
polynucleotides is allowed in a host cell. The polynucleotides can be
introduced into
the host cell with a separate polynucleotide capable of affecting
transcription. Thus,
the second polynucleotide may provide a trans-acting factor interacting with
the cis-
regulatory control region to allow transcription of the ~i-subunit
polynucleotides from
the vector. Alternatively, a trans-acting factor may be supplied by the host
cell.
Finally, a trans-acting factor can be produced from the vector itself.
It is understood, however, that in some embodiments, transcription and/or
translation of the ~-subunit polynucleotides can occur in a cell-free system.
The regulatory sequence to which the polynucleotides described herein can
be operably linked include promoters for directing mRNA transcription. These .
include, but are not limited to, the left promoter from bacteriophage ?~, the
lac, TRP,
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and TAC promoters from E coli, the early and late promoters from SV40, the CMV
immediate early promoter, the adenovirus early and late promoters, and
retrovirus
long-terminal repeats.
In addition to control regions that promote transcription, expression vectors
may also include regions that modulate transcription, such as repressor
binding sites
and enhancers. Examples include the SV40 enhancer, the cytomegalovirus
immediate early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus
LTR enhancers.
In addition to containing sites for transcription initiation and control,
expression vectors can also contain sequences necessary for transcription
termination
and, in the transcribed region a ribosome binding site for translation. Other
regulatory control elements for expression include initiation and termination
codons
as well as polyadenylation signals. The person of ordinary skill in the art
would be
aware of the numerous regulatory sequences that are useful in expression
vectors.
Such regulatory sequences are described, for example, in Sambrook et al.,
Molecular
Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press,
Cold
Spring Harbor, NY (1989).
A variety of expression vectors can be used to express a (3-subunit
polynucleotide. Such vectors include chromosomal, episomal, and virus-derived
vectors, for example vectors derived from bacterial plasmids, from
bacteriophage,
from yeast episomes, from yeast chromosomal elements, including yeast
artificial
chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40,
Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and
retroviruses.
Vectors may also be derived from combinations of these sources such as those
derived from plasmid and bacteriophage genetic elements, eg. cosmids and
phagemids. Appropriate cloning and expression vectors for prokaryotic and
eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A
Laboratory
Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
( 1989).
The regulatory sequence may provide constitutive expression in one or more
host cells (i.e. tissue specific) or may provide for inducible expression in
one or more
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cell types such as by temperature, nutrient additive, or exogenous factor such
as a
hormone or other ligand. A variety of vectors providing for constitutive and
inducible expression in prokaryotic and eukaryotic hosts are well known to
those of
ordinary skill in the art.
The p-subunit polynucleotides can be inserted into the vector nucleic acid by
well-known methodology. Generally, the DNA sequence that will ultimately be
expressed is joined to an expression vector by cleaving the DNA sequence and
the
expression vector with one or more restriction enzymes and then ligating the
fragments together. Procedures for restriction enzyme digestion and ligation
are well
known to those of ordinary skill in the art.
The vector containing the appropriate polynucleotide can be introduced into
an appropriate host cell for propagation or expression using well-known
techniques.
Bacterial cells include, but are not limited to, E. toll, Streptomyces, and
Salmonella
typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect
cells such
as Drosophila, animal cells such as COS and CHO cells, and plant cells.
As described herein, it may be desirable to express the polypeptide as a
fusion protein. Accordingly, the invention provides fusion vectors that allow
for the
production of the ~3-subunit polypeptides. Fusion vectors can increase the
expression
of a recombinant protein, increase the solubility of the recombinant protein,
and aid
in the purification of the protein by acting for example as a ligand for
affinity
purification. A proteolytic cleavage site may be introduced at the junction of
the
fusion moiety so that the desired polypeptide can ultimately be separated from
the
fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa,
thrombin, and enterokinase. Typical fusion expression vectors include pGEX
(Smith
et al. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and
pRITS (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the target
recombinant
protein. Examples of suitable inducible non-fusion E. toll expression vectors
include
pTrc (Amann et al., Gene 69:301-315 (1988)) and pET l ld (Studier et al., Gene
Expression Technology.' Methods in Enrymolo~ 185:60-89 (1990)).
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Recombinant protein expression can be maximized in a host bacteria by
providing a genetic background wherein the host cell has an impaired capacity
to
proteolytically cleave the recombinant protein. (Gottesman, S., Gene
Expression
Technology. Methods in Enzymology 185, Academic Press, San Diego, California
(1990) 119-128). Alternatively, the sequence of the polynucleotide of interest
can
be altered to provide preferential codon usage for a specific host cell, for
example E
coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).
The ~i-subunit polynucleotides can also be expressed by expression vectors
that are operative in yeast. Examples of vectors for expression in yeast e.g.,
S cerevisiae include pYepSecl (Baldari, et al., EMBO J. 6:229-234 (1987)),
pMFa
(Kurjan et al., Cell 30:933-943 (1982)), pJRY88 (Schultz et al., Gene 54:113-
123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, CA).
The ~i-subunit polynucleotides can also be expressed in insect cells using,
for
example, baculovirus expression vectors. Baculovirus vectors available for
expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series
(Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow
et
al., Virology 170:31-39 (1989)).
In certain embodiments of the invention, the polynucleotides described herein
are expressed in mammalian cells using mammalian expression vectors. Examples
of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840 (1987))
and pMT2PC (Kaufman et al., EMBOJ. 6:187-195 (1987)).
The expression vectors listed herein are provided by way of example only of
the well-known vectors available to those of ordinary skill in the art that
would be
useful to express the ~i-subunit polynucleotides. The person of ordinary skill
in the
art would be aware of other vectors suitable for maintenance propagation or
expression of the polynucleotides described herein. These are found for
example in
Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory
Manual. 2nd, ed , Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, 1989.
The invention also encompasses vectors in which the nucleic acid sequences
described herein are cloned into the vector in reverse orientation, but
operably linked
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to a regulatory sequence that permits transcription of antisense RNA. Thus, an
antisense transcript can be produced to all, or to a portion, of the
polynucleotide
sequences described herein, including both coding and non-coding regions.
Expression of this antisense RNA is subject to each of the parameters
described
above in relation to expression of the sense RNA (regulatory sequences,
constitutive
or inducible expression, tissue-specific expression).
The invention also relates to recombinant host cells containing the vectors
described herein. Host cells therefore include prokaryotic cells, lower
eukaryotic
cells such as yeast, other eukaryotic cells such as insect cells, and higher
eukaryotic
cells such as mammalian cells.
The recombinant host cells are prepared by introducing the vector constructs
described herein into the cells by techniques readily available to the person
of
ordinary skill in the art. These include, but are not limited to, calcium
phosphate
transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection, lipofection, and other
techniques such as those found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, 1989).
Host cells can contain more than one vector. Thus, different nucleotide
sequences can be introduced on different vectors of the same cell. Similarly,
the ~3-
subunit polynucleotides can be introduced either alone or with other
polynucleotides
that are not related to the ~i-subunit polynucleotides such as those providing
trans-
acting factors for expression vectors. When more than one vector is introduced
into a
cell, the vectors can be introduced independently, co-introduced or joined to
the ~i-
subunit polynucleotide vector.
In the case of bacteriophage and viral vectors, these can be introduced into
cells as packaged or encapsulated virus by standard procedures for infection
and
transduction. Viral vectors can be replication-competent or replication-
defective. In
the case in which viral replication is defective, replication will occur in
host cells
providing functions that complement the defects.
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Vectors generally include selectable markers that enable the selection of the
subpopulation of cells that contain the recombinant vector constructs. The
marker
can be contained in the same vector that contains the polynucleotides
described
herein or may be on a separate vector. Markers include tetracycline or
ampicillin-
resistance genes for prokaryotic host cells and dihydrofolate reductase or
neomycin
resistance for eukaryotic host cells. However, any marker that provides
selection for
a phenotypic trait will be effective.
While the mature proteins can be produced in bacteria, yeast, mammalian
cells, and other cells under the control of the appropriate regulatory
sequences, cell-
free transcription and translation systems can also be used to produce these
proteins
using RNA derived from the DNA constructs described herein.
Where secretion of the polypeptide is desired, appropriate secretion signals
are incorporated into the vector. The signal sequence can be endogenous to the
~i-
subunit polypeptides or heterologous to these polypeptides.
Where the polypeptide is not secreted into the medium, the protein can be
isolated from the host cell by standard disruption procedures, including
freeze thaw,
sonication, mechanical disruption, use of lysing agents and the like. The
polypeptide
can then be recovered and purified by well-known purification methods
including
ammonium sulfate precipitation, acid extraction, anion or cationic exchange
chromatography, phosphocellulose chromatography, hydrophobic-interaction
chromatography, affinity chromatography, hydroxylapatite chromatography,
lectin
chromatography, or high performance liquid chromatography.
It is also understood that depending upon the host cell in recombinant
production of the polypeptides described herein, the polypeptides can have
various
glycosylation patterns, depending upon the cell, or maybe non-glycosylated as
when
produced in bacteria. In addition, the polypeptides may include an initial
modified
methionine in some cases as a result of a host-mediated process.
Uses of Vectors and Host Cells
The host cells expressing the polypeptides described herein, and particularly
recombinant host cells, have a variety of uses. First, the cells are useful
for
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producing p-subunit proteins or polypeptides that can be further purified to
produce
desired amounts of ~i-subunit protein or fragments. Thus, host cells
containing
expression vectors are useful for polypeptide production.
Host cells are also useful for conducting cell-based assays involving the ~i-
subunit or (3-subunit fragments. Thus, a recombinant host cell expressing a
native ~i-
subunit is useful to assay for compounds that stimulate or inhibit ~3-subunit
function.
This includes ligand binding, gene expression at the level of transcription or
translation, a-subunit interaction, and ability to be phosphorylated.
Accordingly, in preferred embodiments the host cells express both the a and
~i subunits or relevant portions thereof. Therefore, cell-based and cell-free
assays are
provided in which both a and ~i subunits (or relevant portions thereof)
provide assays
useful for detection of ~i-subunit function. In a preferred embodiment, the
invention
provides a cell-based assay in which the cell expresses both the a and ~i
subunits.
Assay end points include ligand binding, a-subunit association or activation,
channel currents, phosphorylation, and conformational changes in either the a
or ~i
subunit. Interaction of the a and ~i subunit can be measured in assays based
on dual
label energy transfer, methods in which reactants are separately labeled with
an
energy transfer donor and acceptor, such that energy transfer results when the
donor
and acceptor are brought into close proximity to each other, producing a
detectable
lifetime change. Assay methods for detection of a complex formed between the a
and (3 subunits include determining fluorescence emission or fluorescence
quenching
or other energy transfer between labels on the two subunits. One example is a
fluorescein homoquenching method in which a subunit is labeled with
fluorescein
positioned such that when the other subunit is bound the fluorescein molecules
quench one another and the fluorescence of the solution decreases. This
analytical
technique is well-known and within the skill of those in the art. See, for
example,
U.S. Patent No. 5,631,169; U.S. Patent No. 5,506,107; U.S. Patent No.
5,716,784;
and U.S. Patent No. 5,763,189.
Host cells are also useful for identifying subunit mutants in which these
functions are affected. If the mutants naturally occur and give rise to a
pathology,
host cells containing the mutations are useful to assay compounds that have a
desired
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effect on the mutant ~i-subunit (for example, stimulating or inhibiting
function)
which may not be indicated by their effect on the native ~i-subunit.
Recombinant host cells are also useful for expressing the chimeric
polypeptides described herein to assess compounds that activate or suppress
activation by means of a heterologous intracellular or extracellular domain.
Alternatively, one or more heterologous transmembrane domains can be used to
assess the effect of a desired extracellular domain on any given host cell. In
this
embodiment, a transmembrane domain compatible with the specific host cell is
used
to make the chimeric polypeptide.
Further, mutant (3-subunits can be designed in which one or more of the
various functions is engineered to be increased or decreased (i.e., ligand
binding or
a-subunit activation) and used to augment or replace ~i-subunit proteins in an
individual. Thus, host cells can provide a therapeutic benefit by replacing an
aberrant
(3-subunit or providing an aberrant (3-subunit that provides a therapeutic
result. In
one embodiment, the cells provide ~i-subunits that are abnormally active.
In another embodiment, the cells provide ~i-subunits that are abnormally
inactive. These ~i-subunits can compete with endogenous ~i-subunits in the
individual.
In another embodiment, cells expressing ~3-subunits that cannot be activated,
are introduced into an individual in order to compete with endogenous (3-
subunits for
ligand or a-subunit. For example, in the case in which excessive ligand is
part of a
treatment modality, it may be necessary to inactivate this ligand at a
specific point in
treatment. Providing cells that compete for the ligand, but which cannot be
affected
by ~i-subunit activation would be beneficial.
Homologously recombinant host cells can also be produced that allow the in
situ alteration of endogenous ~i-subunit polynucleotide sequences in a host
cell
genome. This technology is more fully described in WO 93/09222, WO 91/12650
and U.S. 5,641,670. Briefly, specific polynucleotide sequences corresponding
to the
(3-subunit polynucleotides or sequences proximal or distal to a ~i-subunit
gene are
allowed to integrate into a host cell genome by homologous recombination where
expression of the gene can be affected. In one embodiment, regulatory
sequences are
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introduced that either increase or decrease expression of an endogenous
sequence.
Accordingly, a (3-subunit protein can be produced in a cell not normally
producing it,
or increased expression of a-subunit protein can result in a cell normally
producing
the protein at a specific level. Alternatively, the entire gene can be
deleted. Still
further, specific mutations can be introduced into any desired region of the
gene to
produce mutant ~i-subunit proteins. Such mutations could be introduced, for
example, into the specific functional regions such as the ligand-binding site
or the a-
subunit interaction site.
In one embodiment, the host cell can be a fertilized oocyte or embryonic stem
cell that can be used to produce a transgenic animal containing the altered (3-
subunit
gene. Alternatively, the host cell can be a stem cell or other early tissue
precursor
that gives rise to a specific subset of cells and can be used to produce
transgenic
tissues in an animal. See also Thomas et al., Cell 51:503 (1987) for a
description of
homologous recombination vectors. The vector is introduced into an embryonic
stem cell line (e.g., by electroporation) and cells in which the introduced
gene has
homologously recombined with the endogenous a-subunit gene is selected (see
e.g.,
Li, E. et al., Cell 69:915 (1992)). 'The selected cells are then injected into
a
blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see
e.g.,
Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach,
E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can
then
be implanted into a suitable pseudopregnant female foster animal and the
embryo
brought to term. Progeny harboring the homologously recombined DNA in their
germ cells can be used to breed animals in which all cells of the animal
contain the
homologously recombined DNA by germline transmission of the transgene.
Methods for constructing homologous recombination vectors and homologous
recombinant animals are described further in Bradley, A. ( 1991 ) Current
Opinion in
Biotechnology 2:823-829 and in PCT International Publication Nos. WO 90/11354;
WO 91/01140; and WO 93/04169.
The genetically engineered host cells can be used to produce non-human
transgenic animals. A transgenic animal is preferably a mammal, for example a
rodent, such as a rat or mouse, in which one or more of the cells of the
animal include
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a transgene. A transgene is exogenous DNA which is integrated into the genome
of a
cell from which a transgenic animal develops and which remains in the genome
of
the mature animal in one or more cell types or tissues of the transgenic
animal.
These animals are useful for studying the function of a ~i-subunit protein and
identifying and evaluating modulators of p-subunit protein activity.
Other examples of transgenic animals include non-human primates, sheep,
dogs, cows, goats, chickens, and amphibians.
In one embodiment, a host cell is a fertilized oocyte or an embryonic stem
cell into which ~i-subunit polynucleotide sequences have been introduced.
A transgenic animal can be produced by introducing nucleic acid into the
male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral
infection, and
allowing the oocyte to develop in a pseudopregnant female foster animal. Any
of the
~i-subunit nucleotide sequences can be introduced as a transgene into the
genome of a
non-human animal, such as a mouse.
Any of the regulatory or other sequences useful in expression vectors can
form part of the transgenic sequence. This includes intronic sequences and
polyadenylation signals, if not already included. A tissue-specific regulatory
sequences) can be operably linked to the transgene to direct expression of the
(3-
subunit protein to particular cells.
Methods for generating transgenic animals via embryo manipulation and
microinjection, particularly animals such as mice, have become conventional in
the
art and are described, for example, in U.S. Patent Nos. 4,736,866 and
4,870,009,
both by Leder et al., U.S. Patent No. 4,873,191 by Wagner et al. and in Hogan,
B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1986). Similar methods are used for production of other
transgenic animals. A transgenic founder animal can be identified based upon
the
presence of the transgene in its genome and/or expression of transgenic mRNA
in
tissues or cells of the animals. A transgenic founder animal can then be used
to breed
additional animals carrying the transgene. Moreover, transgenic animals
carrying a
transgene can further be bred to other transgenic animals carrying other
transgenes.
A transgenic animal also includes animals in which the entire animal or
tissues in the
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animal have been produced using the homologously recombinant host cells
described
herein.
In another embodiment, transgenic non-human animals can be produced
which contain selected systems which allow for regulated expression of the
transgene. One example of such a system is the crelloxP recombinase system of
bacteriophage P1. For a description of the crelloxP recombinase system, see,
e.g.,
Lakso et al., PNAS 89:6232-6236 (1992). Another example of a recombinase
system
is the FLP recombinase system of S. cereviriae (O'Gorman et al. Science
251:1351-
1355 (1991). If a crelloxP recombinase system is used to regulate expression
of the
transgene, animals containing transgenes encoding both the Cre recombinase and
a
selected protein is required. Such animals can be provided through the
construction
of "double" transgenic animals, e.g., by mating two transgenic animals, one
containing a transgene encoding a selected protein and the other containing a
transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be
produced according to the methods described in Wilmut, I. et al., Nature
385:810-
813 (1997) and PCT International Publication Nos. WO 97/07668 and WO
97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal
can be
isolated and induced to exit the growth cycle and enter Go phase. The
quiescent cell
can then be fused, e.g., through the use of electrical pulses, to an
enucleated oocyte
from an animal of the same species from which the quiescent cell is isolated.
The
reconstructed oocyte is then cultured such that it develops to morula or
blastocyst and
then transferred to pseudopregnant female foster animal. The offspring borne
of this
female foster animal will be a clone of the animal from which the cell, e.g.,
the
somatic cell, is isolated.
Transgenic animals containing recombinant cells that express the
polypeptides described herein are useful to conduct the assays described
herein in an
in vivo context. Accordingly, the various physiological factors that are
present in
vivo and that could effect ligand binding, a-subunit activation, and ability
to be
phosphorylated may not be evident from in vitro cell-free or cell-based
assays.
Accordingly, it is useful to provide non-human transgenic animals to assay in
vivo ~i-
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subunit function, including ligand and a-subunit interaction, the effect of
specific
mutant ~i-subunits on the a-subunit, channel function, and ligand interaction,
and the
effect of chimeric subunits or channels. It is also possible to assess the
effect of null
mutations, that is mutations that substantially or completely eliminate one or
more (3-
subunit functions.
Pharmaceutical Compositions
The ~i-subunit nucleic acid molecules, protein {particularly fragments such as
the various domains), modulators of the protein, and antibodies (also referred
to
herein as "active compounds") can be incorporated into pharmaceutical
compositions
suitable for administration to a subject, e.g., a human. Such compositions
typically
comprise the nucleic acid molecule, protein, modulator, or antibody and a
pharmaceutically acceptable carrier.
As used herein the language "pharmaceutically acceptable Garner" is intended
to include any and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the like,
compatible
with pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except insofar as
any
conventional media or agent is incompatible with the active compound, such
media
can be used in the compositions of the invention. Supplementary active
compounds
can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be
compatible with its intended route of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral
{e.g., inhalation), transdermal (topical), transmucosal, and rectal
administration.
Solutions or suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent such as
water for
injection, saline solution, fixed oils, polyethylene glycols, glycerine,
propylene
glycol or other synthetic solvents; antibacterial agents such as benzyl
alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate;
chelating
agents such as ethylenediaminetetraacetic acid; buffers such as acetates,
citrates or
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phosphates and agents for the adjustment of tonicity such as sodium chloride
or
dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or
sodium hydroxide. The parenteral preparation can be enclosed in ampules,
disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous solutions (where water soluble) or dispersions and sterile powders for
the
extemporaneous preparation of sterile injectable solutions or dispersion. For
intravenous administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor ELT"" (BASF, Parsippany, NJ) or phosphate
buffered
saline (PBS). In all cases, the composition must be sterile and should be
fluid to the
extent that easy syringability exists. It must be stable under the conditions
of
manufacture and storage and must be preserved against the contaminating action
of
microorganisms such as bacteria and fungi. The can ier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol (for
example,
glycerol, propylene glycol, and liquid polyetheylene glycol, and the like),
and
suitable mixtures thereof. The proper fluidity can be maintained, for example,
by the
use of a coating such as lecithin, by the maintenance of the required particle
size in
the case of dispersion and by the use of surfactants. Prevention of the action
of
microorganisms can be achieved by various antibacterial and antifungal agents,
for
example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the
like. In
many cases, it will be preferable to include isotonic agents, for example,
sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought about by
including in the composition an agent which delays absorption, for example,
aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound (e.g., a ~i-subunit protein or anti-~i-subunit antibody) in the
required
amount in an appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into a sterile
vehicle
which contains a basic dispersion medium and the required other ingredients
from
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those enumerated above. In the case of sterile powders for the preparation of
sterile
injectable solutions, the preferred methods of preparation are vacuum drying
and
freeze-drying which yields a powder of the active ingredient plus any
additional
desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier.
They can be enclosed in gelatin capsules or compressed into tablets. For oral
administration, the agent can be contained in enteric forms to survive the
stomach or
further coated or mixed to be released in a particular region of the GI tract
by known
methods. For the purpose of oral therapeutic administration, the active
compound
can be incorporated with excipients and used in the form of tablets, troches,
or
capsules. Oral compositions can also be prepared using a fluid carrier for use
as a
mouthwash, wherein the compound in the fluid carrier is applied orally and
swished
and expectorated or swallowed. Pharmaceutically compatible binding agents,
and/or
adjuvant materials can be included as part of the composition. The tablets,
pills,
I 5 capsules, troches and the like can contain any of the following
ingredients, or
compounds of a similar nature: a binder such as microcrystalline cellulose,
gum
tragacanth or gelatin; an excipient such as starch or lactose, a
disintegrating agent
such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium
stearate
or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent
such as
sucrose or saccharin; or a flavoring agent such as peppermint, methyl
salicylate, or
orange flavoring.
For administration by inhalation, the compounds are delivered in the form of
an aerosol spray from pressured container or dispenser which contains a
suitable
propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transrr~ucosal or transdermal means.
For transmucosal or transdermal administration, penetrants appropriate to the
barner
to be permeated are used in the formulation. Such penetrants are generally
known in
the art, and include, for example, for transmucosal administration,
detergents, bile
salts, and fusidic acid derivatives. Transmucosal administration can be
accomplished
through the use of nasal sprays or suppositories. For transdennal
administration, the
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active compounds are formulated into ointments, salves, gels, or creams as
generally
known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with
conventional suppository bases such as cocoa butter and other glycerides) or
retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with earners that will
protect the compound against rapid elimination from the body, such as a
controlled
release formulation, including implants and microencapsulated delivery
systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl
acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic
acid.
Methods for preparation of such formulations will be apparent to those skilled
in the
art. The materials can also be obtained commercially from Alza Corporation and
Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted
to
infected cells with monoclonal antibodies to viral antigens) can also be used
as
pharmaceutically acceptable carriers. These can be prepared according to
methods
known to those skilled in the art, for example, as described in U.S. Patent
No.
4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in
dosage unit form for ease of administration and uniformity of dosage. Dosage
unit
form as used herein refers to physically discrete units suited as unitary
dosages for
the subject to be treated; each unit containing a predetermined quantity of
active
compound calculated to produce the desired therapeutic effect in association
with the
required pharmaceutical carrier. The specification for the dosage unit forms
of the
invention are dictated by and directly dependent on the unique characteristics
of the
active compound and the particular therapeutic effect to be achieved, and the
limitations inherent in the art of compounding such an active compound for the
treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and
used as gene therapy vectors. Gene therapy vectors can be delivered to a
subject by,
for example, intravenous injection, local administration (U.S. 5,328,470) or
by
stereotactic injection (see e.g., Chen et al., PNAS 91:3054-3057 (1994)). The
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pharmaceutical preparation of the gene therapy vector can include the gene
therapy
vector in an acceptable diluent, or can comprise a slow release matrix in
which the
gene delivery vehicle is imbedded. Alternatively, where the complete gene
delivery
vector can be produced intact from recombinant cells, e.g. retroviral vectors,
the
pharmaceutical preparation can include one or more cells which produce the
gene
delivery system.
The pharmaceutical compositions can be included in a container, pack, or
dispenser together with instructions for administration.
I 0 EXPERIMENTAL
The pore-forming a-subunit of the high conductance calcium-activated
potassium channel (maxi-K) has been identified and cloned in human (called
hSlo}
and mouse (called mSlo). See, for example, Knaus, J. Biol. Chem.269:3921-3924
(1994), and Butler et al., Science 261:221-224 (1993). Experiments were
conducted
to examine the functional role of the novel human calcium-activated potassium
channel ~i-subunit C7F2 (SEQ ID NO 2) in the high conductance calcium-
activated
potassium channel maxi-K.
These experiments show that a physical interaction of C7F2 with hSlo and
mSlo modifies the channel activity of maxi-K, supporting the claim that C7F2
is a
functional ~i-subunit of maxi-K.
Example 1: Physical Association of (:7F2 with mSlo
The open reading frame of C7F2 (nucleotides 502-1131 of SEQ ID NO 2)
was cloned into the pcDNA3.1/VS/His-TOPO vector (Invitrogen) to provide a VS
epitope tag. This vector and a vector containing mSlo were transiently co-
transfected
into HEK293 cells with lipofectamine or Fugene. mSlo was immunoprecipitated
with antibodies directed against the a-subunit. The immunoprecipitates were
subjected to Western blotting with monoclonal antibody directed against the VS
epitope tag to reveal the presence of the VS-tagged C7F2.
These experiments demonstrate that human C7F2 can associate with mSlo
(data not shown), suggesting a physical interaction of C7F2 with the pore-
forming a-
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subunit of maxi-K. These results confirm the claim that C7F2 is a ~i-subunit
for
maxi-K.
Example 2: Electrophysiological Consequences of Association of C7F2
with hSlo and mSlo on the Channel Activity of Maxi-K
The open reading frame of C7F2 was cloned into the pIRES-EGFP vector
(Clontech) to express both C7F2 and green fluorescent protein (GFP) in
transfected
cells. This vector and vectors containing either hSlo or mSlo were transiently
co-
transfected into HEK293 cells with lipofectamine or Fugene. Cells were
selected for
recording based on the expression of GFP.
Activation and deactivation kinetics of the mouse maxi-K channel (mSlo)
were dramatically different when expressed alone or when co-expressed with
C7F2
(Figure 6). These inside-out patch-clamp experiments revealed that co-
expression of
I S C7F2 with mSlo (horizontal bars labeled mSlo + C71?2) dramatically
increases the
time constants of activation (mSlo + C7F2 activation) and deactivation (mSlo +
C7F2 deactivation) of the mouse maxi-K when compared to expression of mSlo
alone (horizontal bars labeled mSlo activation and mSlo deactivation,
respectfully).
Similar effects were seen for the human maxi-K hSlo.
In the presence of 3 pM Ca~', C7F2 co-expression with mSlo causes a
hyperpolarizing (leftward) shift of 20 mV of half maximal channel activation,
suggesting increased sensitivity of the mouse maxi-K channel to calcium ions
(Figure 7). This is the typical behavior of the previously characterized ~3-
subunit.
However, when C7F2 is co-expressed with hSlo, there is a 20-50 mV depolarizing
(rightward) shift of half maximal channel activation, suggesting decreased
sensitivity
of the human maxi-K channel to calcium ions (Figure 8). This is a unique,
novel
behavior of C7F2 compared to the previously characterized (3-subunit.
Functional interaction of C7F2 with mSlo and hSlo, leading to changes in
activation and deactivation kinetics of the maxi-K channel along with shifts
in the
half maximal channel activation in response to calcium, confirms the claim
that
C7F2 is a ~i-subunit for maxi-K.
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This invention may be embodied in many different forms and should not be
construed as limited to the embodiments set forth herein; rather, these
embodiments
are provided so that this disclosure will fully convey the invention to those
skilled in
the art. Many modifications and other embodiments of the invention will come
to
mind in one skilled in the art to which this invention pertains having the
benefit of
the teachings presented in the foregoing description. .Although specific terms
are
employed, they are used as in the art unless otherwise indicated.
All publications and patent applications mentioned in the specification are
indicative of the level of those skilled in the art to which this invention
pertains. All
publications and patent applications are herein incorporated by reference to
the same
extent as if each individual publication or patent application was
specifically and
individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity of understanding, it will
be
obvious that certain changes and modifications may be practiced within the
scope of
the appended claims.
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SEQUENCE LISTING
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CA 02335643 2001-O1-16
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gaggacaaga gcatccggct cggcttgttt ctcatcatct ccggcgtcgt gtcgctcttc 600
atcttcggct tctgctggct gagtcccgcg ctgcaggatc tgcaagccac ggaggccaat 660
tgcacggtgc tgtcggtgca gcagatcggc gaggtgttcg agtgcacctt cacctgtggc 720
gccgactgca ggggcacctc gcagtacccc tgcgtccagg tctacgtgaa caactctgag 780
tccaactcta gggcgctgct gcacagcgac gagcaccagc tcctgaccaa ccccaagtgc 840
tcctatatcc ctccctgtaa gagagaaaat cagaagaatt tggaaagtgt catgaattgg 900
caacagtact ggaaagatga gattggttcc cagccattta cttgctattt taatcaacat 960
caaagaccag atgatgtgct tctgcatcgc actcatgatg agattgtcct cctgcattgc 1020
2
CA 02335643 2001-O1-16
WO 00/06183 PCT/US99/16949
ttcctctggc ccctggtgac atttgtggtg ggcgttctca ttgtggtcct gaccatctgt 1080
gccaagagct tggcggtcaa ggcggaagcc atgaagaagc gcaagttctc ttaaagggga 1140
aggaggcttg tagaaagcaa agtacagaag ctgtactcat cggcacgcgt ccacctgcgg 1200
aacctgtgtt tcctggcgca ggagatggac agggccacga caggc~ctctg agaggctcat 1260
ccctcagtgg caacagaaac aggcacaact ggaagacttg gaacctcaaa gcttgtattc 1320
catctgctgt agcaatggct aaagggtcaa gatcttagct gtatggagta actatttcag 1380
aaaaccctat aagaagttca ttttctttca aaagtaacag tatattattt gtacagtgta 1940
gtatacaaac cattatgatt tatgctactt aaaaatatta aaatagagtg gtctgtgtta 1500
ttttctattt ccttttttat gcttagaaca ccagggttta aaaaaaaaaa aaaagggcgg 1560
acatctgggt ctcatttgct tctgctaggt taaactttta cttgacaa 1608
<210> 3
<211> 211
<212> PRT
<213> Homo sapiens
<220>
<221> UNSURE
<222> (1)..(211)
<223> Xaa = any amino acid
<400> 3
Met Val Lys Lys Leu Val Met Ala Gln Lys Xaa Arg Gly Glu Thr Arg
1 5 10 15
Ala Leu Cys Leu Gly Val Thr Met Val Val Cys Ala Val Ile Thr Tyr
20 25 30
Tyr Ile Leu Val Thr Thr Val Leu Xaa Pro Leu Tyr Gln Lys Ser Val
35 40 45
Trp Thr Gln Glu Ser Lys Cys His Leu Ile Glu Thr Asn Xaa Ile Arg
50 55 60
Asp Gln Glu Glu Xaa Leu Lys Xaa Xaa Gly Lys Lys Xaa Xaa Xaa Val
65 70 75 80
Pro Gln Tyr Pro Cys Leu Xaa Xaa Trp Val Asn Val Ser Ala Ala Gly
85 90 95
Arg Trp Ala Val Leu Tyr His Thr Glu Asp Thr Arg Asp Gln Asn Gln
100 105 110
Gln Cys Ser Tyr Ile Pro Gly Ser Val Asp Asn Tyr Gln Thr Ala Arg
115 120 125
Ala Asp Val Glu Lys Val Arg Ala Lys Phe Gln Glu Gln Xaa Xaa Xaa
130 135 190
3
CA 02335643 2001-O1-16
WO 00/06183 PCT/US99/16949
Gln Val Phe Tyr Cys Phe Ser Ala Pro Arg Gly Asn Glu Thr Ser Val
145 150 155 160
Leu Phe Gln Arg Leu Tyr Gly Pro Gln AIa Leu Leu Phe Ser Leu Phe
165 170 175
Trp Pro Thr Leu Leu Leu Thr Gly Gly Leu Leu Ile I:le Ala Met Val
180 185 190
Lys Ser Asn Gln Tyr Leu Ser Ile Leu Ala Ala Gln Lys Xaa Xaa Xaa
195 200 205
Xaa Xaa Xaa
210
<210> 4
<211> 213
<212> PRT
<213> Phasianidae gen. sp.
<220>
<221> UNSURE
<222> (1)..(213)
<223> Xaa = any amino acid
<400> 4
Met Leu Ala Lys Lys Leu Val Thr Ala Gln Lys Arg Gly Glu Thr Arg
1 5 10 15
Ala Leu Cys Leu Gly Leu Gly Met Val Ala Cys Ser Met Met Met Tyr
20 25 30
Phe Phe Ile Gly Ile Thr Xaa Ile Val Pro Phe Tyr Thr Lys Ser Val
35 90 45
Trp Thr Thr Glu Thr Ile Cys Lys Val Leu Lys Ala Asn Xaa Ile Lys
50 55 60
Asp Lys Thr His Cys Thr Asn Ser Glu Gly Ser Glu Asp Glu Asp Ile
65 70 75 80
Phe His Tyr Pro Cys Leu Gln Val Trp Val Asn Leu Thr Ala Ser Gly
85 90 95
Gln Glu Val Met Leu Tyr His Thr Xaa Glu Asp Thr Leu Glu Arg Asn
100 105 110
9
CA 02335643 2001-O1-16
WO 00/06183 PCT/US99/16949
Pro Lys Cys Ser Tyr Val Pro Xaa Xaa Gly Xaa Xaa Asn Ser Glu Asn
115 120 125
Ser Lys Glu Val Lys Ala Arg Ile Glu Thr Ile Ala Ser Asn Phe Lys
130 135 190
Lys Tyr Gln Thr Phe Pro Cys Tyr Tyr Asp Pro Gly Gly Met Gln Thr
145 150 155 160
Asn Val Ile Leu Ser Arg Leu Tyr Pro Pro Lys Gly Leu Leu Phe Thr
165 170 175
Phe Leu Trp Pro Thr Leu Met Phe Thr Gly Gly Cys Leu Ile Ile Val
180 185 190
Leu Val Lys Ile Ser Gln Tyr Phe Ser Val Xaa Xaa Xaa Xaa Leu Ser
195 200 205
Ala Arg Gln Xaa Xaa
210
