Note: Descriptions are shown in the official language in which they were submitted.
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NUCLEIC ACIDS ENCODING A
FUNCTIONAL HUMAN PURINORECEPTOR P2X2
AND METHODS OF PRODUCTION AND USE THEREOF
Technical Field
The invention relates generally to receptor proteins and to DNA and RNA
molecules encoding therefor. In particular, the invention relates to a nucleic
acid
sequence that encodes a human receptor P2X2. The invention also relates to
methods of using the receptor encoded thereby to identify compounds that
interact
s with it. This invention further relates to compounds which act as
antagonists and
agonists to compounds which have reactivity with the P2X2 receptor and methods
utilized in determining said reactivity. The invention also involves
therapeutic uses
involving aspects of this receptor.
to Background of the Invention
P2 receptors have been generally categorized as either metabotropic
nucleotide receptors or ionotropic receptors for extrace0ular nucleotides.
Metabotropic nucleotide receptors (usually designated P2Y or P2Y~, where "n"
is a
subscript integer indicating subtype) are believed to differ from ionotropic
receptors
~s (usually designated P2X or P2X~) in that they are based on a different
fundamental
means of transmembrane signal transduction: P2Y receptors operate through a G
protein-coupled system, while P2X receptors are ligand-gated ion channels. The
ligand for these P2X receptors is ATP, and/or other natural nucleotides, for
example,
ADP, UTP, UDP, or synthetic nucleotides, for example 2-methylthioATP.
2o At least seven P2X receptors, and the cDNA sequences encoding them, have
been identified to date. P2X, cDNA was cloned from the smooth muscle of the
rat vas
deferens (Valera et al. (1994) Nature 371:516-519) and P2X2 cDNA was cloned
from
PC12 cells (Brake et al. (1994) Nature 371:519-523}. Five other P2X receptors
have
been found in cDNA libraries by virtue of their sequence similarity to P2X,
and P2X2
2s (P2X3: Lewis et al. (1995) Nature 377:432-435, Chen et al. (1995) Nature
377:428-
431; P2X4: Buell et al. (1996) EMBO J. 15:55-62, Seguela et al. (1996) J.
Neurosci.
16:448-455, Bo et al. (1995) FEBS Lett. 375:129-133, Soto et al. (1996) Proc.
Natl.
Acad. Sci. USA 93:3684-3688, Wang et al. (1996) Biochem. Biophys. Res.
Commun.220:196-202; P2X5: Collo et al. (1996} J. Neurosci. 16:2495-2507,
Garcia-
3o Guzman et al. (1996) FEBS Lett. 388:123-127; P2X6: Collo ef al. (1996),
supra, Soto
et al. (1996) Biochem. Biophys. Res. Commun. 223:456-460; P2X7: Surprenant et
al.
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2
(1996) Science 272:735-738). For a comparison of the amino acid sequences of
rat
P2X receptors see Buell et al. (1996) Eur. J. Neurosci. 8:2221-2228.
Native P2X receptors form rapidly activated, nonselective cationic channels
that are activated by ATP. Rat P2X, and rat P2X2 have equal permeability to
Na+ and
s K+ but significantly less to Cs+. The channels formed by the P2X receptors
generally
have high Ca2+ permeability (P~a/PNa =4). The cloned rat P2X,, P2X2 and P2X4
receptors exhibit the same permeability for Ca2+ observed with native
receptors.
However, the mechanism by which P2X receptors form an ionic pore or bind ATP
is
not known.
io A variety of tissues and cell types, including epithelial, immune, muscle
and
neuronal, express at least one form of P2X receptor. The widespread
distribution of
P2X4 receptors in the rat central nervous system suggests a role for P2X;
mediated
events in the central nervous system. However, study of the role of individual
P2X
receptors is hampered by the lack of receptor subtype-specific agonists and
is antagonists. For example, one agonist useful for studying ATP-gated
channels is a,~3-
methylene-ATP (a,~imeATP). However, the P2X receptors display differential
sensitivity to the agonist with P2X, and P2X2 being a,~3meATP-sensitive and
insensitive, respectively. Furthermore, binding of a,~imeATP to P2X receptors
does
not always result in channel opening. The predominant forms of P2X receptors
in the
2o rat brain, P2X4 and P2X6 receptors, cannot be blocked by suramin or PPADS.
These
two forms of the P2X receptor are also not activated by a,~imeATP and are,
thus,
intractable to study with currently available pharmacological tools.
A therapeutic role for P2 receptors has been suggested, for example, for
cystic
fibrosis (Boucher et al. (1995) in: Belardinelli et al. (eds) Adenosine and
Adenine
2s Nucleotides: From Molecular Biology to Integrative Physiology (Kluwer
Acad., Nonrvell
MA) pp 525-532), diabetes (Loubatieres-Mariani et al. (1995) in: Belardinelli
et al.
(eds), supra, pp 337-345), immune and inflammatory diseases (Di Virgilio et
al. (1995)
in: Belardinelli et aI. (eds), supra, pp 329-335), cancer (Rapaport (1993)
Drug Dev.
Res. 28:428-431}, constipation and diarrhea (Milner et al. (1994) in: Kamm et
al.
30 (eds.) Constipation and Related Disorders: Pathophysiology and Management
in
Adults and Children (Wrightson Biomedical, Bristol) pp 41-49), behavioral
disorders
such as epilepsy, depression and aging-associated degenerative diseases
(Williams
(1993) Drug. Dev. Res. 28:438-444), contraception and sterility (Foresta et
al. (1992)
J. Biol. Chem. 257:19443-19447), and wound healing (Wang et al. (1990)
Biochim.
3s Biophys. Res. Commun. 166:251-258).
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Accordingly, there is a need in the art for specific agonists and antagonists
for
each P2X2 receptor subtype and, in particular, agents that will be effective
in vivo, as
well as for methods for identifying P2X2 receptor-specific agonist and
antagonist
compounds.
s
Summay of the Invention
The present invention relates to a human P2X2 receptor.
In one embodiment, a DNA molecule or fragments thereof is provided, wherein
the DNA molecule encodes a human P2X2 receptor or subunit thereof.
~o In another embodiment, a recombinant vector comprising such a DNA
molecule, or fragments thereof, is provided.
In another embodiment, the subject invention is directed to a human P2X2
receptor polypeptide, either alone or in multimeric form.
In still other embodiments, the invention is directed to messenger RNA
~s encoded by the DNA, recombinant host cells transformed or transfected with
vectors
comprising the DNA or fragments thereof, and methods of producing recombinant
P2X2 polypeptides using such cells.
In yet another embodiment, the invention is directed to a method of expressing
a human P2X2 receptor, or a subunit thereof, in a cell to produce the
resultant P2X2
2o containing receptor.
In a further embodiment, the invention is directed to a method of using such
cells to identify potentially therapeutic compounds that modulate or otherwise
interact
with the above P2X2-containing receptors.
In another embodiment, therapeutic uses involving a P2Xz modulator, such as
2s an ATP agonist or antagonist are contemplated.
These and other embodiments of the present invention will readily occur to
those of ordinary skill in the art in view of the disclosure herein.
Brief Description of the Drawings
3o FIGURE 1 depicts the partial sequence of a cDNA clone (SEQ ID N0:1)
derived from human fetal colon tissue which encodes a polypeptide with
homology to
a region of the rat P2X2 receptor;
FIGURE 2 depicts the full sequence of the cDNA clone (SEQ ID N0:2), the
underlined sequences sequence denotes overlap with the sequence of Figure 1;
3s FIGURE 3 a-a depicts primers designed to the cDNA of Figure 2 and
commercial RACE primers: 3a depicts GSP 1 (SEQ ID N0:3); 3b depicts GSP 2
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4
(SEQ ID N0:4); 3c depicts GSP 3 (SEQ ID N0:5); 3d depicts the anchor primer
(SEQ
ID N0:6); and 3e depicts the universal amplification primer (SEQ ID N0:7);
FIGURE 4 depicts the approximately 600 by product (SEQ ID N0:8) produced
by 5' RACE reactions using poly A RNA from human pituitary tissue;
s FIGURE 5 depicts genomic primers (SEQ ID N0:9 and SEQ ID N0:10);
FIGURE 6 depicts hP2X2 RT-PCR primers (SEQ ID N0:11 and SEQ ID
N0:12);
FIGURE 7 a-d depicts four species of cDNAs (SEQ ID N0:13; SEQ ID N0:14;
SEQ ID N0:15; and SEQ ID N0:16, respectively) containing intact open reading
io frames from the predicted initiation to termination sites;
FIGURE 8 a-d depicts the predicted amino acid sequences (SEQ ID N0:17;
SEQ ID N0:18; SEQ ID N0:19; and SEQ ID N0:20) encoded by the nucleotides of
Figure 7;
FIGURE 9 depicts an alignment of the predicted amino acid sequences (SEQ
is ID N0:17; SEQ ID N0:18; SEQ ID N0:19; and SEQ ID N0:20); and
FIGURE 10 depicts electrophysiological characterization of hP2X2 channels.
Detailed Description of the Invention
The practice of the present invention will employ, unless otherwise indicated,
2o conventional techniques of molecular biology, microbiology, recombinant DNA
technology, electrophysiology, and pharmacology, that are within the skill of
the art.
Such techniques are explained fully in the literature. See, for example,
Sambrook,
Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition
(1989);
DNA Cloning, Vols. I and II (D.N. Glover Ed. 1985); Perbal, B., A Practical
Guide to
Zs Molecular Cloning (1984); the series, Methods In Enzymology (S. Colowick
and N.
Kaplan eds., Academic Press, Inc.); Transcription and Translation (Names et
al. eds.
1984); Gene Transfer Vectors For Mammalian Cells (J. H. Miller et al. eds.
(1987)
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); Scopes, Protein
Purification: Principles and Practice (2nd ed., Springer-Verlag); and PCR: A
Practical
so Approach (McPherson et al. eds. (1991) IRL Press).
All patents, patent applications and publications cited herein, whether supra
or
infra, are hereby incorporated by reference in their entirety and are deemed
representative of the prevailing state of the art.
As used in this specification and the appended claims, the singular forms "a,"
3s "an" and "the" include plural references unless the content clearly
dictates otherwise.
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Thus, for example, reference to "a primer" includes two or more such primers,
reference to "an amino acid" includes more than one such amino acid, and the
like.
In describing the present invention, the following terms will be employed, and
are intended to be defined as indicated below.
s The term "P2 receptor" intends a purinergic receptor for the ligand ATP
and/or
other purine or pyrimidine nucleotides, whether natural or synthetic. P2
receptors are
broadly subclassified as "P2X" or "P2Y" receptors. These types differ in their
pharmacology, structure, and signal transduct(on mechanisms. The P2X receptors
are generally ligand-gated ion channels, while the P2Y receptors operate
generally
~o through a G protein-coupled system. Moreover, and without intending to be
limited by
theory, it is believed that P2X receptors comprise multimers of receptor
polypeptides,
which multimers may be of either the same or different subtypes. Consequently,
the
term "P2X receptor" refers, as appropriate, to the individual receptor subunit
or
subunits, as well as to the homomeric and heteromeric receptors comprised
thereby.
~s The term "P2X~" intends a P2X receptor subtype wherein n is an integer of
at
least 1. At the time of the invention, at least 7 P2X~ receptor subtypes have
been
isolated and/or characterized.
A "P2X2 receptor agonist" is a compound that binds to and activates a P2X2
receptor. By "activates" is intended the elicitation of one or more
pharmacological,
2o physiological, or electrophysiological responses. Such responses may
include, but
are not limited to, an increase in receptor-specific cellular depolarization.
A "P2X2 receptor antagonist" is a substance that binds to a P2X2 receptor and
prevents agonists from activating the receptor. Pure antagonists do not
activate the
receptor, but some substances may have mixed agonist and antagonist
properties.
2s The term "polynucleotide" as used herein means a polymeric form of
nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
This term
refers only to the primary structure of the molecule. Thus, the term includes
double
and single-stranded DNA, as well as double- and single-stranded RNA. It also
includes modifications, such as by methy(ation and/or by capping, and
unmodified
3o forms of the polynucleotide.
The term "variant" is used to refer to an oligonucleotide sequence which
differs
from the related wild-type sequence in the insertion, deletion or substitution
of one or
more nucleotides. When not caused by a structurally conservative mutation (see
below), such a variant oligonucleotide is expressed as a "protein variant"
which, as
3s used herein, indicates a polypeptide sequence that differs from the wild-
type
polypeptide in the insertion, deletion or substitution of one or more amino
acids. The
protein variant differs in primary structure (amino acid sequence), but may or
may not
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6
differ significantly in secondary or tertiary structure or in function
relative to the wild-
type.
The term "mutant" generally refers to an organism or a cell displaying a new
genetic character or phenotype as the result of change in its gene or
chromosome. In
s some instances, however, "mutant" may be used in reference to a variant
protein or
oligonucleotide and "mutation" may refer to the change underlying the variant.
"Polypeptide" and "protein" are used interchangeably herein and indicate a
molecular chain of amino acids linked through peptide bonds. The terms do not
refer
to a specific length of the product. Thus, peptides, oligopeptides, and
proteins are
~o included within the definition of polypeptide. The terms include post-
translational
modifications of the polypeptide, for example, glycosylations, acetylations,
phosphorylations and the like. In addition, protein fragments, analogs,
mutated or
variant proteins, fusion proteins and the like are included within the meaning
of
polypeptide, provided that such fragments, etc. retain the binding or other
~ s characteristics necessary for their intended use.
A "functionally conservative mutation" as used herein intends a change in a
polynucleotide encoding a derivative polypeptide in which the activity is not
substantially altered compared to that of the polypeptide from which the
derivative is
made. Such derivatives may have, for example, amino acid insertions,
deletions, or
2o substitutions in the relevant molecule that do not substantially affect its
properties.
For example, the derivative can include conservative amino acid substitutions,
such
as substitutions which preserve the general charge,
hydrophobicity/hydrophilicity, side
chain moiety, and/or steric bulk of the amino acid substituted, for example,
Gly/Ala,
Val/Ile/Leu, Asp/Glu, Lys/Arg, Asn/Gln, ThNSer, and PhelTrp/Tyr.
2s By the term "structurally conservative mutant" is intended a polynucleotide
containing changes in the nucleic acid sequence but encoding a polypeptide
having
the same amino acid sequence as the polypeptide encoded by the polynucleotide
from which the degenerate variant is derived. This can occur because a
specific
amino acid may be encoded by more than one "codon," or sequence of three
so nucleotides, i.e., because of the degeneracy of the genetic code.
"Recombinant host cells," "host cells," "cells," "cell lines," "cell
cultures," and
other such terms denoting microorganisms or higher eukaryotic cell lines
cultured as
unicellular entities refer to cells which can be, or have been, used as
recipients for
recombinant vectors or other transfer DNA, immaterial of the method by which
the
3s DNA is introduced into the cell or the subsequent disposition of the cell.
The terms
include the progeny of the original cell which has been transfected. Cells in
primary
culture as well as cells such as oocytes also can be used as recipients.
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A "vector" is a replicon in which another polynucleotide segment is attached,
such as to bring about the replication andlor expression of the attached
segment.
The term includes expression vectors, cloning vectors, and the like.
A "coding sequence" is a polynucleotide sequence that is transcribed into
s mRNA and/or translated into a polypeptide. The boundaries of the coding
sequence
are determined by a translation start codon at the 5'-terminus and a
translation stop
codon at the 3'-terminus. A coding sequence can include, but is not limited
to, mRNA,
cDNA, and recombinant polynucleotide sequences. Variants or analogs may be
prepared by the deletion of a portion of the coding sequence, by insertion of
a
~o sequence, and/or by substitution of one or more nucleotides within the
sequence.
Techniques for modifying nucleotide sequences, such as site-directed
mutagenesis,
are well known to those skilled in the art. See, for example, Sambrook et al.,
supra;
DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.
"Operably linked" refers to a situation wherein the components described are
in
is a relationship permitting them to function in their intended manner. Thus,
for
example, a control sequence "operably linked" to a coding sequence is ligated
in such
a manner that expression of the coding sequence is achieved under conditions
compatible with the control sequences. A coding sequence may be operabiy
linked to
control sequences that direct the transcription of the polynucleotide whereby
said
2o polynucleotide is expressed in a host cell.
The term "transfection" refers to the insertion of an exogenous polynucleotide
into a host cell, irrespective of the method used for the insertion, or the
molecular form
of the polynucleotide that is inserted. The insertion of a polynucleotide per
se and the
insertion of a plasmid or vector comprised of the exogenous polynucleotide are
2s included. The exogenous polynucleotide may be directly transcribed and
translated
by the cell, maintained as a nonintegrated vector, for example, a plasmid, or
alternatively, may be stably integrated into the host genome. "Transfection"
generally
is used in reference to a eukaryotic cell while the term "transformation" is
used to refer
to the insertion of a polynucleotide into a prokaryotic cell. "Transformation"
of a
so eukaryotic cell also may refer to the formation of a cancerous or
tumorigenic state.
The term "isolated," when referring to a polynucleotide or a polypeptide,
intends that the indicated molecule is present in the substantial absence of
other
similar biological macromolecules. The term "isolated" as used herein means
that at
least 75 wt.%, more preferably at least 85 wt.%, more preferably still at
least 95 wt.%,
3s and most preferably at least 98 wt.% of a composition is the isolated
polynucleotide or
polypeptide. An "isolated polynucleotide" that encodes a particular
polypeptide refers
to a polynucleotide that is substantially free of other nucleic acid molecules
that do not
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encode the subject polypeptide; however, the molecule may include functionally
and/or structurally conservative mutations as defined herein.
A "test sample" as used herein intends a component of an individual's body
which is a source of a P2X2 receptor. These test samples include biological
samples
s which can be evaluated by the methods of the present invention described
herein and
include body fluids such as whole blood, tissues and cell preparations.
The following single-letter amino acid abbreviations are used throughout the
text:
io Alanine A Arginine R
Asparagine N Aspartic acid D
Cysteine C Glutamine Q
Glutamic acid E Glycine G
Histidine H Isoleucine I
~ s Leucine L Lysine K
Methionine M Phenylalanine F
Proline P Serine S
Threonine T Tryptophan W
Tyrosine Y Valine V
A human P2X2 receptor, a polynucleotide encoding the variant receptor or
polypeptide subunits thereof, and methods of making the receptor are provided
herein. The invention includes not only the P2X2 receptor but also methods for
screening compounds using the receptor and cells expressing the receptor.
Further,
2s poiynucleotides and antibodies which can be used in methods for detection
of the
receptor, as well as the reagents useful in these methods, are provided.
Compounds
and polynucleotides useful in regulating the receptor and its expression also
are
provided as disclosed hereinbelow.
In one preferred embodiment, the polynucleotide encodes a human P2X2
3o receptor polypeptide or a protein variant thereof containing conservative
amino acid
substitutions.
DNA encoding the human P2X2 receptor, and variants thereof, can be derived
from genomic or cDNA, prepared by synthesis, or by a combination of
techniques.
The DNA can then be used to express the human P2X2 receptor or as a template
for
3s the preparation of RNA using methods well known in the art (see, Sambrook
et al.,
supra), or as a molecular probe capable of selectively hybridizing to, and
therefore
detecting the presence of, other P2X2-encoding nucleotide sequences.
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cDNA encoding the P2X2 receptor may be obtained from an appropriate DNA
library. cDNA libraries may be probed using the procedure described by
Grunstein et
al. (1975) Proc. Natl. Acad. Sci. USA 73:3961. The cDNA thus obtained can then
be
modified and amplified using the polymerase chain reaction ("PCR") and primer
s sequences to obtain the DNA encoding the human P2X2 receptor.
More particularly, PCR employs short oligonucleotide primers (generally 10-20
nucleotides in length) that match opposite ends of a desired sequence within
the DNA
molecule. The sequence between the primers need not be known. The initial
template can be either RNA or DNA. If RNA is used, it is first reverse
transcribed to
~o cDNA. The cDNA is then denatured, using well-known techniques such as heat,
and
appropriate oligonucleotide primers are added in molar excess.
Primer extension is effected using DNA polymerase in the presence of
deoxynucleotide triphosphates or nucleotide analogs. The resulting product
includes
the respective primers at their 5'-termini, covalently linked to the newly
synthesized
t s complements of the original strands. The replicated molecule is again
denatured,
hybridized with primers, and so on, until the product is sufficiently
amplified. Such
PCR methods are described in for example, U.S. Patent Nos. 4,965,188;
4,800,159;
4,683,202; 4,683,195; incorporated herein by reference in their entireties.
The
product of the PCR is cloned and the clones containing the P2X2 receptor DNA,
2o derived by segregation of the primer extended strand, selected. Selection
can be
accomplished using a primer as a hybridization probe.
Alternatively still, the P2X2 receptor DNA could be generated using an RT-PCR
(reverse transcriptase - polymerase chain reaction) approach starting with
human
RNA. Human RNA may be obtained from cells or tissue in which the P2X2 receptor
is
2s expressed, for example, brain, spinal cord, uterus or lung, using
conventional
methods. For example, single-stranded cDNA is synthesized from human RNA as
the
template using standard reverse transcriptase procedures and the cDNA is
amplified
using PCR. This is but one example of the generation of P2X2 receptor variant
from a
human tissue RNA template.
30. Synthetic oligonucleotides may be prepared using an automated
oligonucleotide synthesizer such as that described by Warner (1984) DNA 3:401.
if
desired, the synthetic strands may be labeled with 32P by treatment with
polynucleotide kinase in the presence of 32P-ATP, using standard conditions
for the
reaction. DNA sequences, including those isolated from genomic or cDNA
libraries,
3s may be modified by known methods which include site-directed mutagenesis as
described by Zoller (1982) Nucleic Acids Res. 10:6487. Briefly, the DNA to be
modified is packaged into phage as a single stranded sequence, and converted
to a
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double stranded DNA with DNA polymerase using, as a primer, a synthetic
oligonucleotide complementary to the portion of the DNA to be modified, and
having
the desired modification included in its own sequence. Culture of the
transformed
bacteria, which contain replications of each strand of the phage, are plated
in agar to
s obtain plaques. Theoretically, 50% of the new plaques contain phage having
the
mutated sequence, and the remaining 50% have the original sequence. Replicates
of
the plaques are hybridized to labeled synthetic probe at temperatures and
conditions
suitable for hybridization with the correct strand, but not with the
unmodified
sequence. The sequences which have been identified by hybridization are
recovered
to and cloned. Alternatively, it may be necessary to identify clones by
sequence
analysis if there is difficulty in distinguishing the variant from wild type
by hybridization.
In any case, the DNA would be sequence-confirmed.
Once produced, DNA encoding the P2X2 receptor may then be incorporated
into a cloning vector or an expression vector for replication in a suitable
host cell.
Is Vector construction employs methods known in the art. Generally, site-
specific DNA
cleavage is performed by treating with suitable restriction enzymes under
conditions
that generally are specifed by the manufacturer of these commercially
available
enzymes. After incubation with the restriction enzyme, protein is removed by
extraction and the DNA recovered by precipitation. The cleaved fragments may
be
2o separated using, for example, polyacrylamide or agarose gel electrophoresis
methods, according to methods known by those of skill in the art.
Sticky end cleavage fragments may be blunt ended using E. coli DNA
polymerase 1 (Klenow) in the presence of the appropriate deoxynucleotide
triphosphates (dNTPs) present in the mixture. Treatment with S1 nuclease also
may
2s be used, resulting in the hydrolysis of any single stranded DNA portions.
Ligations are performed using standard buffer and temperature conditions
using T4 DNA ligase and ATP. Alternatively, restriction enzyme digestion of
unwanted fragments can be used to prevent ligation.
Standard vector constructions generally include specific antibiotic resistance
3o elements. Ligation mixtures are transformed into a suitable host, and
successful
transformants selected by antibiotic resistance or other markers. Plasmids
from the
transformants can then be prepared according to methods known to those in the
art
usually following a chloramphenicol amplification as reported by Clewell et
al. (1972)
J. Bacteriol. 110:667. The DNA is isolated and analyzed usually by restriction
3s enzyme analysis and/or sequencing. Sequencing may be by the well-known
dideoxy
method of Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463) as further
described by Messing et al. (1981) Nucleic Acid Res. 9:309, or by the method
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11
reported by Maxam et al. (1980) Meth. Enzymol. 65:499. Problems with band
compression, which are sometimes observed in GC rich regions, are overcome by
use of, for example, T-deazoguanosine or inosine, according to the method
reported
by Barr et aI. (1986) Biotechniques 4:428.
s Host cells are genetically engineered with the vectors of this invention,
which
may be a cloning vector or an expression vector. The vector may be in the form
of a
plasmid, a viral particle, a phage, etc. The engineered host cells can be
cultured in
conventional nutrient media modified as appropriate for activating promoters,
selecting transformants/transfectants or amplifying the subunit-encoding
~o polynucleotide. The culture conditions, such as temperature, pH and the
like,
generally are similar to those previously used with the host cell selected for
expression, and will be apparent to those of skill in the art.
Both prokaryotic and eukaryotic host cells may be used for expression of
desired coding sequences when appropriate control sequences that are
compatible
is with the designated host are used. For example, among prokaryotic hosts,
Escherichia coli is frequently used. Also, for example, expression control
sequences
for prokaryotes include but are not limited to promoters, optionally
containing operator
portions, and ribosome binding sites. Transfer vectors compatible with
prokaryotic
hosts can be derived from, for example, the plasmid pBR322 that contains
operons
2o conferring ampicillin and tetracycline resistance, and the various pUC
vectors, that
also contain sequences conferring antibiotic resistance markers. These markers
may
be used to obtain successful transformants by selection. Commonly used
prokaryotic
control sequences include but are not limited to the lactose operon system
(Chang et
al. (1977) Nature 198:1056), the tryptophan operon system (reported by Goeddel
et
2s al. (1980) Nucleic Acid Res. 8:4057) and the lambda-derived PI promoter and
N gene
ribosome binding site (Shimatake et al. (1981) Nature 292:128), the hybrid Tac
promoter (De Boer et al. (1983) Proc. Natl. Acad. Sci. USA 292:128) derived
from
sequences of the trp and lac UV5 promoters. The foregoing systems are
particularly
compatible with E. coli; however, other prokaryotic hosts such as strains of
Bacillus or
3o Pseudomonas may be used if desired.
Eukaryotic hosts include yeast and mammalian cells in culture systems. Pichia
pastoris, Saccharomyces cerevisiae and S. carlsbergensis are commonly used
yeast
hosts. Yeast-compatible vectors carry markers that permit selection of
successful
transformants by conferring protrophy to auxotrophic mutants or resistance to
heavy
3s metals on wild-type strains. Yeast-compatible vectors may employ the 2-N
origin of
replication (Broach et al. (1983) Meth. Enzymol. 101:307), the combination of
CEN3
and ARS1 or other means for assuring replication, such as sequences that will
result
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12
in incorporation of an appropriate fragment into the host cell genome. Control
sequences for yeast vectors are known in the art and include but are not
limited to
promoters for the synthesis of glycolytic enzymes, including the promoter for
3-
phosphoglZ~cerate kinase. See, for example, Hess et al. (1968) J. Adv. Enzyme
Reg.
s 7:149, Holland et al. (1978) Biochemistry 17:4900 and Hitzeman (1980) J.
Biol. Chem.
255:2073. For example, some useful control systems are those that comprise the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter or alcohol
dehydrogenase (ADH) regufatable promoter, or the hybrid yeast promoter
ADH2/GAPDH described in Cousens et al. Gene (1987) 61:265-275, terminators
also
~o derived from GAPDH, and, if secretion is desired, leader sequences from
yeast alpha
factor. In addition, the transcriptional regulatory region and the
transcriptional
initiation region which are operably linked may be such that they are not
naturally
associated in the wild-type organism.
Mammalian cell lines available as hosts for expression are known in the art
and
is are available from depositories such as the American Type Culture
Collection. These
include but are not limited to HeLa cells, human embryonic kidney (HEK) cells,
Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and
others.
Suitable promoters for mammalian cells also are known in the art and include
viral
promoters such as that from Simian Virus 40 (SV40), Rous sarcoma virus (RSV),
2o adenovirus (ADV), bovine papilloma virus (BPV) and cytomegalovirus (CMV).
Mammalian cells also may require terminator sequences and poly A addition
sequences; enhancer sequences which increase expression also may be included,
and sequences which cause amplification of the gene also may be desirable.
These
sequences are known in the art. Vectors suitable for replication in mammalian
cells
2s may include viral replicons, or sequences which ensure integration of the
appropriate
sequences encoding the P2X2 receptor into the host genome. An example of such
a
mammalian expression system is described in Gopalakrishnan et al. (1995), Eur.
J.
Pharmacol.-Mol. Pharmacol. 290: 237-246.
Other eukaryotic systems are also known, as are methods for introducing
3o polynucleotides into such systems, such as amphibian cells, using standard
methods
such as described in Briggs et al. {1995) Neuropharmacol. 34:583-590 or
Stiihmer
(1992) Meth. Enzymol. 207:319-345, insect cells using methods described in
Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555
(1987),
and the like.
3s The baculovirus expression system can be used to generate high levels of
recombinant proteins in insect host cells. This system allows for high level
of protein
expression, while post-translationally processing the protein in a manner
similar to
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13
mammalian cells. These expression systems use viral promoters that are
activated
following baculovirus infection to drive expression of cloned genes in the
insect cells
(O'Reilly et al. (1992) Baculovirus Expression Vectors: A Laboratory Manual,
IRUOxford University Press).
s Transfection may be by any known method for introducing polynucleotides into
a host cell, including packaging the polynucleotide in a virus and transducing
a host
cell with the virus, by direct uptake of the polynucleotide by the host cell,
and the like,
which methods are known to those skilled in the art. The transfection
procedures
selected depend upon the host to be transfected and are determined by the
io rountineer.
The expression of the receptor may be detected by use of a radioligand
selective for the receptor. However, any radioligand binding technique known
in the
art may be used to detect the receptor (see, for example, Winzor et al. (1995)
Quantitative Characterization of Ligand Binding, Wiley-Liss, Inc., NY; Michel
et al.
~s (1997) Mol. Pharmacol. 51:524-532). Alternatively, expression can be
detected by
utilizing antibodies or functional measurements, i.e., ATP-stimulated cellular
depolarization using methods that are well known to those skilled in the art.
For
example, agonist-stimulated Ca2+influx, or inhibition by antagonists of
agonist-
stimulated Ca2''influx, can be measured in mammalian cells transfected with
the
2o recombinant P2X2 receptor cDNA, such as COS, CHO or HEK cells.
Alternatively,
Ca2'influx can be measured in cells that do not naturally express P2
receptors, for
example, the 1321N1 human astrocytoma cell line, have been prepared using
recombinant technology to transiently or stably express the P2X2 receptor.
The P2X2 polypeptide is recovered and purified from recombinant host cell
2s cultures expressing the same by known methods including ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
hydroxyapatite chromatography or lectin chromatography. Protein refolding
steps can
be used, as necessary, in completing configuration of the protein. Finally,
high
3o performance liquid chromatography (HPLC) can be employed for final
purification
steps.
The human P2X2 receptor polypeptide, or fragments thereof, of the present
invention also may be synthesized by conventional techniques known in the art,
for
example, by chemical synthesis such as solid phase peptide synthesis. In
general,
3s these methods employ either solid or solution phase synthesis methods. See,
for
example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd
Ed.,
Pierce Chemical Co., Rockford, IL (1984) and G. Barany and R. B. Merrifield,
The
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Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer,
Vol. 2,
Academic Press, New York, (1980), pp. 3-254, for solid phase peptide synthesis
techniques; and M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag,
Berlin
(1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis,
Synthesis,
s Biology, supra, Vol. 1, for classical solution synthesis.
In one preferred system, either the DNA or the RNA derived therefrom, each of
which encode the human P2X2 receptor, may be expressed by direct injection
into a
cell, such as a Xenopus laevis oocyte. Using this method, the functionality of
the
human P2X2 receptor encoded by the DNA or the mRNA can be evaluated as
follows.
~o A receptor-encoding polynucleotide is injected into an oocyte for
translation into a
functional receptor subunit. The function of the expressed variant human P2X2
receptor can be assessed in the oocyte by a variety of techniques including
electrophysiological techniques such as voltage-clamping, and the like.
Receptors expressed in a recombinant host cell may be used to identify
~s compounds that modulate P2X2 activity. In this regard, the specificity of
the binding of
a compound showing affinity for the receptor is demonstrated by measuring the
affinity of the compound for cells expressing the receptor or membranes from
these
cells. This may be done by measuring specific binding of labeled (for example,
radioactive) compound to the cells, cell membranes or isolated receptor, or by
2o measuring the ability of the compound to displace the specific binding of a
standard
labeled ligand. See, Michel et al., supra. Expression of variant receptors and
screening for compounds that bind to, or inhibit the binding of labeled ligand
to these
cells or membranes, provide a method for rapid selection of compounds with
high
affinity for the receptor. These compounds may be agonists, antagonists or
2s modulators of the receptor.
Expressed receptors also may be used to screen for compounds that modulate
P2X2 receptor activity. One method for identifying compounds that modulate
P2X2
activity, comprises providing a cell that expresses a human P2X2 receptor
polypeptide, combining a test compound with the cell and measuring the effect
of the
3o test compound on the P2X2 receptor activity. The cell may be a bacterial
cell, a
mammalian cell, a yeast cell, an amphibian cell, an insect or any other cell
expressing
the receptor. Preferably, the cell is a mammalian cell or an amphibian cell.
Thus, for
example, a test compound is evaluated for its ability to elicit an appropriate
response,
for example, the stimulation of cellular depolarization, or for its ability to
modulate the
3s response to an agonist or antagonist.
Additionally, compounds capable of modulating P2X2 receptors are considered
potential therapeutic agents in several disorders including, without
limitation, central
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nervous system or peripheral nervous system conditions, for example, epilepsy,
pain,
depression, neurodegenerative diseases, and the like, and in disorders of
skeletal
muscle such as neuromuscular diseases.
)n addition, the DNA, or RNA derived therefrom, can be used to design
s oligonucleotide probes for DNAs that express P2X2 receptors. As used herein,
the
term "probe" refers to a structure comprised of a polynuc(eotide, as defined
above,
which contains a nucleic acid sequence complementary to a nucleic acid
sequence
present in a target polynucleotide. The polynucleotide regions of probes may
be
composed of DNA, and/or RNA, and/or synthetic nucleotide analogs. Such probes
~o could be useful in in vitro hybridization assays to distinguish P2X2
variant from wild-
type message, with the proviso that it may be difficult to design a method
capable of
making such a distinction given the small differences that may exist between
sequences coding the wild-type and a variant P2X2 receptor. Alternatively, a
PCR-
based assay could be used to amplify the sample RNA or DNA for sequence
analysis.
~s Furthermore, the P2X2 polypeptide or fragments) thereof can be used to
prepare monoclonal antibodies using techniques that are well known in the art.
The
P2X2 receptor or relevant fragments can be obtained using the recombinant
technology outlined below, i.e., a recombinant cell that expresses the
receptor or
fragments can be cultured to produce quantities of the receptor or fragment
that can
2o be recovered and isolated. Alternatively, the P2X2 polypeptide or
fragments) thereof
can be synthesized using conventional polypeptide synthetic techniques as
known in
the art. Monoclonal antibodies that display specificity and selectivity for
the P2X2
polypeptide can be labeled with a measurable and detectable moiety, for
example, a
fluorescent moiety, radiolabels, enzymes, chemiluminescent labels and the
like, and
2s used in in vitro assays. It is theorized that such antibodies could be used
to identify
wild-type or variant P2X2 receptor polypeptides for irnmuno-diagnostic
purposes. For
example, antibodies have been generated to detect amyloid b1-40 v. 1-42 in
brain
tissue (Wisniewski ef al. (1996) Biochem. J. 313:575-580; also see, Suzuki et
al.
(1994) Science 264:1336-1340; Gravina et al. (1995) J. Biol. Chem. 270:7013-
7016;
3o and Turnet et al. (1996) J. Biol. Chem. 271:8966-8970).
Therapeutic Indications for Modulators of the Human P2X2 Receptor
Activation of the P2X2 receptor by ATP and other nucleotides regulates ion
gradients across the cell membrane, modulates the cytosolic concentrations of
3s cations, including Ca2+, Na+ and K+, and has a role in the regulation of
cell
membrane potential which in turn has specific physiological effects.
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Pain
The rat P2X2 receptor is expressed in the spinal cord, and in the nodose and
dorsal root ganglia (Brake et al., Nature 371:519-523 (1994)), a distribution
consistent
with a role in pain transmission. Specifically, the P2X2 receptor subunit
forms
s functional channels when expressed alone, and it can also form a functional
heteromultimeric channel that has properties similar to currents seen in
native sensory
channels when co-expressed with the P2X3 receptor, another P2X receptor which
is
expressed in sensory neurons (Lewis et al., Nature 377:432-435 (1995)).
Evidenced
from studies in rat nodose ganglia indicate that both P2X2/P2X3 heteromeric
channels
~o and P2X2 homomeric channels contribute to ATP currents (Virginio et al., J.
Physiol
(Lone) 510:27-35 (1998); Thomas, et al., J. Physiol (Lond) 509 (Pt 2):411-417
(1998}). ATP, which activates P2X2 and P2X~/P2X3 receptors, functions as an
excitatory neurotransmitter in the spinal cord dorsal horn and in primary
afferents from
sensory ganglia (Holton and Holton, J. Physiol. (Lond) 126:124-140 (1954)).
ATP-
~s induced activation of P2X receptors on dorsal root ganglion nerve terminals
in the
spinal cord stimulates the release of glutamate, a key neurotransmitter
involved in
nociceptive signaling (Gu and MacDermott, Nature 389:749-753 (1997}). Thus,
ATP
released from damaged cells evokes pain by activating P2X2 or P2X~/P2X3
receptors
on nociceptive nerve endings or sensory nerves. This is consistent with the
induction
Zo of pain by intradermally applied ATP in the human blister-base model
(Bleehen, Br J.
Pharmacol fi2:573-577 (1978)), and with reports that P2X receptor antagonists
are
analgesic in animal models (Driessen and Starke, Naunyn Schmiederbergs Arch
Pharmacol 350:618-625 (1994)). This evidence clearly suggests that P2X2
functions
in nociception, and that modulators of the human P2X2 receptor are useful as
2s analgesics.
Thus, compounds which block or inhibit activation of P2X2 receptors serve to
block the pain stimulus. Antagonists to compounds which normally activate the
P2X2
receptor, such as ATP, could successfully block the transmission of pain.
3o Diseases of the Neuroendocrine System
Extracellular ATP induces secretion of hormones, including prolactin and
leuteinizing hormone, from cells of the pituitary gland (Chen et aL, Proc Natl
Acad Sci
USA 92:5219-5223 (1995); Nunez et al, Am J. Physiol 272:E1117-E1123 (1997)).
(Carew et al., Cell Calcium 16:227-235 (1994)) (Villalobos et al., Am J
Physiol
3s 273:C1963-C1971 (1997)). In addition, since ATP is co-released with
hormones such
as insulin, prolactin, and leuteinizing hormone, as well as with
catecholamines from
adrenal chromaffin cells, it may act as a paracrine regulator of hormone
release in
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17
these tissues (Chen et al., Proc Natl Acad Sci USA 92:5219:5223 (1995); Tomic
et al.,
J Biol Chem 271:21200-21208 (1996); Nunez et al., Am J Physiol 272:E1117-E1123
(1997)) (Leitner et al., Endocrinology 96:662-677 (1975)); Hollins and Ikeda,
J
Neurophysiol 78:3069-3076 (1997)). The human P2X2 receptor has been found in
s neuroendocrine tissue and, specifically, the human P2X2 receptor cDNAs was
cloned
from pituitary tissue RNA. In addition, the P2X2 receptor RNA and protein have
been
detected in rat pituitary tissue (Brake et al., Nature 371:519-523 (1994))
(Housley et
al., Biochem Biophys Res Commun 212:501-508 (1995); Tomic et al., J Biol Chem
271:21200-21208 (1996); Vulchanova et al., Proc Natl Acad Sci USA 93:8063-8067
~o (1996)). Clearly, the P2X2 receptor is involved in hormone secretion via
activation by
ATP. Thus, an agonist or antagonist to ATP would be effective in modulating
hormone release. Thus, pharmaceutical agents that act on the P2X2 receptor may
be
useful to modulate hormonal secretion from this gland.
~s Auditory and Vestibular Disorders
Extracellular ATP acts as a stimulus for neurons and epithelial cells of the
inner
ear (Housley, Mol Neurobial 16:21-48 (1998)). Perfusion of ATP into the guinea
pig
cochlear perilymphatic compartment inhibits auditory parameters such as
auditory-
nerve compound action potential and sound tranduction current across the
apical
2o surface of sensory hair cells. (Bobbin and Thompson, Ann Otol Rhinol
Laryngol
87:185-190 (1978)). Perfusion of ATP into the cochlear endolymph also inhibits
sensory current transduction and endocochlear potential, and these effects are
blocked by the P2 receptor antagonists suramin and reactive blue 2 (Munoz et
al.,
Hear Res 90:119-125 (1995)). Suramin also blocks the decline in quadratic
2s electrophysiological and mechanical coupling of the organ of Corti which
occurs
during continuous sound stimulation, suggesting that P2 activation plays a
role in this
event (Kujawa et al., Hear Res 78:181-188 (1994); (Housley, Mol Neurogiol
16:21-48
(1998)). ATP also affects vestibular system function. ATP stimulates
vestibular
afferent nerve discharge, and these responses are blocked by the P2 antagonist
3o suramin and reactive blue 2 (Aubert et al., Neuroscience 62-963-974 (1994);
Aubert
et al., Neuroscience 64:1153-1160 (1995)). Autoradiographic binding studies
using
ATP analogs indicate the presence of P2 receptors on auditory tissues (Mockett
et al.,
Hear Res 84:177-193 (1995)). P2X2 receptor messenger RNA has been localized in
tissues of the rat auditory system. Several message variants for this receptor
have
3s been found in various vestibular and auditory tissues, including the
cochlea, spiral
ganglia, Dieter's cells, christa ampuliaris, and the organ of Corti (Glowatzki
et al., Proc
R Soc Lond B Biol Sci 262:141-147 (1995); Housley et al., Biochem Biophys Res
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Commun 212:501-508 (1995); Salih et al., Neuroreport 9:279-282 (1998); Chen
and
Bobbin, Br J Pharmacol 124:337-344 (1998); Housley et al., J Comp Neurol
393:403-
414 (1998)). Evidence of the expression of P2X2 receptors in those tissues of
the
auditory and vestibular systems which are functionally modulated by ATP
indicates a
s role for this receptor in auditory and vestibufar function. Altered function
of P2
receptors in the ear have pathological implications, as exposure to noise has
been
shown to after the response of outer hair cells to ATP (Chen et al., Hear Res
88:215-
221 (1995)), and P2X2 receptor modulators may have utility in disorders of
auditory
and vestibular function. Thus, ATP agonists and antagonists have effects on
io modulation of the P2Xz receptor, in auditory and vestibular functions.
Other
ATP is a potent neurotransmitter in neurons of the gastrointestinal tract, and
ATP-mediated signals from enteric neurons appears to be characteristic of P2X2
Is receptors (Zhou and Galligan, J Physiol (Loud) 496 (Pt 3):719-729 (1996)).
Additionally, the discovery of the human P2X2 EST from a library derived from
colon
tissue suggests that this receptor plays a role in gastrointestinal function.
P2X2 is also
expressed in vascular smooth muscle tissue, where ATP has been shown to
influence
vascular tone (Nori et al., J. Vasc Res 35:179-185 (1998)) (Kennedy et al.,
Eur J
2o Pharmacol 107:161-168 (1985)).
Below are examples of specific embodiments for carrying out the present
invention. The examples are offered for illustrative purposes only, and are
not
intended to limit the scope of the present invention in any way.
2s Exam~~le 1
Identification of a Human cDNA Sequence Likel~i to Encode P2X~ Poly peptide
The predicted amino acid sequence of the rat P2X2 receptor (Genbank
accession number 1352688) was used to search for human DNA sequences which
so would code for similar polypeptides. The TBLASTN database search tool
(Altschul
(1993) J. Mol. Evol. 36:290-300) was used, which allows querying nucleotide
databases with a protein sequence by dynamically translating the DNA sequences
into all 6 possible reading frames. A search of the Lifeseq database (Incyte
Pharmaceuticals, Inc., Palo Alto California, CA) revealed a partial sequence
of cDNA
ss clone derived from human fetal colon tissue which encoded a polypeptide
having a
high degree of homology to a region of the rat P2X2 receptor. The database
entry for
this sequence is shown in Figure 1 and SEQ. fD N0:1.
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The position of this sequence with respect to that of the rat P2Xz sequence
predicted that this cDNA clone would only contain a partial coding sequence
for the
receptor. The cDNA clone was ordered and the clone was fully sequenced as
shown
in Figure 2 and SEQ ID N0:2. Note that in Figure 2 the underlined sequence
denotes
s overlap with the original database entry.
Primers were designed to the non-coding sequence of this cDNA to enable 5'
RACE procedures in an attempt to identify the missing coding sequence, shown
in
Figure 3 and SEQ.ID. NOS:3-7. Using poly A plus RNA derived from human
pituitary
tissue, 5' RACE reactions were performed using a commercially available system
~o (GibcoBRL, Gaithersburg, MD). A product of approximately 600 by was cloned
and
sequenced, shown in Figure 4 and SEQ ID N0:8. This product was found to
contain
additional sequence information for an open reading frame with homology to the
P2X
receptors, but did not extend to what would be the predicted initiation codon
of an
intact receptor cDNA.
is A pair of primers were designed and synthesized based on the sequence
compiled from Incyte clone 1310493 and the RACE product, and are shown in
Figure
5. These primers were sent to Genome Systems (St. Louis, MO) and used in PCR
reactions to probe a P1 bacteriophage library of human genomic DNA. Two clones
were identified and obtained from Genome systems. The human P2X2 gene
2o contained in clone 18860 was sequenced both directly and after subcloning
into the
vector pBluescript II SK+.
Example 2
Isolation of Human cDNAs Encoding Novel P2 Receptors
2s
Using information on the sequence surrounding the predicted initiation and
termination codons of the human P2X2 message, oligonucleotide primers were
designed and synthesized to enable RT-PCR of the intact open reading frame of
the
mRNA. The sequence of these primers, hP2X2 5' and hP2X2 3', are shown in
Figure
30 6. The primers were used to amplify the open reading frames of human P2Xz
receptors in reverse transcription- PCR reactions as follows: Poly A+ RNA (1
microgram) derived from pituitary gland tissue (Clontech, Inc. Palo Alto, CA)
and 10
picomoles oligo dT primer were combined in a final volume of 12 NI dH20. This
mixture was heated to 70°C for 10 min. and chilled on ice for 1 min.
The following
3s components were added: 2 NI 10X PCR buffer (200 mM Tris-HCI pH 8.4, 500mM
KCI), 2 NI 25 mM MgCl2, 1 NI 10mM dNTP mix, and 2 NI 0.1M dithiothreitol. The
reaction was equilibrated to 42°C for 2 minutes after which 1 NI (200
units)
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Superscript II reverse transcriptase was added and incubation continued at
42°C for
50 minutes. The reaction was terminated by incubation at 70°C for 15
min. and
chilled on ice. Rnase H (1 ul; 2 units) was added and the mixture was
incubated for
20 minutes at 37°C, then stored on ice.
s A proofreading thermostable polymerase (Cloned Pfu DNA Polymerase,
Stratagene Inc. La Jolla, CA) was used in the amplification to ensure high-
fidelity
amplification. The reaction mixture consisted of: 2 NI cDNA, 5 NI 10x cloned
Pfu
polymerase reaction buffer (200 mM Tris-HCI (pH 8.8), 100mMKCl,
100mM(NH4)2S04,
20mM MgS04, 1 % Triton X-100, 1 mg/ml nuclease-free bovine serum albumin), 1
NI
io dNTP mix, 1 NI (10picomoles) 5'hP2X2 primer, 1 NI (10 picomoles) 3'hP2X2
primer, and
39 NI dH20. The reaction was heated to 95°C for 1 min., then held at
80°C for 2 min.,
during which 1 ~I (2.5 units) cloned Pfu polymerase was added. The reaction
was
cycled 35 times under these conditions; 94°C for 15 sec., 60°C
for 20 sec., and 72°C
for 5 minutes. After cycling, the reaction was incubated for 10 minutes at
70°C. The
~s reaction products were separated on a 0.8 % agarose gel and products of
approximately 1.5 kilobases were excised and purified via the Qiaquick gel
purification
system (Qiagen, Inc., Chatsworth, CA). The DNA was eluted with 50 NI dH20,
lyophilized and resuspended in 10 NI dH20. The DNA was eluted with 50 NI dH20,
lyophilized and resuspended in 15 NI dH20. Three microliters of the purified
PCR
2o product was used in a ligation reaction using the pCRscript cloning system
(Stratagene) which also included: 0.5N1 (5 ng) of the pCRscript Amp SK(+)
vector, 1 NI
of pCRscript 1 Ox Reaction Buffer, 0.5 NI of 10mM ATP, 1 NI (5 units) Srf I
restriction
enzyme, 1 NI (4 units) T4 DNA ligase, and 3 NI dH20. The reaction was
incubated at
room temperature for one hour, then at 65°C for 10 minutes. One
microliter of this
2s reaction was used to transform ultracompetent DH-5-a(Gibco BRL) as per
standard
manufacturer's protocols. Resulting clones were screened by restriction
analysis and
sequenced using fluorescent dye-terminator reagents (Prism, Perkin Elmer
Applied
Biosystems) and an Applied Biosystems 310 DNA sequencer. Three species of
cDNAs containing intact open reading frames from the predicted initiation to
3o termination codons were isolated (Figure 7, hP2X2b, c, d). Based on
structural
similarity to the rat P2X2 receptor, a fourth species, (hP2X2a, Figure 7a) was
created
by joining nucleotides 1-666 (using adenine of the initiation codon as
nucleotide #1 ) of
hP2X2d with nucleotides 595-1349 of hP2X2~. The predicted polypeptides encoded
by
these cDNAs are shown in Figure 8. An alignment of the predicted amino acid
ss sequences are shown in Figure 9.
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Example 3
Expression and Electrophysiological Analysis of
Recombinant P2X~ Receptors in Xenopus Oocytes
s To assess function of the human P2Xz receptors, RNA was synthesized from
the clones using the T, bacterial promoter present on the pCRscript vector and
reagents from Ambion (Message Machine; Ambion, Inc., Austin Tx.).
1. Preaaration and injection of ooc~~tes
~o Adult female frogs (Xenopus laevis) were anesthetized with 0.2% tricaine
before surgery. During surgery, sections of one ovary were removed and oocytes
were denuded of overlying follicle cells by agitation for 1-2 hours in 2 mg/ml
collagenase (Sigma type IA) in tow-Ca2' Barth's solution containing (in mM):
88 NaCI,
2.5 KCI, 1.0 MgCl2 10 Na-HEPES (pH 7.4) plus 100 Ng/ml gentamicin. Selection
of
~s stage V and VI oocytes was begun after approximately 50% of the cells were
denuded. Cytoplasmic injections of 50 ng hP2X2a-d RNA were performed on
denuded
oocytes using a glass microelectrode. Only one receptor subtype RNA was
injected
per cell. Oocytes were used for recording 1-2 days after injection and were
maintained at 16-19°C in normal Barth's solution (incubation medium in
mM): 90
2o NaCI, 1.0 KCI, 0.66 NaN03, 0.74 CaCl2, 0.82 MgCl2, 2.4 NaHC03, 2.5 Na-
pyruvate, 10
Na-HEPES (pH 7.4) plus 100 trg/ml gentamicin.
2. Recording solutions and chemicals
The standard recording solution contained (in mM): 96 NaCI, 2.0 KCI, 1.8
2s BaCl2, 1.0 MgCl2, 5.0 Na-pyruvate, and 5.0 Na-HEPES (pH 7.4). BaCl2 was
replaced
with CaCl2 (1 mM) in some experiments without significant effects on the
pharmacological properties of the receptors. All oocyte solutions were diluted
in
distilled H20 from 10X stock solutions. Concentrated stocks of agonists and
antagonists were made in distilled H20 and then serially diluted in recording
solution to
3o desired final concentrations. All chemicals and agonists (ATP and a,(3me-
ATP) were
obtained from Sigma Chemical Company.
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3. Electroahysiolopical recordings
Transmembrane currents were recorded using two-electrode voltage-clamp
techniques with an Axoclamp-2A amplifier, and were collected and analyzed
using
pCLAMP software (Axon Instruments). Electrodes (1.5 - 2.0 M'S2) were filled
with 120
s mM KCI. Responses to ATP and a,~3me-ATP were routinely recorded at room
temperature while the oocyte membrane was voltage-clamped at -60 mV. Agonists
were applied using a computer-controlled small diameter drug application
pipette
positioned close to the oocyte in the perfusion chamber. Application duration
typically
lasted 5-10 sec. The peak amplitude of the ATP-activated inward current was
used
~ o for determining ECSO values.
4. Results
hP2X2a and hP2X2b receptors - Transient external application of ATP to oocytes
expressing hP2X2a or hP2X2b receptors produced a concentration-dependent
increase
~s in net inward current (Figure 10, panels A and B). Peak inward current
increased with
increasing ATP concentrations, consistent with an increase in probability of
agonist
binding, and therefore receptor activation. Concentration-response curves for
four
hP2X2a cells revealed a mean ATP ECSO of 16 ~M, and a Hill coefficient (nH) of
1.5.
Concentration-response curves for three hP2X2b cells revealed a mean ATP ECso
of
20 20 ~,M, and a nH of 1.5. Both receptor subtypes exhibited reversible non-
desensitizing
response kinetics.
Application of another P2X receptor agonist, a(3Methylene-ATP (a~Me-ATP)
had no effect on hP2X2a or hP2X2b receptors at a concentration of 100 ~,M.
Zs 5. hP2X2~ and hP2X2~receptors
Transient external application of ATP (30 pM) to oocytes injected with hP2X2d
or hP2Xzd RNA had no effect (Figure 10, panels C and D).
6. Conclusions
3o Using an electrophysiological approach to analyze hP2X2a_d receptor
function,
we have shown that two receptor subtypes (hP2Xza and hP2X2b) can be
selectively
activated by ATP, but not a~iMe-ATP. These responses are also non-
desensitizing.
The hP2X2~ and hP2X2d subtypes expressed alone did not respond to ATP. These
data support the formation of functional homomeric recombinant hP2X2a and
hP2X2b
3s ion channel receptors.
