# Patent

## Title
Canine cold- and menthol-sensitive receptor 1

## Abstract

The present invention provides nucleic acid and polypeptide sequences describing a novel canine cold- and menthol-sensitive receptor, herein named as canine CMR1 (cCMR1). The isolated nucleic acid or polypeptide molecule of the invention can be used in detection assays and screening assays.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Application Nos. 60/560,400 filed on Apr. 8, 2004 and 60/621,223 filed on Oct. 22, 2004, the entire contents of which are incorporated by reference herein.


FIELD OF THE INVENTION
The present invention relates to thermal receptor ion channel proteins. In particular, the present invention relates to isolated nucleic acid molecules and polypeptides of a novel canine cold- and menthol-sensitive receptor, CMR1, and uses thereof.
BACKGROUND
Considerable efforts have been put into elucidating the biochemical mechanisms involved in the detection, transduction and transmission of hot and cold sensations in neuronal tissues. Thermal stimuli activate specialized receptors located on sensory neurons, such as those deriving from the dorsal root ganglion (DRG) and the trigeminal ganglion (TG). When these stimuli are in the noxious range (i.e, very hot or cold), they activate a certain subset of thermal receptors on a sub-population of sensory neurons called nociceptors (pain-sensing neurons). Upon activation, the thermal receptors (e.g., ion channels) transduce the noxious stimulus into an electrical signal that is propagated along the sensory neuron to the spinal cord, where it is relayed to the brain, ultimately leading to the perception of pain. Accordingly, these thermal receptors represent highly promising targets for developing drugs for the treatment of various painful conditions.
Several temperature-activated receptors have been implicated in sensing heat. TRPV1 (VR1: a capsaicin- and heat-activated channel) is activated near 43° C., a temperature most mammals perceive as noxious. Other TRPV channels with greater than 40% amino acid level identity to TRPV1 also have been cloned and characterized as thermosensors. These channels are activated at various heat thresholds, ranging from 39° C. (warm) for TRPV3 to 55° C. (high-threshold noxious heat) for TRPV2/VRL1 (See Story et al., Cell, 2003, 112:819-829, and references therein). In contrast, TRPV4 is constitutively opened at room temperature being activated at temperatures greater than approximately 27° C. (Güler et al., J. Neurosci. 2002). These temperature-activated receptors belong to the transient receptor potential (TRP) family of non-selective cation channels, which in C. elegans and D. melanogaster are involved in mechano- and osmoregulation. TRP channels are divided into three subfamilies designated TRPC (canonical or capacitive subfamily), TRPV (vanilloid subfamily), and TRPM (melanostatin subfamily). All have six putative transmembrane domains with a proposed pore region between transmembrane domains five and six. TRP channels are thought to have cytoplasmic N- and C termini (See Story et al., supra, and references therein).
More recently, proteins have been discovered that fall within the TRP family of proteins and modulate responses to cold stimuli. A rat CMR1 protein (for “cold- and menthol-sensitive receptor”; McKemy, D. D., et al., Nature, 416:52-58, 2002) and a mouse TRPM8 protein (for “transient receptor potential channel, melanostatin subfamily, type 8”; Peier, A. M. et al., Cell 108:705-715, 2002) appear to function as excitatory ion channels that are activated upon exposure to relatively low temperatures. The threshold of TRPM8 activation is approximately about 23° C. The rat CMR1 and mouse TRPM8 are also sensitive to compounds that provoke cold sensations, such as menthol and icilin. Interestingly, the rat CMR1 and mouse TRPM8 share over 90% sequence identity over the entire length of their amino acid sequences.
There is a need to identify additional thermal receptors, as they are potential targets for the treatment of pain. There is also a need to identify thermal receptors in different species, as they can be used as model systems to investigate the effects of test compounds. Particularly, there is a need for systems that can be used to test compounds that potentially increase or decrease the activity of a thermal receptor responding to cold stimuli. Identification and testing of such compounds would enable the treatment of various disorders associated with chronic pain or for uses in other conditions in which tissue cooling is desirable.
SUMMARY
It has now been discovered that a canine protein, designated canine CMR1 (cCMR1) herein, modulates responses to cold stimuli and belongs to the TRP family of proteins.
In one general aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide capable of detecting and transducing cold stimuli and having at least 96% sequence identity to SEQ ID NO: 2. In one embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a cCMR1 protein having an amino acid sequence of SEQ ID NO: 2. The invention also provides expression vectors or recombinant host cells comprising a nucleic acid molecule of the invention. The invention further provides a nucleic acid probe that selectively hybridizes to the nucleic acid molecule of the invention under stringent hybridization conditions, and a kit comprising such a probe.
In another general aspect, the invention provides a substantially purified polypeptide capable of detecting and transducing cold stimuli and having at least 96% sequence identity to SEQ ID NO: 2. In one embodiment, the invention provides a substantially purified polypeptide comprising a cCMR1 protein having an amino acid sequence of SEQ ID NO: 2. The invention also provides a method of expressing the polypeptide of the invention, comprising the steps of: a) introducing an expression vector capable of encoding a polypeptide of the invention into a cell; and b) culturing the cells under conditions that allow expression of the polypeptide from the expression vector. The invention further provides an antibody that binds selectively to a polypeptide of the invention, and a kit comprising such an antibody.
The invention provides methods of detecting a nucleic acid molecule or polypeptide of the invention, comprising the step of contacting the nucleic acid molecule or polypeptide with an agent capable of binding specifically to the nucleic acid molecule or polypeptide.
The invention provides a method of identifying a compound that increases or decreases the expression of a cCMR1 protein, comprising the steps of:

    (a) contacting a test compound with a cell comprising a mechanism for regulating the expression of the cCMR gene; and (b) determining whether the test compound increases or decreases the expression of a gene controlled by said mechanism from the cell.


The invention also provides a method of identifying a compound that increases or decreases the conductivity of a cCMR1 ion channel, comprising the steps of: (a) contacting a test compound with the ion channel; and (b) determining whether the test compound increases or decreases the conductivity of the ion channel.
Other aspects of the invention include a method of identifying a compound that increases or decreases the conductivity of a mammalian CMR1 ion channel, comprising the steps of: (a) incubating the ion channel in a buffer solution containing a sub-inactivating amount of calcium; (b) activating the ion channel; (c) contacting the ion channel with a test compound; (d) increasing the amount of calcium in the buffer solution; and c) determining the intracellular amount of calcium, and comparing the amount with that of a control wherein the ion channel was not contacted with the test compound.
In addition, the invention provides a method of identifying a compound useful for treating pain, comprising the steps of: (a) contacting a test compound with a cCMR1 ion channel; and (b) determining whether the test compound increases or decreases the conductivity of the ion channel. In some embodiments, the method further comprises the steps of: (a) administering the test compound to an animal; and (b) determining the extent to which the test compound alters the nociceptive/nocifensive response of the animal.
Other aspects, features and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments and the appended claims.



DESCRIPTION OF THE FIGURES
FIG. 1 illustrates results of a cell-based calcium influx assay on recombinant cells stably transfected with a canine CMR1 expression vector. The cells showed an increase in calcium-mediated fluorescence in response to 10 μM of icilin (filled circle); or 100 μM of (−)-menthol (open circle). The compounds were added to the cells at time point 200 seconds. No calcium influx was observed upon the addition of buffer only to the cells (open triangle).
FIG. 2 illustrates results of a cell-based calcium influx assay using a loading buffer that is substantially free of calcium. Recombinant cells stably transfected with a rat CMR1 expression vector (filled circle) showed an increase in calcium-mediated fluorescence upon the addition of 4 mM Ca2+. The non-transformed cell (open circle) had less Ca2+ influx upon the addition of 4 mM Ca2+. The Ca2+ was added to the cells at time point 10 seconds.
FIG. 3 illustrates that cCMR1 is activated by mustard oil, a pungent compound. Recombinant cells stably transfected with cCMR1 showed an increase in calcium-mediated fluorescence upon the addition of 1 mM mustard oil (dash line) or 100 nM icilin as the positive control (solid line). Buffer alone was used as the negative control (dot line).
FIG. 4 illustrates that cCMR1 is strongly outwardly rectifying and non-selective to cations. The solid line represents the whole-cell patch clamp recording of cCMR1 performed in the presence of 100 μM menthol, whereas the dashed line represents the buffer control.
FIG. 5 illustrates the temperature sensitivity of cCMR1. The current passing through the cell was significantly increased as the temperature of the solution perfusing the cCMR1-expressing cell was lowered, demonstrating an activation threshold of about 17° C.
FIG. 6 illustrates that extracellular Ca2+ desensitizes the cCMR1 channel. The lowest trace represents the whole-cell patch clamp recording of cCMR1 in the presence of 100 μM menthol and in the absence of extracellular Ca2+, whereas the upper most, black trace, normalized to the Ca2+-free trace for display clarity, represents current activated by 100 μM menthol in the presence of 1.8 mM extracellular Ca2+.
FIG. 7 illustrates the concentration dependence of the inhibition of the current amplitude of cCMR1 channel by extracellular Ca2+. The channel was voltage-clamped at −80 mV and activated by 1 mM menthol. The dashed line is a logistic function representing the best fit to the data, with an IC50 value of 1.6 mM.
FIG. 8 illustrates the voltage dependence of the inhibition of the current amplitude of cCMR1 channel by extracellular Ca2+. The channel was activated by 1 mM menthol.



DETAILED DESCRIPTION
All publications cited herein are hereby incorporated by reference. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains.
As used herein, the terms “comprising”, “containing”, “having” and “including” are used in their open, non-limiting sense.
The following are abbreviations that are at times used in this specification:
bp=base pair
cDNA=complementary DNA
CMR1=cold- and menthol-sensitive receptor 1;
cCMR1=canine cold- and menthol-sensitive receptor 1;
DRG=dorsal root ganglion
ELISA=enzyme-linked immunoabsorbent assay
FLIPR=fluorescence imaging plate reader
kb=kilobase; 1000 base pairs
nt=nucleotide
PAGE=polyacrylamide gel electrophoresis
PCR=polymerase chain reaction
RT-PCR=Reverse transcription polymerase chain reaction
SDS=sodium dodecyl sulfate
SSC=sodium chloride/sodium citrate
TG=trigeminal ganglion
TRPM8=transient receptor potential channel, melanostatin subfamily, type 8
UTR=untranslated region
“An activity”, “a biological activity”, or “a functional activity” of a polypeptide or nucleic acid refers to an activity exerted by a polypeptide or nucleic acid molecule as determined in vivo, or in vitro, according to standard techniques. Such activities can be a direct activity, such as an ion channel activity, an association with or an enzymatic activity on a second protein, or an indirect activity, such as a cellular signaling activity mediated by interaction of the protein with one or more than one additional protein or other molecule(s), including but not limited to, interactions that occur in a multi-step, serial fashion.
A “biological sample” as used herein refers to a sample containing or consisting of cell or tissue matter, such as cells or biological fluids isolated from a subject. The “subject” can be a mammal, such as a rat, a mouse, a monkey, or a human, that has been the object of treatment, observation or experiment. Examples of biological samples include, for example, sputum, blood, blood cells (e.g., white blood cells), amniotic fluid, plasma, semen, bone marrow, tissue or fine-needle biopsy samples, urine, peritoneal fluid, pleural fluid, and cell cultures. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. A test biological sample is the biological sample that has been the object of analysis, monitoring, or observation. A control biological sample can be either a positive or a negative control for the test biological sample. Often, the control biological sample contains the same type of tissues, cells and/or biological fluids of interest as that of the test biological sample.
A “cell” refers to at least one cell or a plurality of cells appropriate for the sensitivity of the detection method. Cells suitable for the present invention may be bacterial, but are preferably eukaryotic, and are most preferably mammalian.
A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a primary cell, that derives clonal expansion of cells and is capable of stable growth in vitro for many generations.
A “cold- and menthol-sensitive receptor”, a “CMR1”, a “transient receptor potential channel, melanostatin subfamily, type 8”, or a “TRPM8” protein, each refers to a protein that is capable of sensing and transducing cold stimuli, such as cold temperatures or compounds that provoke cold sensations including, but not limited to, menthol and icilin. A “CMR1” can form an excitory ion channel, the CMR1 channel, which can be activated by low temperature or compounds that provoke cold sensations. An activated CMR1 channel gates the influx of Ca++ ions through the channel, resulting in membrane depolarization. A CMR1 protein can, (1) have greater than about 80% amino acid sequence identity to a canine CMR1 (cCMR1) protein depicted in SEQ ID NO: 2; or (2) bind to antibodies, e.g., polyclonal or monoclonal antibodies, raised against a cCMR1 protein depicted in SEQ ID NO: 2. In some embodiments, the CMR1 has greater than about 85, 90, or 95 percent amino acid sequence identity to SEQ ID NO: 2. Exemplary CMR1 includes cCMR1, which includes structural and functional polymorphisms of the cCMR1 protein depicted in SEQ ID NO: 2. “Polymorphism” refers to a set of genetic variants at a particular genetic locus among individuals in a population. CMR1 also includes orthologs of the canine CMR1 in other animals including human, rat, mouse, pig, dog and monkey, for example, the structural and functional polymorphisms of the rat CMR1 (GenBank protein ID: NP—599198), or mouse TRPM8 (GenBank protein ID: NP—599013). CMR1 genes are naturally expressed in certain neuronal tissues, such as DRG and TG.
“CMR1 activation temperature” is the temperature at which a CMR1 channel exhibits at least a 10% increase in its conductivity compared to the baseline. A person skilled in the art can experimentally determine the activation temperature for a CMR1 channel. In some embodiments, “CMR1 activation temperature” is the temperature at which a CMR1 channel exhibits at least a 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% increase in its conductivity compared to the baseline. “CMR1 activation temperature” is typically of about 6° C.-28° C. In some embodiments, the CMR1 activation temperature is about 15° C.-28° C., 19° C.-28° C., 23° C.-28° C., or, 19° C.-24° C.
“CMR1 non-activation temperature” is the temperature that falls outside of the range for a “CMR1 activation temperature”. An exemplary CMR1 non-activation temperature is 37° C.
A “gene” is a segment of DNA involved in producing a peptide, polypeptide, or protein, and the mRNA encoding such protein species, including the coding region, non-coding regions preceding (“5′ UTR”) and following (“3′ UTR”) the coding region. A “gene” may also include intervening non-coding sequences (“introns”) between individual coding segments (“exons”). “Promoter” means a regulatory sequence of DNA that is involved in the binding of RNA polymerase to initiate transcription of a gene. Promoters are often upstream (“5′ to”) the transcription initiation site of the gene. A “regulatory sequence” refers to the portion of a gene that can control the expression of the gene. A “regulatory sequence” can include promoters, enhancers and other expression control elements such as polyadenylation signals, ribosome binding site (for bacterial expression), and/or, an operator. An “enhancer” means a regulatory sequence of DNA that can regulate the expression of a gene in a distance- and orientation-dependent fashion. A “coding region” refers to the portion of a gene that encodes amino acids and the start and stop signals for the translation of the corresponding polypeptide via triplet-base codons.
“Nucleic acid sequence” or “nucleotide sequence” refers to the arrangement of either deoxyribonucleotide or ribonucleotide residues in a polymer in either single- or double-stranded form. Nucleic acid sequences can be composed of natural nucleotides of the following bases: thymidine, adenine, cytosine, guanine, and uracil; abbreviated T, A, C, G, and U, respectively, and/or synthetic analogs.
The term “oligonucleotide” refers to a single-stranded DNA or RNA sequence of a relatively short length, for example, less than 100 residues long. For many methods, oligonucleotides of about 16-25 nucleotides in length are useful, although longer oligonucleotides of greater than about 25 nucleotides may sometimes be utilized. Some oligonucleotides can be used as “primers” for the synthesis of complimentary nucleic acid strands. For example, DNA primers can hybridize to a complimentary nucleic acid sequence to prime the synthesis of a complimentary DNA strand in reactions using DNA polymerases. Oligonucleotides are also useful for hybridization in several methods of nucleic acid detection, for example, in Northern blotting or in situ hybridization.
A “polypeptide sequence” or “protein sequence” refers to the arrangement of amino acid residues in a polymer. Polypeptide sequences can be composed of the standard 20 naturally occurring amino acids, in addition to rare amino acids and synthetic amino acid analogs. Shorter polypeptides are generally referred to as peptides.
An “isolated” nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid. An “isolated” nucleic acid molecule can be, for example, a nucleic acid molecule that is free of at least one of the nucleotide sequences that naturally flank the nucleic acid molecule at its 5′ and 3′ ends in the genomic DNA of the organism from which the nucleic acid is derived. Isolated nucleic acid molecules include, without limitation, separate nucleic acid molecules (e.g., cDNA or genomic DNA fragments produced by PCR or restriction endonuclease treatment) independent of other sequences, as well as nucleic acid molecules that are incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid molecule can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid molecule. An isolated nucleic acid molecule can be a nucleic acid sequence that is: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) synthesized by, for example, chemical synthesis; (iii) recombinantly produced by cloning; or (iv) purified, as by cleavage and electrophoretic or chromatographic separation.
An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a “contaminating protein”). When the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest. Isolated biologically active polypeptide can have several different physical forms. The isolated polypeptide can exist as a full-length nascent or unprocessed polypeptide, or as a partially processed polypeptide or as a combination of processed polypeptides. The full-length nascent polypeptide can be postranslationally modified by specific proteolytic cleavage events that result in the formation of fragments of the full-length nascent polypeptide. A fragment, or physical association of fragments can have the biological activity associated with the full-length polypeptide; however, the degree of biological activity associated with individual fragments can vary. An isolated or substantially purified polypeptide, can be a polypeptide encoded by an isolated nucleic acid sequence, as well as a polypeptide synthesized by, for example, chemical synthetic methods, and a polypeptide separated from biological materials, and then purified, using conventional protein analytical or preparatory procedures, to an extent that permits it to be used according to the methods described herein.
“Recombinant” refers to a nucleic acid, a protein encoded by a nucleic acid, a cell, or a viral particle, that has been modified using molecular biology techniques to something other than its natural state. For example, recombinant cells can contain nucleotide sequence that is not found within the native (non-recombinant) form of the cell or can express native genes that are otherwise abnormally expressed, under-expressed, or not expressed at all. Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means. The term also encompasses cells that contain an endogenous nucleic acid that has been modified without removing the nucleic acid from the cell; such modifications include those obtained, for example, by gene replacement, and site-specific mutation.
A “recombinant host cell” is a cell that has had introduced into it a recombinant DNA sequence. Recombinant DNA sequence can be introduced into host cells using any suitable method including, for example, electroporation, calcium phosphate precipitation, microinjection, transformation, biolistics and viral infection. Recombinant DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. For example, the recombinant DNA can be maintained on an episomal element, such as a plasmid. Alternatively, with respect to a stably transformed or transfected cell, the recombinant DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the stably transformed or transfected cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA. Recombinant host cells may be prokaryotic or eukaryotic, including bacteria such as E. coli, fungal cells such as yeast, mammalian cells such as cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells such as Drosophila- and silkworm-derived cell lines. It is further understood that the term “recombinant host cell” refers not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
As used herein, “operably linked”, refers to a functional relationship between two nucleic acid sequences. For example, a promoter sequence that controls expression (for example, transcription) of a coding sequence is operably linked to that coding sequence. Operably linked nucleic acid sequences can be contiguous, typical of many promoter sequences, or non-contiguous, in the case of, for example, nucleic acid sequences that encode repressor proteins. Within a recombinant expression vector, “operably linked” is intended to mean that the coding sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the coding sequence, e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell.
“Vector” or “construct” refers to a nucleic acid molecule into which a heterologous nucleic acid can be or is inserted. Some vectors can be introduced into a host cell allowing for replication of the vector or for expression of a protein that is encoded by the vector or construct. Vectors typically have selectable markers, for example, genes that encode proteins allowing for drug resistance, origins of replication sequences, and multiple cloning sites that allow for insertion of a heterologous sequence. Vectors are typically plasmid-based and are designated by a lower case “p” followed by a combination of letters and/or numbers. Starting plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by application of procedures known in the art. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well-known and readily available to those of skill in the art. Moreover, those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure.
“Sequence” means the linear order in which monomers occur in a polymer, for example, the order of amino acids in a polypeptide or the order of nucleotides in a polynucleotide.
“Sequence identity or similarity”, as known in the art, is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. As used herein, “identity”, in the context of the relationship between two or more nucleic acid sequences or two or more polypeptide sequences, refers to the percentage of nucleotide or amino acid residues, respectively, that are the same when the sequences are optimally aligned and analyzed. For purposes of comparing a queried sequence against, for example, the amino acid sequence SEQ ID NO 2, the queried sequence is optimally aligned with SEQ ID NO 2 and the best local alignment over the entire length of SEQ ID NO 2 (1104 amino acids) is obtained.
Analysis can be carried out manually or using sequence comparison algorithms. For sequence comparison, typically one sequence acts as a reference sequence, to which a queried sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, sub-sequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
Optimal alignment of sequences for comparison can be conducted, for example, by using the homology alignment algorithm of Needleman & Wunsch, J Mol. Biol., 48:443 (1970). Software for performing Needleman & Wunsch analyses is publicly available through the Institut Pasteur (France) Biological Software website. The NEEDLE program uses the Needleman-Wunsch global alignment algorithm to find the optimum alignment (including gaps) of two sequences when considering their entire length. The identity is calculated along with the percentage of identical matches between the two sequences over the reported aligned region, including any gaps in the length. Similarity scores are also provided wherein the similarity is calculated as the percentage of matches between the two sequences over the reported aligned region, including any gaps in the length. Standard comparisons utilize the EBLOSUM62 matrix for protein sequences and the EDNAFULL matrix for nucleotide sequences. The gap open penalty is the score taken away when a gap is created; the default setting using the gap open penalty is 10.0. For gap extension, a penalty is added to the standard gap penalty for each base or residue in the gap; the default setting is 0.5.
Hybridization can also be used as a test to indicate that two polynucleotides are substantially identical to each other. Polynucleotides that share a high degree of identity will hybridize to each other under stringent hybridization conditions. “Stringent hybridization conditions” has the meaning known in the art, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989). An exemplary stringent hybridization condition comprises hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC and 0.1% SDS at 50-65° C., depending upon the length over which the hybridizing polynucleotides share complementarity.
A “reporter gene” refers to a nucleic acid sequence that encodes a reporter gene product. As is known in the art, reporter gene products are typically easily detectable by standard methods. Exemplary suitable reporter genes include, but are not limited to, genes encoding luciferase (lux), β-galactosidase (lacZ), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-glucuronidase, neomycin phosphotransferase, and guanine xanthine phosphoribosyl-transferase proteins.
A “compound that increases the conductivity of a CMR1 channel” includes any compound that results in increased passage of ions through the CMR1 channel. In one embodiment, such a compound is an agonist for the CMR channel that binds to the CMR1 channel to increase its conductivity. In another embodiment, such a compound is a positive allosteric modulator, which interacts with the CMR1 channel at allosteric sites different from the agonist binding-site, but potentiates the response of the channel to an agonist.
A “compound that decreases the conductivity of a CMR1 channel” includes any compound that results in decreased passage of ions through the CMR1 channel. In one embodiment, such a compound is an antagonist for the CMR channel that binds to the CMR1 channel to counter, decrease or limit the action of an agonist in either a competitive or non-competitive fashion. In another embodiment, such a compound is a negative allosteric modulator, which interacts with the CMR1 channel at allosteric sites different from the agonist or antagonist binding-site, and decreases the response of the channel to an agonist. In yet another embodiment, such a compound is an inverse agonist that binds to the CMR1 channel and decreases the conductivity of the channel in the absence of any other compound, such as an agonist.
“Membrane potential”, “transmembrane potential” or “transmembrane potential difference” as used herein, each refers to the electrical potential difference across the plasma membrane, the external, limiting lipid bilayer membrane of cells. Almost all animal cells are negative inside, with resting potentials in the range −20 to −100 mV. “Resting potential” as used herein refers to the electrical potential of the inside of a cell relative to its surroundings when the cell is at rest.
“Depolarization” as used herein refers to the tendency of the cell membrane potential to become more positive, for example from −90 mV to −50 mV.
“Hyperpolarization” as used herein refers to the tendency of the cell membrane potential to become more negative, for example from −50 mV to −90 mV. In practicing the present invention, many conventional techniques in molecular biology, microbiology and recombinant DNA are used. These techniques are well-known and are explained in, for example, Current Protocols in Molecular Biology, Vols. I, II, and III, F. M. Ausubel, ed. (1997); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).
In one aspect, the present invention relates to novel cCMR1 (cCMR1) nucleic acids, polypeptides encoded by these nucleic acids, recombinant cCMR1 materials, and methods involving the production, detection, and utilization of these materials.
The CMR1 nomenclature was established by McKemy, D. D., et al. (Nature, 416:52-58, 2002) and was used to describe a cold- and menthol-sensitive receptor expressed in DRG and TG neurons of rats. The human CMR1 (also known as human TRPM8) is 92% identical to the amino acid sequence of rat CMR1 and has previously been identified as a prostate-specific transcript and has also been found to be expressed in various tumor tissue, including prostate, melanoma, colorectal and breast carcinoma (Tsavaler, L., et al. Cancer Res. 61:3760-3769, 2002). Mouse CMR1 (also known as mouse TRPM8) was cloned from a mouse DRG cDNA preparation and was shown to be 93% identical to the human CMR1 amino acid sequence (Peier, A. M. et al., Cell 108:705-715, 2002).
In the present invention, the canine cCMR1 gene was cloned from a cDNA library prepared from canine DRG tissue. The cCMR1 cDNA was sequenced, including the cCMR1 open reading frame (ORF) and 5′ and 3′ untranslated regions of the corresponding mRNA. The cCMR1 cDNA sequence is shown as SEQ ID NO: 1 (Table 1). SEQ ID NO: 1 encodes a 1104 residue polypeptide (SEQ ID NO: 2), also shown in Table 1, which is aligned with the CMR1 protein sequences from human, mouse, and rat (see Table 2). Based on this alignment, the cCMR1 polypeptide shares the greatest amino acid identity with the human CMR1 at 95.23%.
In the present invention, the cCMR1 nucleic acid was also subcloned into an expression vector and transformed into a host cell for expression of the cCMR1 protein. This recombinant cCMR1 cell system was shown to express a functional cCMR1 protein that allowed influx of Ca++ ions when the recombinant cCMR1 cells were incubated at low temperatures or exposed to menthol or icilin. The recombinant cCMR1 system is useful for screening and for identifying compounds that modulate cCMR1 function or expression. Compounds that modulate CMR1 function or expression can be therapeutically useful. These compounds can be identified using, for example, a recombinant system expressing the cCMR1 protein and then tested in vivo in dogs or any other suitable mammals, to establish dosing parameters that can be useful in humans.
Modulation of the function or expression of CMR1 proteins can be advantageous for the treatment of various painful conditions. Since the CMR1 receptor is responsive to cold and compounds, such as menthol and icilin, that mimic a cold-like sensation, it is anticipated that modulation of cCMR1 activity is also relevant for therapeutic applications where cold or menthol treatment is used as a method of pain relief or other relief, such as congestive rhinitis, cough or asthmatic bronchitis. For example, modulation of function or expression of CMR1 proteins can be useful for patients having dermal or mucus membrane conditions, such as skin inflammation and dermal burns, including sunburn and razor burn, or sore throat. Modulation of CMR1 activity can also be relevant in patients suffering from hypersensitivity to cold that causes cold allodynia. Modulation of CMR1 activity can also be relevant for treating acute pain, for example, toothache (odontalgia) and other trigeminally distributed pains, such as trigeminal neuralgia (tic douleureux) and temperomandibular joint pain.
In addition, since human CMR1 has been identified as a marker that is associated with tumor growth (Tsavaler, L., et al. Cancer Res. 61:3760-3769, 2002), cCMR1 can also be useful for the diagnosis of various cellular proliferation disorders in dogs.
In attempts to clone the cCMR1 homologue, a PCR-based strategy was employed. Oligonucleotide primers were synthesized according to the sequences set forth in SEQ ID NO: 3 (cmr1-23) and SEQ ID NO: 4 (cmr1-26). These primers were able to successfully amplify a portion of the cCMR1 sequence from position 1761 to position 2886 of SEQ ID NO: 1. The PCR product, which was approximately 1.1 kb in size, was purified and then subcloned into a sequencing vector. Based on the sequence of the 1.1 kb cCMR1 fragment, new primers were developed and used in separate PCR reactions with RACE (rapid amplification of cDNA ends)-modified canine DRG cDNA. The complete sequence of the cCMR1 cDNA, including both 5′ and 3′ untranslated regions, was obtained (SEQ ID NO: 1). The open reading frame of cCMR1 encodes a 1104 residue polypeptide (SEQ ID NO: 2), as shown in Tables 1.
Therefore, in one embodiment, the invention provides an isolated nucleic acid sequence comprising a sequence from position 69 to 3380 of SEQ ID NO: 1. Position 69 to 3380 of SEQ ID NO 1 is an open-reading frame sequence (coding region), which can encode a CMR1 polypeptide according to SEQ ID NO: 2. The invention also provides isolated nucleic acids sequences corresponding to the region upstream from the cCMR1 open-reading frame, for example, from position 1 to 69 of SEQ ID NO: 1 and isolated nucleic acid sequences corresponding to the region downstream from the cCMR1 open-reading frame, for example, from position 3380 to 3815 of SEQ ID NO: 1. Therefore, in another embodiment, the invention provides an isolated nucleic acid sequence that includes a sequence from position 1 to 69 of SEQ ID NO: 1, and in another embodiment from position 3380 to 3815 of SEQ ID NO: 1.
Isolated nucleic acids comprising fragments of SEQ ID NO: 1 are useful for a variety of purposes. For example, these sequences can be used as oligonucleotide probes for the detection of CMR1 nucleic acids or for the detection of sequences that flank CMR1 nucleic acids. They can be used as oligonucleotide primers for the amplification of CMR1 nucleic acids. They can also be used for the preparation of chimeric nucleic acids that encode a portion or all of the cCMR1 polypeptide fused to another polypeptide sequence, for example, one or more motifs or domains of the cCMR1 sequence recombined with one or more motifs or domains from one or more heterologous sequences. Further, they can be used for manipulating the structure of the cCMR1 gene.
In yet another embodiment, the invention provides an isolated nucleic acid comprising a nucleic acid sequence that encodes a polypeptide comprising SEQ ID NO: 2. Due to the degeneracy of the genetic code, more than one codon may be used to encode a particular amino acid, and therefore, a cCMR1 amino acid sequence (for example, SEQ ID NO: 2) can be encoded by any one of a plurality of nucleic acid sequences. Isolated nucleic acid includes sequences wherein one or more codons in the sequence are replaced by codons of a different sequence but that code for the same amino acid residue are herein referred to as “conservative codon substitutions”. Therefore, the invention encompasses nucleic acid sequences encoding SEQ ID NO: 2 that have one or more than one conservative codon substitution. One of skill in the art would be able to determine a particular nucleic acid sequence having one or more than one conservative codon substitution and encoding SEQ ID NO: 2, based on the sequence information provided herein. Conservative codon substitutions can be made in the nucleic acid sequence encoding the CMR1 polypeptide, for example, the codons TTT and TTC (collectively referred to as TTT/C) can encode a Phe (phenylalanine) residue; other codon substitutions are as follows: TTA/G and CTT/C/A/G: Leu; ATT/C: Ile; ATG: Met; GTT/C/A/G: Val; TCT/C/A/G: Ser; CCT/C/A/G: Pro; ACT/C/A/G: Thr; GCT/C/A/G: Ala; TAT/C: Tyr; CAT/C: His; CAA/G: Gln; AAT/C: Asn; AAA/G: Lys; GAT/C: Asp; GAA/G Glu; TGT/C: Cys; CGT/C/A/G: Arg; AGT/C: Ser; AGA/G; Arg; GGT/C/A/G:Gly. Conservative codon substitutions can be made at any position in the nucleic acid sequence that encodes the cCMR1 polypeptide.
As shown herein, position 69 to position 3380 of SEQ ID NO: 1 encodes a 1104 amino acid residue polypeptide (SEQ ID NO: 2), which is the predicted sequence of the canine CMR1 as naturally expressed. As shown in Table 2, SEQ ID NO: 2 was aligned to the human, mouse and rat CMR1 protein sequences. By alignment, cCMR1 polypeptide sequence (SEQ ID NO: 2) is most identical to the human CMR1 protein sequence, sharing 1052 out of 1104 residues (95.23% identity). The cCMR1 protein sequence shares a lower degree of identity with the mouse (1042/1104: 94.38% identity) and rat (1043/1104: 94.47% identity) CMR1 polypeptide sequences.
As indicated, the dog, human, mouse and rat CMR1 sequences, as shown in Table 2, generally share greater than 90% amino acid identity. However, at certain amino acid positions, the canine sequence differs from one or more of the human, mouse or rat sequences. The amino acid positions wherein the canine residue differs from one or more than one other species are more variable as compared to positions wherein the residue is identical in the human, dog, mouse and rat sequences. For example, based on the sequence alignment, the amino acid residues at positions 1, 2 and 3 of SEQ ID NO: 2 are identical to those of the human, mouse and rat sequence. However, the amino acid residues at position 4 and 5 of SEQ ID NO: 2 vary with regard to the human sequence, and the amino acid residue at position 28 of SEQ ID NO: 2 varies with regard to human, mouse and rat sequences. Amino acid positions wherein there is at least one difference between the canine sequence and any one of the human, mouse or rat CMR1 sequences are herein referred to as “CMR-family variant positions”. A list of CMR-family variant positions is provided in Table 3.
Based on this analysis, a cCMR1 polypeptide having a substitution of one or more CMR-family variant amino acids is anticipated to have CMR1 biological activity. That is, SEQ ID NO: 2 can be substituted at one or more CMR-family variant amino acid positions with an amino acid selected from amino acid residues found in the human, mouse, or rat sequences, or an equivalent amino acid, at that same position. The amino acid that replaces a cCMR1 amino acid is herein referred to as a “CMR-family variant amino acid”. A “CMR-family variant amino acid” consists of an amino acid that differs from the cCMR1 amino acid and that is the amino acid present in the CMR1 sequence of other mammals, such as human, mouse or rat. A list of suitable CMR-family variant amino acids, any of which can be use to replace an original cCMR1 amino acid residue can also be found in Table 3. For example, a CMR-family variant amino acid at position 4 suitable for the replacement of the glutamic acid (E) of SEQ ID NO: 2, is arginine (R, as occurs in human CMR1).
At some CMR-family variant amino acid positions, the canine, human, mouse and rat amino acid residues share a common chemical property. For example, CMR-family variant amino acids at positions 18 and 34 of SEQ ID NO: 2 can include a hydrophobic amino acid residue, for example, methionine (M) or leucine (L). Other hydrophobic amino acids include glycine, valine, isoleucine and proline. Other amino acid groups include “basic amino acids,” which include histidine, lysine, and arginine; “acidic amino acids,” which include glutamic acid and aspartic acid; “aromatic amino acids,” which include phenylalanine, tryptophan, and tyrosine; “small amino acids,” which include glycine and alanine; “nucleophilic amino acids,” which include serine, threonine, and cysteine; and “amide amino acids,” which include aparagine and glutamine.
Therefore, in another aspect, the invention provides a nucleic acid encoding a CMR1 polypeptide according to SEQ ID NO: 2 that includes a CMR-family variant amino acid. In some embodiments, cCMR1 polypeptides include CMR-family variant amino acids in less than 4% of the original cCMR1 amino acid residues. Preferably the cCMR1 polypeptides include CMR-family variants in less than about 2% of the original cCMR1 amino acid residues, and most preferably less than about 1% of the original cCMR1 amino acid residues.
The invention also provides isolated nucleic acid molecules that are complementary to any isolated nucleic acid molecules, as described herein.
The isolated nucleic acid of the invention can also include nucleic acid sequences that encode the cCMR1 polypeptide having additional amino acid residues. In some embodiments, the additional amino acids are present at the amino terminus, the carboxyl terminus, within the cCMR1 sequence or combinations of these locations. cCMR1 polypeptides having these types of additional amino acid sequences can be referred to as “cCMR1 fusion proteins”. In some cases, it may be more appropriate to refer to them otherwise as “chimeric” or “tagged” cCMR1 proteins, or the like, depending on the nature of the additional amino acid sequences. Nonetheless, one will be able to discern a CMR1 polypeptide having additional amino acid sequences given the sequence information provided herein. The additional amino acid residues can be short, for example, from one to about 20 additional amino acid residues, or longer, for example, greater than about 20 additional amino acid residues. The additional amino acid residues can serve one or more functions or purposes including, for example, serving as epitopes for protein (e.g., antibody) or small molecule binding; serving as tags for intracellular and extracellular trafficking; providing additional enzymatic or other activity; or providing a detectable signal.
For example, a nucleic acid sequence can encode a cCMR1 fusion protein, which can include additional amino acid residues providing coordinates for bonding (such as ionic, covalent, coordinative, hydrogen or Van der Waals bonding or combinations thereof with organic or inorganic compounds. Useful additional amino acid sequences include, for example, poly-histidine residues useful for protein purification via Ni+-coupled residue, constant domains of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3), albumin, hemagluttinin (HA) or myc affinity epitope tags useful for the formation of immuno-complexes for detection or purification (antibodies against these moieties can be obtained commercially), polypeptides useful for detection such as the green fluorescent protein (GFP), enzymes such as beta-galactosidase (B-Gal) chloramphenicol acetyltransferase (CAT), luciferase, and alkaline phosphatase (A), signal sequences for protein trafficking and protease cleavage sequences useful for separating additional amino acid sequences from the cCMR1 sequence, if desired.
In another aspect, diagnostic assays are provided which are capable of detecting the expression of cCMR1, such as cCMR1 protein or nucleic acid. Expression of the cCMR1 proteins can be detected by a probe, which is detectably labeled or which can be subsequently labeled. Typically, the probe is an antibody that recognizes the expressed protein, as described above, especially a monoclonal antibody. Accordingly, in one embodiment, an assay capable of detecting the expression of cCMR1 protein comprises contacting a canine tissue sample with one or more than one monoclonal and/or polyclonal antibody that binds to cCMR1.
cCMR1 nucleic acids and proteins, antibodies directed against CMR1 and biological systems containing any of these components can be labeled with a detectable reagent, or a compound having specificity for cCMR1 can be labeled with a detectable reagent and used to detect the cCMR1 entity. Detectable reagents include compounds and compositions that can be detected by spectroscopic, biochemical, photochemical, bioelectronic, immunochemical, electrical, optical or chemical techniques. Examples of detectable moieties include, but are not limited to, radioisotopes (e.g., 32P 33P, 35S), chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, such as fluorescent markers and dyes, linked enzymes, mass spectrometry tags and magnetic labels.
Immunoassay methods that utilize antibodies include, but are not limited to, dot blotting, Western blotting, competitive and non-competitive protein binding assays, enzyme-linked immunosorbant assays (ELISA), immunohistochemistry, fluorescence-activated cell sorting (FACS), immuno-PCR, immunoprecipitation and others commonly used.
The level of expression of mRNA corresponding to the cCMR1 gene can be detected utilizing commonly used molecular biological methods, for example, Northern blotting, in situ hybridization, nuclease protection assays, RT-PCR (including real-time, quantitative PCR), high density arrays and other hybridization methods. Accordingly, in another embodiment, an assay capable of detecting the expression of one or more than one cCMR1 gene in a sample of canine tissue is provided, which comprises contacting a canine tissue sample with an oligonucleotide capable of hybridizing to a cCMR1 nucleic acid. The oligonucleotide primer is generally from 10-20 nucleotides in length for PCR/primer extension experiments. Longer oligonucleotides of approximately 40-50 nucleotides are more regularly utilized for in situ or blot hybridizations. Sequences even longer than 50 nucleotides can also be employed for the detection experiment. RNA can be isolated from the tissue sample by methods well-known to those skilled in the art as described, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1996). One preferred method for detecting the level of mRNA transcribed from the cCMR1 genes is by RT-PCR. Details of RT-PCR techniques are well known and also described herein.
Another preferred method for detecting the level of mRNA transcripts obtained from more than one of the disclosed genes involves hybridization of labeled mRNA to an ordered array of oligonucleotides or tissue. Such a method allows the level of transcription of a plurality of these genes to be determined simultaneously to generate gene expression profiles or patterns.
The oligonucleotides utilized in this hybridization method typically are bound to a solid support. Examples of solid supports include, but are not limited to, membranes, filters, slides, paper, nylon, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, polymers, polyvinyl chloride dishes, etc. Any solid surface to which the oligonucleotides can be bound, either directly or indirectly, either covalently or noncovalently, can be used. A particularly preferred solid substrate is a high-density array or DNA chip. These high-density arrays contain a particular oligonucleotide probe in a preselected location on the array. Each pre-selected location can contain more than one molecule of the particular probe. Because the oligonucleotides are at specified locations on the substrate, the hybridization patterns and intensities (which together result in a unique expression profile or pattern) can be interpreted in terms of expression levels of particular genes.
The oligonucleotide probes are preferably of sufficient length to specifically hybridize only to complementary transcripts of the above identified gene(s) of interest.
Optionally, all or a portion of the cCMR1 nucleic acid sequence can be used to probe nucleic acid preparations from other species to determine the presence of similar sequences. For example, all or a portion of the cCMR1 nucleic acid can be used as a probed to identify cDNA or genomic nucleic acid sequences from other species that are similar to the cCMR1 sequence. Positive clones can be identified as those that hybridize to the cCMR1 probe.
In addition, all or a portion of the cCMR1 nucleic acid or polypeptide sequence as provided by the invention can be used in computer-aided programs to identify other useful information, for example, proteins having homology to the cCMR1 sequence or molecules that bind to the cCMR1 sequence. For example, all or portions of the cCMR1 sequence can be used to screen various electronic databases to determine whether a member of the electronic database has homology to the cCMR1 sequence. Numerous genetic databases that are species-specific can be queried using any portion of the canine nucleic acid or polypeptide sequences as set forth herein. Either or both nucleic acid and protein searches can be performed.
In another aspect, a three-dimensional model of the cCMR1 polypeptide can be determined and used to identify molecules that bind to various portions of the protein structure. For example, using an isolated cCMR1 nucleic acid as described herein, the cCMR1 protein can be expressed in a cell system, purified and then crystallized in order to obtain information regarding the structure of the protein. Structural information can be obtained by performing, for example, X-ray diffraction or nuclear magnetic resonance spectroscopy. The location of amino acid residues and their side chains can be expressed as coordinates in a three-dimensional model. This information can then be provided to a computer program.
Molecular modeling programs can be used to determine whether a small molecule can fit into a functionally relevant portion, for example, an active site, of the cCMR1 polypeptide. Basic information on molecular modeling is provided in, for example, M. Schlecht, Molecular Modeling on the PC, 1998, John Wiley & Sons; Gans et al., Fundamental Principals of Molecular Modeling, 1996, Plenum Pub. Corp.; N. C. Cohen (editor), Guidebook on Molecular Modeling in Drug Design, 1996, Academic Press; and W. B. Smith, Introduction to Theoretical Organic Chemistry and Molecular Modeling, 1996. U.S. patents that provide detailed information on molecular modeling include U.S. Pat. Nos. 6,093,573; 6,080,576; 5,612,894; and 5,583,973.
Programs that can be useful for molecular modeling studies include, for example, GRID (Goodford, P. J., “A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules” J. Med. Chem., 28, pp. 849-857, 1985), available from Oxford University, Oxford, UK; MCSS (Miranker, A. and M. Karplus, “Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method.” Proteins: Structure, Function and Genetics, 11, pp. 29-34, 1991), available from Molecular Simulations, Burlington, Mass.; AUTODOCK (Goodsell, D. S. and A. J. Olsen, “Automated Docking of Substrates to Proteins by Simulated Annealing” Proteins: Structure. Function, and Genetics, 8, pp. 195-202, 1990); available from Scripps Research Institute, La Jolla, Calif.; and DOCK (Kuntz, I. D. et al., “A Geometric Approach to Macromolecule-Ligand Interactions” J. Mol. Biol., 161, pp. 269-288, 1982), available from University of California, San Francisco, Calif.
In addition to nucleic acid sequences encoding cCMR1 polypeptides, the invention also includes cCMR1 polypeptides, cCMR1 polypeptide variants, fragments of cCMR1 polypeptides and cCMR1 polypeptides having additional amino acids. Aspects of cCMR1 polypeptides encoded by nucleic acids are described herein, and these aspects can also apply to cCMR1 polypeptides.
In one embodiment, the invention provides an isolated polypeptide that includes the sequence of SEQ ID NO: 2.
In another embodiment, the invention provides an isolated polypeptide that includes the sequence of SEQ ID NO: 2 having CMR-family variant amino acids in less than 4% of the original cCMR1 amino acid residues. Preferably the cCMR1 polypeptides include CMR-family variants in less than about 2% of the original cCMR1 amino acid residues, and most preferably less than about 1% of the original cCMR1 amino acid residues.
As described herein, the cCMR1 polypeptide can also have additional amino acid residues at its amino terminus, its carboxyl terminus or both. Such additional residues are useful for a variety or purposes, including, for example, immunodetection, purification, cellular trafficking, enzymatic activity, etc.
The invention also provides fragments of the cCMR1 polypeptide. Fragments of the cCMR1 polypeptide can be useful for a number of purposes including, for example, antibody production. Portions of the cCMR1 polypeptide sequence, or the entire sequence itself, can be used to generate anti-CMR1 antibodies.
In another aspect, the present invention relates to antibodies that specifically recognize epitopes within the amino acid sequence of SEQ ID NO: 2. Useful antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, and biologically functional antibody fragments that are able to bind to a portion of the cCMR1 protein. Antibodies specific for proteins encoded by the aforementioned sequences have utilities in several types of applications. These antibodies can be used in diagnostic kits, for example, for any sort of assay wherein detection of cCMR1 is desired. They can also be used in the preparation of therapeutic agents, for example, wherein the anti-cCMR1 antibody itself is therapeutic or wherein the anti-cCMR1 antibody is coupled to a therapeutic agent. It is anticipated that anti-cCMR1 antibodies could be used for treating pain. In these cases an anti-cCMR1 antibody could modulate the activity of cCMR1, for example, providing either an agonistic (e.g., catalytic) or antagonistic activity.
The invention also provides methods for the production of canine-specific monoclonal anti-CMR1 antibodies. For the production of these monoclonal antibodies, peptides that provide unique anti-cCMR1 determinants can be used. Monoclonal antibodies are homogeneous clonal populations of antibodies that are directed to a specific antigen (i.e., epitope). To prepare anti-cCMR1 monoclonal antibodies, a peptide having cCMR1-specific sequence or a “cCMR1 epitope” is used. A cCMR1 sequence is one that is different at one or more positions relative to the dog, mouse and rat CMR1 sequences. In order to determine a cCMR1 specific sequence, one can refer to Table 2 provided herein.
Monoclonal antibodies (mAbs) can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, for example, the hybridoma technique (Kohler and Milstein, Nature, 256:495-497, 1975); the human B-cell hybridoma technique (Kosbor et al., Immunology Today, 4:72, 1983); and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD or any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo.
For the production of antibodies to the CMR1 protein, various host animals can be immunized by injection with the cCMR1 polypeptide, or a portion thereof. If the entire cCMR1 polypeptide is used, antibodies specific to cCMR1 along with anti-CMR antibodies that are cross-reactive with other CMR1 proteins from different species may be generated. For example, polyclonal antibody preparations are a heterogeneous population of antibody molecules derived from the sera of animals immunized with an antigen, such as the CMR1 polypeptide. In this polyclonal population, antibodies will be cross-reactive with different portions of the CMR1 polypeptide, with some of those antibodies being specifically reactive with cCMR1 and others being cross-reactive with CMR1 polypeptides of other species. For the production of polyclonal antibodies, host animals are immunized with the cCMR1 protein, or a portion thereof, typically repeatedly to boost antibody titer in the animal and typically supplemented with adjuvants as described herein. Commonly used host animals for the production of anti-CMR1 antibodies include rabbits, mice and rats; however, other animals can be used if desired. Various adjuvants may be used to increase the immunological response, depending on the host species, for example, Freund's (complete and incomplete) adjuvant and mineral gels such as aluminum hydroxide. Conjugates (e.g., KLH) can also be included for the immunization, especially in cases where shorter cCMR1 peptides are used for the purposes of immunization and antibody production.
cCMR1 polypeptides or cCMR1 polypeptide fragments can be generated using any sort of synthetic or molecular biological technique. Standard synthetic peptide techniques can be used to generate smaller cCMR1 polypeptide fragments, for example peptide fragments that are 30 amino acids in length or shorter. Techniques for the synthesis of peptides fragments are well known and are described in, for example, Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al., J. Am. Chem. Soc., 85: 2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984).
Recombinant techniques can be used for the expression of cCMR1, including, for example, portions of cCMR1, variants and fusions from prokaryotic or eukaryotic host cells transformed with a cCMR1 nucleic acid. These methods include, for example, in vitro recombinant DNA techniques and in vivo genetic recombination (see, for example, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3'd Edition, Cold Spring Harbor Press, NY (2001); and Ausubel et al., eds., Short Protocols in Molecular Biology, 4th Edition, John Wiley & Sons, Inc., NY (1999)).
Therefore, cCMR1 can be produced by (a) providing a nucleic acid comprising a cCMR1 sequence, (b) inserting the nucleic acid into a host cell and (c) maintaining the host cell under conditions that allow for the expression of the cCMR1 polypeptide. When a purified cCMR1 polypeptide is desired, a step can also be performed to isolate and, if desired, purify the cCMR1 polypeptide.
In another embodiment, the invention provides a heterologous nucleic acid construct that includes the entire or a portion of the cCMR1 coding sequence operably linked to a regulatory sequence. These heterologous nucleic acid constructs include recombinant expression vectors suitable for expression of the cCMR1 nucleic acid in a host cell. Recombinant expression vectors include one or more regulatory sequences, which can be selected based on the type of host cells used for cCMR1 expression, operably linked to the cCMR1 nucleic acid sequence. Regulatory sequences include promoters, enhancers and other expression control elements, for example, poly (A)+ sequences. Regulatory sequences can be specific for prokaryotic cells, for example, bacterial cells, such as E. coli, or for eukaryotic cells, such as yeast cells, insect cells or mammalian cells (for example, HEK, CHO or COS cells). Regulatory sequences can be located cis or trans relative to the cCMR1 nucleic acid sequence. Regulatory sequences can include constitutive expression sequences that typically drive expression of the nucleic acid under a wide variety of growth conditions and in a wide variety of host cells, tissue-specific regulatory sequences that drive expression in particular host cells or tissues and inducible regulatory sequences that drive expression in response to a secondary factor. Choice and design of the expression vector can depend on such factors as the particular host cell utilized and the desired levels of polypeptide expression. Other expression vector components can include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selection genes and a transcription termination sequence. Selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example, ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies or (c) supply critical nutrients not available from complex media.
Heterologous nucleic acid constructs used for expression of the cCMR1 polypeptide can also include constructs that can be transcribed and translated in vitro, for example, constructs having a T7 promoter regulatory sequence.
Vectors suitable for the expression of cCMR1 are known in the art and commercially available. Suitable vectors include, for example, pET-14b, pCDNAIAmp and pVL1392, which are available from Novagen and Invitrogen and can be used for expression in E. Coli, COS cells and baculovirus infected insect cells, respectively.
In another embodiment, the invention provides a recombinant cell that includes a cCMR1 nucleic acid. Recombinant cells include those wherein a nucleic acid sequence has been introduced. Typically, recombinant cells are created by introducing a particular nucleic acid into cells using molecular biological techniques. However, recombinant cells also include cells that have been manipulated in other ways to promote the expression of a desired nucleic acid sequence. For example, regions that are proximal to a target nucleic acid sequence can be altered to promote expression of the target nucleic acid, or genes that act to regulate the expression of a target nucleic acid can be introduced into a cell.
Recombinant cells, after periods of growth and division, may not be identical to the starting parent cell; however, these cells are still referred to as recombinant cells and are included within the scope of the term as used herein.
Host cells suitable for harboring and providing the machinery for cCMR1 expression include both prokaryotic and eukaryotic cells. Examples of suitable prokaryotic host cells are eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, for example, E. coli, Enterobacter, Salmonella, for example, Salmonella typhimurium, as well as Bacilli such as B. subtilis, Pseudomonas, and Streptomyces. 
Eukaryotic cells, such as filamentous fungi or yeast, are suitable cloning or expression hosts for cCMR1 expression vectors. Saccharomyces cerevisiae, also known as baker's yeast, is a commonly used expression system and offers a variety of promoter and selectable marker sequences. Other fungi or yeast useful as host cells include Schizosaccharomyces pombe, Kluyveromyces lactis, Pichia pastoris, Candida, Neurospora crassa and Aspergillus nidulans. 
Many higher eukaryotic host cells can be used, including insect cells, such as Drosophila S2 and Spodoptera Sf9 cells, mammalian cells, such as Chinese Hampster Ovary (CHO) cells, monkey kidney (COS) cells, canine kidney (MDCK) cells, human cervical carcinoma (HeLa) cells, and human embryonic kidney (HEK) cells as well as plant cells.
Growth of the transformed host cells can occur under conditions that are known in the art. The conditions will generally depend upon the host cell and the type of vector used. Suitable induction conditions, such as temperature and chemicals, may be used and will depend on the type of promoter utilized. Examples of suitable media include Minimal Essential Medium ((MEM), RPMI-1640 and Dulbecco's Modified Eagle's Medium (DMEM).
Nucleic acids, including expression constructs, can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing a foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, biolistics or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.
Mammalian cells can be stably transfected with an expression construct having a selectable marker and with the gene of interest. Typically selectable markers for mammalian cells include antibiotic-resistance genes, for example, genes that allow the transformed cell to grow in the presence of compounds such as G418, hygromycin or methotrexate.
Recombinant cells can be useful for the production of a cCMR1 polypeptide for purification purposes or for functional studies involving the cCMR1 polypeptide. For example, a recombinant cCMR1 cell can be used to test a number of compounds for their ability to alter the activity of the cCMR1 polypeptide. The recombinant cCMR1 cell can also be used to test how altering various properties of the cCMR1 polypeptide, for example, altering the amino acid sequence of the cCMR1 polypeptide, affects cCMR1 activity.
Recombinant cells having a cCMR1 nucleic acid sequence can also be used to produce non-human transgenic animals. 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, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. For example, a nucleic acid containing a cCMR1 nucleic acid sequence can be introduced into a host cell such as a fertilized oocyte or an embryonic stem cell, using a suitable technique, such as microinjection. These cCMR1-containing host cells can then be used to create non-human transgenic animals. Particularly useful animals include transgenic mice or rats having a cCMR1 gene, which can also have physical or genetic characteristics making them useful for study as, for example, a pain model.
cCMR1 transgenic animals can be used to identify, screen or test potentially useful compounds, or known compounds that modulate cCMR1 function or expression. These transgenic animals can also be used to study the function of the cCMR1 polypeptide by altering its amino acid sequence.
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. Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan, Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley (Current Opinion in Bio/Technology, 2:823-829, 1991) and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169. Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (Nature, 385:810-813, 1997) and PCT Publication Nos. WO 97/07668 and WO 97/07669.
In some cases, it can be desirable to reduce the amount of CMR1 present in a system, for example, in order to test the specificity of compounds that are suspected of being CMR1 modulators. The recombinant cells or transgenic animals, as described herein, can be manipulated in order to reduce the amount of CMR1 expressed or present on its surface. For example, the cell can include molecules that reduce the amount of cCMR1 RNA present in the cell, thereby reducing cCMR1 protein expression. Suitable molecules include antisense nucleotides, ribozymes, double-stranded RNAs, interfering RNA (iRNA) and antagonists or agonists.
A variety of methods can be used for purification of the cCMR1 polypeptide. For example, crude purification can be performed using ammonium sulfate precipitation, centrifugation or other known techniques. A higher degree of purification can be achieved by suitable chromatographic techniques, including, for example, anion exchange, cation exchange, high performance liquid chromatography (HPLC), gel filtration, hydrophobic interaction chromatography and affinity chromatography, for example, immunoaffinity chromatography using antibodies directed against the cCMR1 protein. If needed, steps for refolding the cCMR1 proteins may be used to obtain the active conformation of the protein when the protein is denatured during intracellular synthesis, isolation or purification.
The invention also encompasses kits for detecting the presence of a polypeptide or nucleic acid of the invention in a biological sample (a test sample). Such a kit preferably comprises a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier can contain a means for detection such as labeled antigen or enzyme substrates or the like. For example, the kit can comprise a labeled compound or agent capable of detecting the polypeptide or mRNA encoding the polypeptide and means for determining the amount of the polypeptide or mRNA in a sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide). The kits can also include instructions for determining whether a test subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide if the amount of the polypeptide or mRNA encoding the polypeptide is above or below a normal level.
For antibody-based kits, the kit can comprise, for example: (1) a first antibody (for example, an antibody attached to a solid support), which binds selectively to a polypeptide comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 2; and, optionally; (2) a second antibody which binds to either the first antibody or the polypeptide that the first antibody binds to, but at a different epitope, and which is conjugated to a detectable agent; and (3) a purified recombinant cCMR1 protein as a positive control. Preferably, the first antibody only binds to a cCMR1, but not a CMR1 from other species, such as human, rat, or mouse.
For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes under stringent condition to SEQ ID NO: 1, or (2) a pair of primers useful for amplifying a nucleic acid molecule encoding a polypeptide having at least 96% sequence identity to SEQ ID NO: 2. The kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit can also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples that can be assayed and compared to the test sample. Each component of the kit is usually enclosed within an individual container and all of the various containers are preferably contained within a single package.
Because CMR1 is activated at cool to cold temperatures and is expressed in nerve tissue, this gene can serve as a therapeutic target for the identification of drugs useful in treating pain, inflammation and skin disorders, for example, those associated with sunburn and other sensitized states. Therefore, in another general aspect, the present invention relates to the use of cCMR1 nucleic acids and proteins in methods for identifying therapeutic compounds, for example, compounds useful in treating pain, modulating responses to cold temperature and compounds that provide a cool sensation to the skin. These types of compounds can be identified using a system that includes a cCMR1 polypeptide or a cCMR1 nucleic acid. Compounds can also be tested directly in vivo in an animal model system, for example, a rat, mouse or canine model system. Particularly useful systems include animal models of pain. These methods comprise assaying for the ability of various compounds to increase or decrease the expression of the cCMR1 protein, the conductivity of the cCMR1 channel or the nociceptive behaviors of an animal.
The compound identification methods can be performed using conventional laboratory formats or in assays adapted for high throughput. The term “high throughput” refers to an assay design that allows easy screening of multiple samples simultaneously and/or in rapid succession, and can include the capacity for robotic manipulation. Another desired feature of high throughput assays is an assay design that is optimized to reduce reagent usage, or minimize the number of manipulations in order to achieve the analysis desired. Examples of assay formats include 96-well or 384-well plates, levitating droplets, and “lab on a chip” microchannel chips used for liquid handling experiments. It is well known by those in the art that as miniaturization of plastic molds and liquid handling devices are advanced, or as improved assay devices are designed, greater numbers of samples can be processed using the design of the present invention.
Candidate compounds encompass numerous chemical classes, including but not limited to, small organic or inorganic compounds, natural or synthetic molecules, such as antibodies, proteins or fragments thereof, antisense nucleotides, interfering RNA (iRNA) and ribozymes. Preferably, they are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500. Candidate compounds comprise functional chemical groups necessary for structural interactions with polypeptides, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups. The candidate compounds can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Candidate compounds also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the compound is a nucleic acid, the compound typically is a DNA or RNA molecule, although modified nucleic acids having non-natural bonds or subunits are also contemplated.
Candidate compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Candidate compounds can also be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries; spatially addressable parallel solid phase or solution phase libraries: synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection (Lam (1997) Anticancer Drug Des. 12:145). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means.
Further, known pharmacological agents can be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidation, etc. to produce structural analogs of the agents. Candidate compounds can be selected randomly or can be based on existing compounds that bind to and/or modulate the function of CMR1 activity. Therefore, a source of candidate agents is one or more than one library of molecules based on one or more than one known compound that increases or decreases CMR1 channel conductivity in which the structure of the compound is changed at one or more positions of the molecule to contain more or fewer chemical moieties or different chemical moieties. The structural changes made to the molecules in creating the libraries of analog activators/inhibitors can be directed, random, or a combination of both directed and random substitutions and/or additions. One of ordinary skill in the art in the preparation of combinatorial libraries can readily prepare such libraries based on the existing compounds.
A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. that can be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent can also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as nuclease inhibitors, antimicrobial agents, and the like can also be used.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: Zuckermann et al. (1994). J. Med. Chem. 37:2678. Libraries of compounds can be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. No. 5,571,698), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) orphage (see e.g., Scott and Smith (1990) Science 249:3 86-390).
In one aspect, the invention provides a method of identifying a compound that increases or decreases the expression of a cCMR1 protein, comprising the steps of: (a) contacting a test compound with a cell comprising a mechanism for regulating the expression of a cCMR gene; and (b) determining whether the test compound increases or decreases the expression of a gene controlled by said mechanism from the cell. The mechanism for regulating the expression of a cCMR gene includes the mechanism by which nuclear, cytoplasmic, or intracellular factors influence the control of gene action at the level of transcription or translation. For example, the mechanism includes gene activation or gene repression. The cell comprising a mechanism for regulating the expression of a cCMR gene can be a native host cell that expresses cCMR endogenously, such as a canine DRG cell. The cell can also be a recombinant cell containing a recombinant DNA sequence having a regulatory sequence for a cCMR gene, and the regulatory sequence is operably linked to a gene, preferably a reporter gene.
The effect of the compound on the expression of a gene controlled by the regulatory sequence of CMR1 can be measured by a variety of means. For example, the effect can be measured by the amount of mRNA or protein of the gene from the cell, or by the activity of the gene product from the cell. When a reporter gene is used, the effect can be measured as the level of reporter gene product from the cell. For example, when the CMR1 regulatory sequence is operably linked to a GFP gene, the effect of the compound on gene expression can be measured as the effect of the compound on emissions of green fluorescence from the cell using a fluorometer. When an endogenous cCMR 1 cell is used, the effect of the compound on gene expression can be measured by the amount of cCMR1 mRNA or protein inside the cell using methods described infra (i.e., Northern Blot, RT-PCR, SDS-PAGE, Western Blot, immunohisto- or immunocytochemistry, radioreceptor ligand binding, etc). Alternatively, the conductivity of the cCMR1 channel can be used to measure the effect of the compound on the expression of the cCMR1 protein.
The cell-based method described herein not only identifies compounds that regulate cCMR1 expression directly via binding to one or more than one regulatory sequence of the cCMR1 gene, but also identifies compounds that regulate cCMR1 expression indirectly via binding to other cellular components whose activities influence cCMR1 expression or protein stability. For example, compounds that regulate the activity of a transcriptional activator or inhibitor for cCMR1 genes can be identified using the method described herein. Compounds that regulate the activity of a protease that degrades the cCMR1 protein in vivo can also be identified.
The invention also provides a method of identifying a compound that increases or decreases the conductivity of a cCMR1 ion channel, comprising the steps of: (a) contacting a test compound with the ion channel; and (b) determining whether the test compound increases or decreases the conductivity of the ion channel. In some embodiments, the cCMR1 ion channel is expressed on the surface of a host cell. The cell can be a native host cell for cCMR1 that expresses the cCMR1 endogenously, for example, a dog DRG or TG cell. The cell can also be a recombinant host cell for cCMR1, for example, a CHO or COS cell expressing a cCMR1 recombinantly.
In some other embodiments, the cCMR1 ion channel is associated with an isolated membrane preparation. The membrane preparation can be isolated from a native host cell that expresses cCMR1 on its cell surface, or from a recombinant host cell that expresses cCMR1 on its cell surface. It can also be prepared from the biological membranes, such as the tissue membrane, plasma membrane, cell membrane, or internal organelle membrane comprising the cCMR1 channel. Methods are known to those skilled in the art for isolation and preparation of biological membrane preparations. For example, such a method can include the steps of mechanical or enzymic disruption of the tissue or cells, centrifugation to separate the membranes from other components, and resuspending the membrane fragments or vesicles in suitable buffer solution. Alternatively, the membrane-containing preparation can also be derived from artificial membranes. Purified cCMR1 protein can be reconstituted into lipid bilayers to form the artificial membrane vesicles (see Chen et al., 1996, J. Gen. Physiol. 108:237-250). Such type of membrane vesicle can be very specific to the channel of interest, avoiding the problem of contamination with other channels. Methods are known to those skilled in the art to prepare artificial membrane vesicles.
In some embodiments, membrane vesicles comprising the cCMR1 can provide an easier format for the inventive assays and methods, because cell lysis and/or shear is not as much of a concern during the assay. In other embodiments, however, cells expressing the cCMR1 are preferred, for example, when the cell membrane preparation procedure destroys or inactivates the channel of interest.
The test compound can be evaluated for its ability to increase or decrease the ion conductivity of a cCMR1 channel. Known to those skilled in the are methods for measuring a CMR1 channel conductivity, for example, via the stimulation of cellular depolarization or an increase in intracellular calcium ion levels. The level of intracellular calcium can be assessed using a calcium ion-sensitive fluorescent indicator, such as a calcium ion-sensitive fluorescent dye. Suitable calcium ion-sensitive fluorescent dyes include, for example, quin-2 (see, e.g., Tsien et al., J. Cell Biol., 94:325, 1982), fura-2 (see, e.g., Grynkiewicz et al., J. Biol. Chem., 260:3440, 1985), fluo-3 (see, e.g., Kao et al., J. BioL—43 Chem., 264:8179, 1989) and rhod-2 (see, e.g., Tsien et al., J. Biol. Chem., Abstract 89a, 1987). Suitable calcium ion-sensitive fluorescent dyes are commercially available from, for example, Molecular Probes (Eugene, Oreg.). Cellular fluorescence can also be monitored using a fluorometer or a flow cytometer having a fluorescence lamp and detector.
The cCMR1 cation channels function to transport not only divalent cations, for example, Ca++, but also monovalent cations, for example, Na+ or K+. Therefore, assays for determining changes in the transport of monovalent cation can also be performed to measure the conductivity of a cCMR1 channel. Na+- and K+-sensitive dyes are known in the art and commercially available from, for example, Molecular Probes (Eugene, Oreg.).
The conductivity of a cCMR1 channel can also be measured by electrophysiologic techniques such as patch-clamp. Patch-clamp techniques are routinely used for studying electrical activities in cells, cell membranes, and isolated tissues. It involves forming an electrically tight, high-resistance seal between a micropipette and the plasma membrane. The current flowing through individual ion channels within the plasma membrane can then be measured. Different variants on the techniques allow different surfaces of the plasma membrane to be exposed to the bathing medium. The four most common variants include on-cell patch, inside-out patch, outside-out patch, and whole-cell clamp.
A patch-clamp method is commonly used with a voltage clamp that controls the voltage across the membrane and measures current flow. During the voltage clamp process, a microelectrode is inserted into a cell and current injected through the electrode so as to hold the cell membrane potential at some predefined level. A patch-clamp method can also be used with current-clamp methods, in which the current is controlled and the voltage is measured.
The assays to identify a compound that decreases cCMR1 channel conductivity are preferably performed under conditions in which the particular ion channel is activated. For example, such assays can be performed at a temperature at which CMR1 is activated . . . Studies from whole-cell patch clamp recordings indicated that cCMR1 is activated at cool temperatures at or below about 17° C. (Example 7 infra). Alternatively, such assays can be performed in the presence of a compound that activates the cCMR1, such as the cool compound menthol or icilin, or the pungent compound mustard oil. In addition, such assay can be performed at conditions when the cCMR1 channel is depolarized, such as by clamping the channel at a depolarized potential.
Conversely, when seeking to identify a compound that increases cCMR1 channel conductivity, test conditions are preferably adjusted wherein the cCMR1 channel is not active or is otherwise blocked. For example, such assays can be performed at a CMR1 non-activation temperature. Unlike the rat CMR1 that is still active at room temperature, cCMR1 is inactivated at room temperature (Example 7 infra). Alternatively, such assays can be performed in the presence of a compound that decreases the conductivity of the cCMR1 channel. In addition, such assays can be performed in the presence of extracellular Ca2+ that is sufficient to desensitize the cCMR1 channel. A person of ordinary skill in the art is able to determine the appropriate concentration of extracellular Ca2+ that is sufficient to desensitize the cCMR1 channel by routine experimentation. Furthermore, such assays can be performed at conditions when the cCMR1 channel is hyperpolarized, such as by clamping the channel at a hyperpolarized potential.
Assays for the identification of cCMR1 modulators can be carried out manually or using an automated system. Automated systems are preferred if high throughput screenings are performed. For example, one type of automated system utilizes multi-well culture plates, for example, 96-well, 384-well or 1536-well culture plates, wherein each well contains recombinant cells having a nucleic acid encoding the cCMR1 protein. The plate is loaded into a fluorometer, for example, the FlexStationTM (from Molecular Devices Corp., Sunnyvale, Calif.), that can measure the calcium flux and/or membrane potential of the cells in each of the wells. Solutions containing the calcium ion-sensitive fluorescent indicator dye or test compounds can be automatically added to each of the wells. The temperature in the fluorometer can be controlled according to the type of assay that is performed, for example, temperatures can be adjusted to a temperature above the CMR-activating temperature, for example, above 28° C., to test compounds suspected of being CMR1 agonists. Likewise, temperatures can be adjusted to a CMR-activating temperature, for example, at or below 28° C., to test compounds suspected of being CMR1 antagonists.
After the CMR1 channel has been activated and allows the influx of cations (such as Ca++ ions), the intracellular accumulation of the Ca++ ions promotes a negative feedback and inactivation of the CMR1 channel. The CMR1 becomes reactivated after intracellular Ca++ levels decrease by, for example, Ca++ being pumped out of the cell or taken up into intracellular organelles.
Although the CMR1 channel can allow the influx of Ca++ ions in response to cool to cold temperatures, it is somewhat of a leaky ion channel. Some CMR1 channels will permit the influx of Ca++ ions even at non-activating temperatures, for example, at above 28° C. In conventional assay systems, extracellular Ca++ concentrations in the mM range are typically used, which can lead to the intracellular accumulation of calcium even at non-activating temperatures, causing the negative feedback inactivation of CMR1.
Therefore, another aspect of the invention is a method of identifying a compound that increases or decreases the conductivity of a mammalian CMR1 ion channel, comprising the steps of: (a) incubating the ion channel in a buffer solution containing a sub-inactivating amount of calcium; (b) activating the ion channel; (c) contacting the ion channel with a test compound; (d) increasing the amount of calcium in the buffer solution; and e) determining the intracellular amount of calcium, and comparing the amount with that of a control wherein the ion channel was not contacted with the test compound. A “sub-inactivating amount of calcium” is the amount of extracellular Ca++ that would not cause intracellular accumulation of the Ca++ ions to an extent that promotes a negative feedback and inactivation of the CMR1 channel. A person skilled in the art can determine the “sub-inactivating amount of calcium” for a particular CMR1 channel experimentally. In some embodiments, the “sub-inactivating amount of calcium’ is essentially zero calcium in the buffer solution. In other embodiments, the “sub-inactivating amount of calcium’ is in the μM range of calcium in the buffer solution. The method of the invention includes a method comprising steps (a) to (e) as described herein, wherein step (c) precedes step (b).
After a compound has been identified that meets the desired criteria for modulating CMR1 activity or expression, the compound can then be administered to live animal. This can be useful to establish toxicity and other pharmacological parameters important for establishing dosing regimens. For example, after a compound is identified using an ex vivo system that contains a cCMR1 polypeptide, the compound can be administered to a dog to examine various pharmacological aspects of the compound in the dog. The cCMR1 systems as described herein are particularly advantageous for identifying and establishing dosing regimens in humans, because dogs, particularly large breeds, are closer in weight to humans as compared to rats or mice and therefore provide a more suitable animal model for estimating human dosing.
The compound can also be administered to animals to assess the ability of the compound to alter nociceptive processes. Various animal models of pain exist, for example, the spinal nerve ligation (SNL) model of nerve injury, which is a neuropathic pain model in rats developed by Kim and Chung (Pain, 50:355-363, 1992).
Other suitable animal models of pain can be utilized in connection with the teachings herein. Commonly studied rodent models of neuropathic pain include the chronic constriction injury (CCI) or Bennett model; neuroma or axotomy model; and the partial sciatic transection or Seltzer model (Shir et al., Neurosci. Lett., 115:62-67, 1990). Exemplary neuropathic pain models include several traumatic nerve injury preparations (Bennett et al., Pain 33: 87-107, 1988; Decosterd et al., Pain 87: 149-58, 2000; Kim et al., Pain 50: 355-363, 1992; Shir et al., Neurosci Lett 115: 62-7, 1990), neuroinflammation models (Chacur et al., Pain 94: 231-44, 2001; Milligan et al., Brain Res 861: 105-16, 2000) diabetic neuropathy (Calcutt et al., Br J Pharmacol 122: 1478-82, 1997), virus-induced neuropathy (Fleetwood-Walker et al., J Gen Virol 80: 2433-6, 1999), vincristine neuropathy (Aley e t al., Neuroscience 73: 259-65, 1996; Nozaki-Taguchi et al., Pain 93: 69-76, 2001), and paclitaxel neuropathy (Cavaletti et al., Exp Neurol 133: 64-72, 1995), as well as acute nociceptive tests models and inflammatory models (Brennan, T. J. et al. Pain 64:493, 1996; D'Amour, F. E. and Smith, D. L. J Pharmacol 72: 74-79, 1941; Eddy, N. B. et al. J Pharmacol Exp Ther 98:121, 1950; Haffner, F. Dtsch Med Wochenschr 55:731, 1929; Hargreaves, K. et al. Pain 32: 77-88, 1988; Hunskaar, S. et al. J Neurosci Meth 14:69, 1985; Randall, L. O. and Selitto, J. J. Arch. Int. Pharmacodyn 111: 409-419, 1957; Siegmund, E. et al. Proc Soc Exp Bio Med 95:729, 1957).
Therefore, in another embodiment, the invention provides a method of identifying a compound useful for treating pain, comprising the steps of: (a) contacting a test compound with a cCMR1 ion channel; and (b) determining whether the test compound increases or decreases the conductivity of the ion channel. In some embodiments, the method further comprises the steps of: (a) administering the test compound to an animal; and (b) determining the extent to which the test compound alters the nociceptive/nocifensive response of the animal.
In some embodiments, the animal model of pain involves a rodent, for example, a rat or mouse; in another aspect the animal model of pain involves a dog, for example, the skin twitch test (Kamerling et al. Pharmacol. Biochem. Behav. 17:733-740, 1982; also, see Burns J C et al. Perspect Biol Med. Autumn; 35(1): 68-73, 1991).
Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals by calculating, for example, the ED50 (the dose therapeutically effective in 50% of the population) and the LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. The data obtained from cell culture assays using recombinant CMR1 and animal studies, such as canine studies, is used in formulating a range of dosage for human use. The dosage contained in such compositions preferably gives rise to a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient and the route of administration. The exact dosage will be determined by the one administering the dose, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active agent or to maintain the desired effect, for example, effective pain relief. Factors that may be taken into account include the severity of the pain and other factors, including the general health of the subject, age, weight and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance/response to therapy.
The pharmaceutical compositions containing a compound that has been identified as modulating CMR1 activity can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarticular, intraarterial, intramedullary, intrathecal, epidural, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, inhalational, intraocular, intra-aural or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable, pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations that can be used pharmaceutically or which facilitate absorption or distribution of the active compounds. Further details on techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, Maack Publishing Co., Easton, Pa.
Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well-known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the patient.











TABLE 1








ACGCGGGGAAGGCCGGCAGGATCTTTCCAGGGAAAGCAAATCCTGCCTCACAAACCTCAA

SEQ1



1
---------+---------+---------+---------+---------+---------+
60



TGCGCCCCTTCCGGCCGTCCTAGAAAGGTCCCTTTCGTTTAGGACGGAGTGTTTGGAGTT


 



CCGGAGAGATGTCCTTCGAGGGGGCCAGGCTCAGCATGAGGAACAGAAGGAACGGCACGC


61
---------+---------+---------+---------+---------+---------+
120



GGCCTCTCTACAGGAAGCTCCCCCGGTCCGAGTCGTACTCCTTGTCTTCCTTGCCGTGCG



        M  S  F  E  G  A  R  L  S  M  R  N  R  R  N  G  T  L
-
SEQ2



TGGACAGCACCCGGACCCTGTACTCCAGCACGTCTCGGAGCACCGACGTGTCCTACAGCG


121
---------+---------+---------+---------+---------+---------+
180



ACCTGTCGTGGGCCTGGGACATGAGGTCGTGCAGAGCCTCGTGGCTGCACAGGATGTCGC



  D  S  T  R  T  L  Y  S  S  T  S  R  S  T  D  V  S  Y  S  E
-



AAAGCGACTTGGTGAATTTTATTCAAGCAAATTTTAAGAAACGAGAATGTGTCTTCTTCA


181
---------+---------+---------+---------+---------+---------+
240



TTTCGCTGAACCACTTAAAATAAGTTCGTTTAAAATTCTTTGCTCTTACACAGAAGAAGT



  S  D  L  V  N  F  I  Q  A  N  F  K  K  R  E  C  V  F  F  T
-



CCAAAGATTCCAAGGCCACGGAAAATGTGTGCAAGTGTGGCTATGCCCAGAGCCAGCACA


241
---------+---------+---------+---------+---------+---------+
300



GGTTTCTAAGGTTCCGGTGCCTTTTACACACGTTCACACCGATACGGGTCTCGGTCGTGT



  K  D  S  K  A  T  E  N  V  C  K  C  G  Y  A  Q  S  Q  H  I
-



TAGAAGGCACCCAGATCAACTCAAACGAGAAATGGAATTACAAGAAACACACCAAGGAAT


301
---------+---------+---------+---------+---------+---------+
360



ATCTTCCGTGGGTCTAGTTGAGTTTGCTCTTTACCTTAATGTTCTTTGTGTGGTTCCTTA



  E  G  T  Q  I  N  S  N  E  K  W  N  Y  K  K  H  T  K  E  F
-



TTCCGACTGACGCCTTTGGGGATATTCAGTTTGAGACTCTGGGGAAGAAAGGGAAGTATA


361
---------+---------+---------+---------+---------+---------+
420



AAGGCTGACTGCGGAAACCCCTATAAGTCAAACTCTGAGACCCCTTCTTTCCCTTCATAT



  P  T  D  A  F  G  D  I  Q  F  E  T  L  G  K  K  G  K  Y  I
-



TCCGCCTGTCCTGTGACACGGATGCGGAGACCCTCTATGAGCTGCTGACCCAGCACTGGC


421
---------+---------+---------+---------+---------+---------+
480



AGGCGGACAGGACACTGTGCCTACGCCTCTGGGAGATACTCGACGACTGGGTCGTGACCG



  R  L  S  C  D  T  D  A  E  T  L  Y  E  L  L  T  Q  H  W  H
-



ACCTGAAAACGCCCAACCTGGTCATATCTGTCACCGGCGGCGCCAAGAACTTCGCCCTGA


481
---------+---------+---------+---------+---------+---------+
540



TGGACTTTTGCGGGTTGGACCAGTATAGACAGTGGCCGCCGCGGTTCTTGAAGCGGGACT



  L  K  T  P  N  L  V  I  S  V  T  G  G  A  K  N  F  A  L  K
-



AGCCGAGGATGCGCAAGATCTTCAGCCGCCTCATCTACATCGCGCAGTCCAAAGGTGCTT


541
---------+---------+---------+---------+---------+---------+
600



TCGGCTCCTACGCGTTCTAGAAGTCGGCGGAGTAGATGTAGCGCGTCAGGTTTCCACGAA



  P  R  M  R  K  I  F  S  R  L  I  Y  I  A  Q  S  K  G  A  W
-



GGATTCTCACTGGAGGAACCCATTATGGCCTGATGAAGTACATCGGGGAGGTGGTGAGAG


601
---------+---------+---------+---------+---------+---------+
660



CCTAAGAGTGACCTCCTTGGGTAATACCGGACTACTTCATGTAGCCCCTCCACCACTCTC



  I  L  T  G  G  T  H  Y  G  L  N  K  Y  I  G  E  V  V  R  D
-



ACAACACCATCAGCAGGAATTCAGAGGAGAACATTGTGGCCATTGGCATAGCGGCTTGGG


661
---------+---------+---------+---------+---------+---------+
720



TGTTGTGGTAGTCGTCCTTAAGTCTCCTCTTGTAACACCGGTAACCGTATCGCCGAACCC



  N  T  I  S  R  N  S  E  E  N  I  V  A  I  G  I  A  A  W  G
-



GCATGGTCTCCAACAGGGACACTCTCCTCAGGAATTGCGATGCTGAGGGATATTTTTCAG


721
---------+---------+---------+---------+---------+---------+
780



CGTACCAGAGGTTGTCCCTGTGAGAGGAGTCCTTAACGCTACGACTCCCTATAAAAAGTC



  M  V  S  N  R  D  T  L  L  R  N  C  D  A  E  G  Y  F  S  A
-



CTCAGTACATAATGGATGACTTCAAGAGAGACCCTCTGTATATCTTGGACAACAACCACA


781
---------+---------+---------+---------+---------+---------+
840



GAGTCATGTATTACCTACTGAAGTTCTCTCTGGGAGACATATAGAACCTGTTGTTGGTGT



  Q  Y  I  M  D  D  F  K  R  D  P  L  Y  I  L  D  N  N  H  T
-



CCCATCTGCTGCTTGTGGACAACGGCTGCCATGGACATCCTACAGTTGAAGCAAAACTCC


841
---------+---------+---------+---------+---------+---------+
900



GGGTAGACGACGAACACCTGTTGCCGACGGTACCTGTAGGATGTCAACTTCGTTTTGAGG



  H  L  L  L  V  D  N  G  C  H  G  H  P  T  V  E  A  K  L  R
-



GGAATCAGCTGGAGAAGTACATCTCCGAGCGCACTATTCAAGATTCCAACTATGGTGGCA


901
---------+---------+---------+---------+---------+---------+
960



CCTTAGTCGACCTCTTCATGTAGAGGCTCGCGTGATAAGTTCTAAGGTTGATACCACCGT



  N  Q  L  E  K  Y  I  S  E  R  T  I  Q  D  S  N  Y  G  G  K
-



AGATCCCCATTGTGTGTTTTGCCCAAGGAGGTGGCAGAGAAACTTTGAAAGCCATCAACA


961
---------+---------+---------+---------+---------+---------+
1020



TCTAGGGGTAACACACAAAACGGGTTCCTCCACCGTCTCTTTGAAACTTTCGGTAGTTGT



  I  P  I  V  C  F  A  Q  G  G  G  R  E  T  L  K  A  I  N  T
-



CCTCCATCAAAAGCAAAATCCCCTGTGTGGTGGTGGAAGGCTCAGGGCAGATTGCAGACG


1021
---------+---------+---------+---------+---------+---------+
1080



GGAGGTAGTTTTCGTTTTAGGGGACACACCACCACCTTCCGAGTCCCGTCTAACGTCTGC



  S  I  K  S  K  I  P  C  V  V  V  E  G  S  G  Q  I  A  D  V
-



TGATCGCGAGCCTGGTGGAGGTGGAGGACGTCCTGACGTCATCTGTGGTCAAGGAGAAGT


1081
---------+---------+---------+---------+---------+---------+
1140



ACTAGCGCTCGGACCACCTCCACCTCCTGCAGGACTGCAGTAGACACCAGTTCCTCTTCA



  I  A  S  L  V  E  V  E  D  V  L  T  S  S  V  V  K  E  K  L
-



TGGTGCGCTTCTTACCCCGCACAGTGTCCCGGCTGCCTGAGGAGGAGACCGAGAGTTGGA


1141
---------+---------+---------+---------+---------+---------+
1200



ACCACGCGAAGAATGGGGCGTGTCACAGGGCCGACGGACTCCTCCTCTGGCTCTCAACCT



  V  R  F  L  P  R  T  V  S  R  L  P  E  E  E  T  E  S  W  I
-



TCAAATGGCTCAAAGAAATTCTCGAAAGTTCTCACCTATTAACAGTTATTAAAATGGAAG


1201
---------+---------+---------+---------+---------+---------+
1260



AGTTTACCGAGTTTCTTTAAGAGCTTTCAAGAGTGGATAATTGTCAATAATTTTACCTTC



  K  W  L  K  E  I  L  E  S  S  H  L  L  T  V  I  K  M  E  E
-



AAGCTGGAGACGAAATTGTGAGCAATGCTATTTCTTATGCTTTGTACAAAGCCTTTAGCA


1261
---------+---------+---------+---------+---------+---------+
1320



TTCGACCTCTGCTTTAACACTCGTTACGATAAAGAATACGAAACATGTTTCGGAAATCGT



  A  G  D  E  I  V  S  N  A  I  S  Y  A  L  Y  K  A  F  S  T
-



CCAATGAACAAGATAAGGATAACTGGAATGGGCAGCTGAAGCTTCTGCTGGAATGGAACC


1321
---------+---------+---------+---------+---------+---------+
1380



GGTTACTTGTTCTATTCCTATTGACCTTACCCGTCGACTTCGAAGACGACCTTACCTTGG



  N  E  Q  D  K  D  N  W  N  G  Q  L  K  L  L  L  E  W  N  Q
-



AGCTGGACCTAGCCAATGAGGAGATATTCACCAACGACCGCCGATGGGGGTCTGCTGATC


1381
---------+---------+---------+---------+---------+---------+
1440



TCGACCTGGATCGGTTACTCCTCTATAAGTGGTTGCTGGCGGCTACCCCCAGACGACTAG



  L  D  L  A  N  E  E  I  F  T  N  D  R  R  W  G  S  A  D  L
-



TGCAAGAGGTCATGTTTACAGCTCTCATAAAGGACAGACCCAAGTTTGTCCGCCTCTTCC


1441
---------+---------+---------+---------+---------+---------+
1500



ACGTTCTCCAGTACAAATGTCGAGAGTATTTCCTGTCTGGGTTCAAACAGGCGGAGAAGG



  Q  E  V  M  F  T  A  L  I  K  D  R  P  K  F  V  R  L  F  L
-



TGGAGAATGGGTTGAACCTGCGCAAGTTTCTCACCAATGACGTCCTCACTGAACTCTTCT


1501
---------+---------+---------+---------+---------+---------+
1560



ACCTCTTACCCAACTTGGACGCGTTCAAAGAGTGGTTACTGCAGGAGTGACTTGAGAAGA



  E  N  G  L  N  L  R  K  F  L  T  N  D  V  L  T  E  L  F  S
-



CCAACCACTTCAGCACCCTTGTCTACCGGAACCTGCAGATTGCCAAGAATTCCTATAACG


1561
---------+---------+---------+---------+---------+---------+
1620



GGTTGGTGAAGTCGTGGGAACAGATGGCCTTGGACGTCTAACGGTTCTTAAGGATATTGC



  N  H  F  S  T  L  V  Y  R  N  L  Q  I  A  K  N  S  Y  N  D
-



ATGCCCTCCTCACATTCGTCTGGAAACTGGTGGCCAACTTCCGGAGAGGCTTCCGAAAGG


1621
---------+---------+---------+---------+---------+---------+
1680



TACGGGAGGAGTGTAAGCAGACCTTTGACCACCGGTTGAAGGCCTCTCCGAAGGCTTTCC



  A  L  L  T  F  V  W  K  L  V  A  N  F  R  R  G  F  R  K  E
-



AAGACAGAAGTAGCAGGGATGACATAGATGTAGAACTTCACGATGTGTCTCCTATCACTC


1681
---------+---------+---------+---------+---------+---------+
1740



TTCTGTCTTCATCGTCCCTACTGTATCTACATCTTGAAGTGCTACACAGAGGATAGTGAG



  D  R  S  S  R  D  D  I  D  V  E  L  H  D  V  S  P  I  T  R
-



GGCACCCGCTGCAAGCACACTTCATCTGGGCCATTCTTCAGAACAAGAAGGAACTGTCCA


1741
---------+---------+---------+---------+---------+---------+
1800



CCGTGGGCGACGTTCGTGTGAAGTAGACCCGGTAAGAAGTCTTGTTCTTCCTTGACAGGT



  H  P  L  Q  A  H  F  I  W  A  I  L  Q  N  K  K  E  L  S  K
-



AGGTCATTTGGGAGCAGACCAGGGGCTGCACGTTGGCAGCCCTGGGAGCCAGCAAGCTTC


1801
---------+---------+---------+---------+---------+---------+
1860



TCCAGTAAACCCTCGTCTGGTCCCCGACGTGCAACCGTCGGGACCCTCGGTCGTTCGAAG



  V  I  W  E  Q  T  R  G  C  T  L  A  A  L  G  A  S  K  L  L
-



TGAAGACTCTGGCCAAGGTGAAGAATGACATCAATGCTGCAGGGGAGTCCGAGGAGCTGG


1861
---------+---------+---------+---------+---------+---------+
1920



ACTTCTGAGACCGGTTCCACTTCTTACTGTAGTTACGACGTCCCCTCAGGCTCCTCGACC



  K  T  L  A  K  V  K  N  D  I  N  A  A  G  E  S  E  E  L  A
-



CAAATGAGTATGAGACCCGTGCAGTTGAGCTGTTCACGGAGTGCTACAGCAGCGACGAGG


1921
---------+---------+---------+---------+---------+---------+
1980



GTTTACTCATACTCTGGGCACGTCAACTCGACAAGTGCCTCACGATGTCGTCGCTGCTCC



  N  E  Y  E  T  R  A  V  E  L  F  T  E  C  Y  S  S  D  E  D
-



ACCTGGCCGAGCAGCTGCTGGTGTACTCCTGCGAAGCCTGGGGCGGGAGCAACTGCTTGG


1981
---------+---------+---------+---------+---------+---------+
2040



TGGACCGGCTCGTCGACGACCACATGAGGACGCTTCGGACCCCGCCCTCGTTGACGAACC



  L  A  E  Q  L  L  V  Y  S  C  E  A  W  G  G  S  N  C  L  E
-



AGCTGGCGGTGGAGGCCACGGACCAGCACTTCATCGCCCAGCCCGGGGTCCAGAATTTTC


2041
---------+---------+---------+---------+---------+---------+
2100



TCGACCGCCACCTCCGGTGCCTGGTCGTGAAGTAGCGGGTCGGGCCCCAGGTCTTAAAAG



  L  A  V  E  A  T  D  Q  H  F  I  A  Q  P  G  V  Q  N  F  L
-



TTTCCAAGCAATGGTATGGAGAGATTTCCCGAGACACCAAGAACTGGAAGATTATCCTGT


2101
---------+---------+---------+---------+---------+---------+
2160



AAAGGTTCGTTACCATACCTCTCTAAAGGGCTCTGTGGTTCTTGACCTTCTAATAGGACA



  S  K  Q  W  Y  G  E  I  S  R  D  T  K  N  W  K  I  I  L  C
-



GTTTGTTTATTATACCCTTGGTGGGCTGTGGCTTTGTATCCTTTAGGAAGAGGCCCATCG


2161
---------+---------+---------+---------+---------+---------+
2220



CAAACAAATAATATGGGAACCACCCGACACCGAAACATAGGAAATCCTTCTCCGGGTAGC



  L  F  I  I  P  L  V  G  C  G  F  V  S  F  R  K  R  P  I  D
-



ACAAGCACAAGAAGATCCTGTGGTACTACGTGGCGTTCTTCACCTCCCCCTTTGTGGTCT


2221
---------+---------+---------+---------+---------+---------+
2280



TGTTCGTGTTCTTCTAGGACACCATGATGCACCGCAAGAAGTGGAGGGGGAAACACCAGA



  K  H  K  K  I  L  W  Y  Y  V  A  F  F  T  S  P  F  V  V  F
-



TCGCCTGGAACGTGGTCTTCTACATCGCCTTCCTCCTGCTCTTTGCCTACGTGCTGCTCA


2281
---------+---------+---------+---------+---------+---------+
2340



AGCGGACCTTGCACCAGAAGATGTAGCGGAAGGAGGACGAGAAACGGATGCACGACGAGT



  A  W  N  V  V  F  Y  I  A  F  L  L  L  F  A  Y  V  L  L  M
-



TGGATTTTCACTCAGTGCCACACTCCCCCGAGCTGGTCCTCTACGCACTGGTCTTTGTCC


2341
---------+---------+---------+---------+---------+---------+
2400



ACCTAAAAGTGAGTCACGGTGTGAGGGGGCTCGACCAGGAGATGCGTGACCAGAAACAGG



  D  F  H  S  V  P  H  S  P  H  L  V  L  Y  A  L  V  F  V  L
-



TGTTCTGTGATGAAGTGAGACAGTGGTACATGAATGGGGTGAATTATTTTACCGACCTGT


2401
---------+---------+---------+---------+---------+---------+
2460



ACAAGACACTACTTCACTCTGTCACCATGTACTTACCCCACTTAATAAAATGGCTGGACA



  F  C  D  E  V  R  Q  W  Y  M  N  G  V  N  Y  F  T  D  L  W
-



GGAATGTCATGGACACACTTGGGCTTTTTTACTTCATAGCAGGCATTGTGTTTCGGCTCC


2461
---------+---------+---------+---------+---------+---------+
2520



CCTTACAGTACCTGTGTGAACCCGAAAAAATGAAGTATCGTCCGTAACACAAAGCCGAGG



  N  V  M  D  T  L  G  L  F  Y  F  I  A  G  I  V  F  R  L  H
-



ACCCTTCTAATAAAACCTCTTTGTATTCCGGACGAGTCATCTTTTGCCTGGATTACATTA


2521
---------+---------+---------+---------+---------+---------+
2580



TGGGAAGATTATTTTGGAGAAACATAAGGCCTGCTCAGTAGAAAACGGACCTAATGTAAT



  P  S  N  K  T  S  L  Y  S  G  R  V  I  F  C  L  D  Y  I  I
-



TATTCACCCTAAGGTTGATCCACATTTTCACCGTAAGCAGAAATTTGGGACCGAAGATTA


2581
---------+---------+---------+---------+---------+---------+
2640



ATAAGTGGGATTCCAACTAGGTGTAAAAGTGGCATTCGTCTTTAAACCCTGGCTTCTAAT



  F  T  L  R  L  I  H  I  F  T  V  S  R  N  L  G  P  K  I  I
-



TAATGTTGCAGAGGATGCTGATCGACGTGTTTTTCTTCCTGTTTCTGTTTGCCGTGTGGA


2641
---------+---------+---------+---------+---------+---------+
2700



ATTACAACGTCTCCTACGACTAGCTGCACAAAAAGAAGGACAAAGACAAACGGCACACCT



  M  L  Q  R  M  L  I  D  V  F  F  F  L  F  L  F  A  V  W  M
-



TGGTGGCCTTCGGCGTGGCCAGGCAAGGGATCCTCAGGCAAAATGAGCATCGCTGGAGGT


2701
---------+---------+---------+---------+---------+---------+
2760



ACCACCGGAAGCCGCACCGGTCCGTTCCCTAGGAGTCCGTTTTACTCGTAGCGACCTCCA



  V  A  F  G  V  A  R  Q  G  I  L  R  Q  N  E  H  R  W  R  W
-



GGATATTCCGCTCGGTTATCTACGAGCCCTACCTGGCCATGTTCGGCCAAGTGCCCAGCG


2761
---------+---------+---------+---------+---------+---------+
2820



CCTATAAGGCGAGCCAATAGATGCTCGGGATGGACCGGTACAAGCCGGTTCACGGGTCGC



  I  F  R  S  V  I  Y  E  P  Y  L  A  M  F  G  Q  V  P  S  D
-



ACGTGGATGGTACCACATATGACTTTGCCCACTGCACTTTCACTGGGAATGAGTCCAAGC


2821
---------+---------+---------+---------+---------+---------+
2880



TGCACCTACCATGGTGTATACTGAAACGGGTGACGTGAAAGTGACCCTTACTCAGGTTCG



  V  D  G  T  T  Y  D  F  A  H  C  T  F  T  G  N  E  S  K  P
-



CGCTGTGTGTGGAGCTGGATGAGCACAACCTCCCCCGGTTCCCCGAGTGGATCACCATCC


2881
---------+---------+---------+---------+---------+---------+
2940



GCGACACACACCTCGACCTACTCGTGTTGGAGGGGGCCAAGGGGCTCACCTAGTGGTAGG



  L  C  V  E  L  D  E  H  N  L  P  R  F  P  E  W  I  T  I  P
-



CTCTGGTGTGCATCTACATGCTCTCCACCAACATCCTGCTGGTCAATCTGCTCGTTGCCA


2941
---------+---------+---------+---------+---------+---------+
3000



GAGACCACACGTAGATGTACGAGAGGTGGTTGTAGGACGACCAGTTAGACGAGCAACGGT



  L  V  C  I  Y  M  L  S  T  N  I  L  L  V  N  L  L  V  A  M
-



TGTTTGGCTACACAGTGGGAACGGTCCAGGAGAACAACGATCAGGTCTGGAAGTTCCAGA


3001
---------+---------+---------+---------+---------+---------+
3060



ACAAACCGATGTGTCACCCTTGCCAGGTCCTCTTGTTGCTAGTCCAGACCTTCAAGGTCT



  F  G  Y  T  V  G  T  V  Q  E  N  N  D  Q  V  W  K  F  Q  R
-



GGTACTTCTTGGTGCAGGAGTACTGCAACCGCCTGAACATCCCCTTCCCCTTTGTGGTCT


3061
---------+---------+---------+---------+---------+---------+
3120



CCATGAAGAACCACGTCCTCATGACGTTGGCGGACTTGTAGGGGAAGGGGAAACACCAGA



  Y  F  L  V  Q  E  Y  C  N  R  L  N  I  P  F  P  F  V  V  F
-



TCGCCTACTTCTACATGGTGGTCAAGAAGTGCTTCGGATGCTGCTGCAGGGAGAAACACG


3121
---------+---------+---------+---------+---------+---------+
3180



AGCGGATGAAGATGTACCACCAGTTCTTCACGAAGCCTACGACGACGTCCCTCTTTGTGC



  A  Y  F  Y  M  V  V  K  K  C  F  G  C  C  C  R  E  K  H  A
-



CCGAGCCTTCTGCCTGCTGTTTCAGAAATGAAGACAATGAGACTCTGGCATGGGAGGGTG


3181
---------+---------+---------+---------+---------+---------+
3240



GGCTCGGAAGACGGACGACAAAGTCTTTACTTCTGTTACTCTGAGACCGTACCCTCCCAC



  E  P  S  A  C  C  F  R  N  E  D  N  E  T  L  A  W  E  G  V
-



TCATGAAAGAAAATTACCTTGTCAAGATCAACACGGAGGCCAATGACACCTCACAGGAAA


3241
---------+---------+---------+---------+---------+---------+
3300



AGTACTTTCTTTTAATGGAACAGTTCTAGTTGTGCCTCCGGTTACTGTGGAGTGTCCTTT



  M  K  E  N  Y  L  V  K  I  N  T  E  A  N  D  T  S  Q  E  M
-



TGAGGCATCGGTTTAGACAGCTGGATACAAAGATTAATGATCTCAAGGGCCTTCTGAAAG


3301
---------+---------+---------+---------+---------+---------+
3360



ACTCCGTAGCCAAATCTGTCGACCTATGTTTCTAATTACTAGAGTTCCCGGAAGACTTTC



  R  H  R  F  R  Q  L  D  T  K  I  N  D  L  K  G  L  L  K  E
-



AGATCGCTAATAAAATCAAATAGAACTTCATGGACTGTACTGGAGAAAAACCTAATTATA


3361
---------+---------+---------+---------+---------+---------+
3420



TCTAGCGATTATTTTAGTTTATCTTGAAGTACCTGACATGACCTCTTTTTGGATTAATAT



  I  A  N  K  I  K  * (SEQ ID NO: 2)



GCAAGGTGACACCAGAAATCGAAGTGGGAACCAGTCAAGAAAAGCTGATGAACAGTTTTG


3421
---------+---------+---------+---------+---------+---------+
3480



CGTTCCACTGTGGTCTTTAGCTTCACCCTTGGTCAGTTCTTTTCGACTACTTGTCAAAAC



TTACTGACTGCTCAGTAAGAACTGTTCAGGCCGTGGGTATTTAGCAGATGGCTTTCATCA


3481
---------+---------+---------+---------+---------+---------+
3540



AATGACTGACGAGTCATTCTTGACAAGTCCGGCACCCATAAATCGTCTACCGAAAGTAGT



CCCCAGTGTGCTCAAATCTGGGAAACAGACGTGTGATTGGTTTCCCCCGAGAAGATAGAC


3541
---------+---------+---------+---------+---------+---------+
3600



GGGGTCACACGAGTTTAGACCCTTTGTCTGCACACTAACCAAAGGGGGCTCTTCTATCTG



ACCCAGGAAGAGCTTCCCCTGAAGGCCACCCTGTTACTTCCTGAGTCTCCACCACTCATA


3601
---------+---------+---------+---------+---------+---------+
3660



TGGGTCCTTCTCGAAGGGGACTTCCGGTGGGACAATGAAGGACTCAGAGGTGGTGAGTAT



CCCACTGCGGGTCATCTTAGAGTGTGTTCCTGCACTCTTCTTCTTTCTTCACTTTTCCTA


3661
---------+---------+---------+---------+---------+---------+
3720



GGGTGACGCCCAGTAGAATCTCACACAAGGACGTGAGAAGAAGAAAGAAGTGAAAAGGAT



CTTCTAACTCTGTGCATATTACATCTCTCCTGCAAGGGGGTCATGCCTTCCCTCCCATAA


3721
---------+---------+---------+---------+---------+---------+
3780



GAAGATTGAGACACGTATAATGTAGAGAGGACGTTCCCCCAGTACGGAAGGGAGGGTATT



AAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 1)


3781
---------+---------+---------+----
-
3815



TTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
















TABLE 2













 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 



























TABLE 3






cCMR1


cCMR1



Position
residue
Variant
Position
residue
Variant




















4
E
R
353
V
Hydrophobic (e.g., A, M)


5
G
A
374
T
I


12
N
S
387
S
C


16
G
D
388
S
P


18
L
Hydrophobic (e.g., M)
403
I
Hydrophobic (e.g., V)


19
D
G
406
N
S


28
T
A, V
419
N
S


34
V
Hydrophobic (e.g., L)
443
N
S


38
E
D
444
E
D


58
T
I
449
N
(Amine-containing, E.g. H)


59
K
Basic (e.g., R)
454
G
E


64
T
M
485
R
(Amine-containing, E.g. Q)


66
N
S
490
N
(Amine-containing, E.g. H)


67
V
Hydrophobic (e.g., I)
491
D
E


78
I
Hydrophobic (e.g., M)
499
N
T


85
S
Q
534
G
S


86
N
S
536
R
W


118
I
Hydrophobic (e.g., L)
541
S
N


126
A
S
542
S
G


128
T
I
544
D
E


204
N
S
545
D
E


227
L
Hydrophobic (e.g., I)
546
I
Hydrophobic (e.g., M, L)


229
N
S
548
V
Hydrophobic (e.g., I)


232
A
D
553
V
Hydrophobic (e.g., A)


235
Y
H
555
P
Hydrophobic (e.g., L)


237
S
L
556
I
T


241
I
L
564
H
L


246
K
T, M
585
R
(Basic, e.g., K)


290
I
S
635
S
N


310
R
(Basic, e.g., K)
709
F
L


320
I
Hydrophobic (e.g., V)
715
R
(Basic, e.g., K)


322
S
N
717
I
Hydrophobic (e.g., V)


348
V
Hydrophobic (e.g., A)
723
I
Hydrophobic (e.g., L)


739
A
S


766
S
(Nucleophillic, e.g., T or




P)


770
V
Hydrophobic (e.g., I)


773
A
S


788
M
Hydrophobic (e.g., V)


819
P
S


823
T
(Nucleophillic, e.g., S)


894
H
(Amine-containing, eg.,




Q)


921
G
S


927
A
S


932
T
(Nucleophillic, e.g., S)


946
H
Y


985
T
I


1007
N
S


1016
V
Hydrophobic (e.g., I)


1030
G
K


1034
R
(Basic, e.g., K)


1037
H
(Amine-containing, eg.,




N)


1038
A
M, T


1040
P
S


1041
S
N


1042
A
Hydrophobic (e.g., V)


1046
R
(Basic, e.g., K)


1070
E
K


1074
T
N


1075
S
A


1076
Q
E


1087
T
(Nucleophillic, e.g., S)


1089
I
Hydrophobic (e.g., L)


1094
G
S


1102
K
(Basic, e.g., N)


















TABLE 4





SEQ ID NO
Description
Sequence


















SEQ ID NO
Upstream primer
ttcatctgggccattcttcag



3
(cmr1-23)


 


SEQ ID NO
Downstream primer
cacagtggcttggactcatt


4
(cmr1-26)


 


SEQ ID NO
Forward primer for
gcccatcgacaagcacaagaagatc


5
3′ RACE-PCR (dcmr1-



3)


 


SEQ ID NO
Reverse primer for
gatcttcttgtgcttgtcgatgggc


6
5′-RACE-PCR (dcmr1-



1)


 


SEQ ID NO
Universal Primer
ccatcctaatacgactcactatagggc


7


 


SEQ ID NO
Forward primer
aagcttcatatgtccttcgagggggccaggctcagcatgaggaa


8
(dcmr1-7)


 


SEQ ID NO
Reverse primer
ctcgagctatttgattttattagcgatctctttcagaaggccc


9
(dcmr1-8)









EXAMPLE 1
Cloning of CMR1 from Dog DRG Neurons
A. Isolation of poly(A+) RNA
As a first step in the cloning of cCMR1, poly(A+) RNA was isolated from 100 μg of total RNA from canine DRG [(Custom made by Analytical Biological Service Inc. DE)] using an OligotexTM spin column (Qiagen Inc., CA). Briefly, 150 μl of RNase-free water, 250 μl of buffer OBB [20 mM Tris, pH7.5, 1M NaCl, 2 mM EDTA and 0.2% SDS] and 15 μl of a suspension of Oligotex beads were added to 100 μl of total RNA solution (1 μg/μl). The RNA/Oligotex bead mixture was then heated at 70° C. for 3 min to disrupt any secondary structure of the RNA followed by incubation at room temperature for 10 min. The poly(A+) RNA/Oligotex particle complex was centrifuged and washed twice with 400 μl of buffer OW2 [10 mM Tris, pH7.5, 150 nM NaCl, and 1 mM EDTA] and then transferred to a spin column for the elution step. The poly(A+) RNA was eluted from Oligotex bead using 200 μl of prewarmed (70° C.) Buffer OEB [5 mM Tris, pH 7.5]. Finally, canine DRG poly(A+) RNA was precipitated by ethanol in the presence of 20 μg of glycogen and 150 mM sodium acetate and resuspended in 10 μl of RNase free water.
B. Synthesis of Double-stranded cDNA
4 μl (1 μg) of canine DRG poly(A+) RNA and 1 μl of cDNA synthesis primer, a 52-mer oligo with sequence of 5′-TTCTAGAATTCAGCGGCGC(T)30N−1N-3′, N−1=G, A or C; and N=G, A, C or T (Clontech, CA SEQ ID NO:10) were mixed, incubated at 70° C. for 2 min and then cooled on ice for 2 min. The first strand cDNA synthesis (reverse transcription) was performed at 42° C. for 1 hour using 20 units of AMV reverse transcriptase in the presence of 1 mM dNTP mixture and first strand synthesis buffer (50 mM Tris, pH 8.5, 8 mM MgCl2, 30 mM KCl and 1 mM DTT) in 10 μl. The second strand cDNA synthesis was performed by adding an enzyme cocktail consisting of 24 units of E. coli DNA polymerase I, 5 units of E. coli DNA ligase I unit of E. coli RNase H, 0.25 mM of dNTP mixture (0.25 mM of each dATP, dCTP, dGTP, and dTTP), and second strand buffer (100 mM KCl, 10 mM ammonium sulfate, 5 mM MgCl2, 0.15 mM β-NAD, 20 mM Tris pH 7.5, and 50 μM/ml bovine serum albumin) in 80 μl. The reaction was first carried out at 16° C. for 90 min followed by addition of 20 units of T4 DNA polymerase with continued incubation at the same temperature for 45 min. The reaction was terminated by adding 10 mM EDTA and 8 μg of glycogen. Phenol and chloroform extractions were performed, followed by ethanol precipitation. Double-stranded cDNA was then suspended in 200 μl of TE buffer and stored at −20° C.
C. PCR Amplification of Near Carboxyl Terminus of Dog CMR1
A portion of the cCMR1 sequence was successfully amplified by PCR using two primers designated cmr1-23 (5′-ttcatctgggccattcttcag-3′ (SEQ ID NO: 3), which hybridizes to nucleotides 1761-1781 of SEQ ID NO 1 and cmr1-26 (5′-cacagtggcttggactcatt-3′ (SEQ ID NO: 4), which hybridizes to nucleotides 2868-2886 of SEQ ID NO: 1. The PCR reaction was performed in final volume of 50 μl, containing 5 μl of canine DRG double-stranded cDNA, 5 μl of 10× reaction buffer provided with Advantage2 DNA polymerase, 200 μM dNTPs, 200 nM forward primer cmr1-23, 200 nM reverse primer cmr1-26 and 1 μl of 50× AdvantageTM-HF2 DNA polymerase mixture (Clontech, CA). PCR was performed by an initial denaturing step at 94° C. for 1 min, followed by 30 cycles of: (a) denaturing at 94° C. for 30 sec, (b) annealing at 55° C. for 30 sec and (c) extension at 72° C. for 60 sec.
Agarose gel electrophoresis was performed, which revealed that the PCR product was approximately 1.1 kb. After PCR, the 1.1 kb PCR fragment was purified and subcloned into pPCRscript (Stratagene) following the vendor's protocol. Two independent clones were picked and subjected to DNA sequencing analysis.
The sequence results revealed that the PCR amplified fragment was 83% 84%, and 87% identical to the near the carboxyl termini of mouse, rat and human CMR1, respectively.
D. RACE-PCR of 5′ and 3′ Ends of cCMR1 Sequence
To obtain the complete 5′ and 3′ cDNA sequences of the cCMR1 gene, RACE-PCR technology was performed. First, both 5′- and 3′-RACE-Ready cDNAs were synthesized separately with SMARTTM RACE DNA Amplification Kit (BD Clontech, CA), according to the manufacturer's instructions. To prepare cDNA for 5′ RACE, in one 0.5 ml tube, 3 μl of dog DRG poly(A+) RNA obtained in A. was mixed with 1 μl of 5′-CDS primer and 1 ml of SMART II A oligo. To prepare cDNA for 3′ RACE, 3 μl of dog DRG poly(A+) RNA was mixed with 1 μl 3′-CDS primer and 1 μl RNase free water in another 0.5 ml tube and then incubated at 70° C. for 2 min followed by cooling on ice for 2 min. Next, 2 μl of 5× First Strand buffer, 1 μl 20 mM DTT, 1 μl of 10 mM dNTP mix and 1 μl PowerScript Reverse Transcriptase were added to each tube, and synthesis was performed at 42° C. for 90 min. The reactions were stopped by adding 200 μl of TE buffer and heating the sample to 72° C. for 7 min. The reaction products were stored at −20° C.
For RACE-PCR, two primers were synthesized based on the 1.1 kb cDNA sequence proximal to the 5′ portion of the cCMR1 cDNA. The forward primer for 3′ RACE-PCR was named dcmr1-3 and had the following sequence: 5′-GCCCATCGACAAG CACAAGAAGATC-3′ (SEQ ID NO: 5), which hybridizes to nucleotides 2213-2237 of SEQ ID NO: 1 (complementary strand); the reverse primer for 5′-RACE-PCR was named dcmr1-1 and had the following sequence: 5′-GATCTTCTTGTGCTTGTCGATGGGC-3′ (SEQ ID NO: 6), which hybridizes to nucleotides 2213-2237 of SEQ ID NO: 1. Both 5′ and 3′-RACE PCRs were performed in a final volume of 50 μl containing 5 μl of cDNA template (either 5′- or 3′-RACE-Ready cDNA, as described above), 5 μl of 10× reaction buffer, 200 μM dNTPs, 200 nM Universal Primer Mix (UPM) (Clontech), SEQ ID NO: 7 (5′-CCA TCC TAA TAC GAC TCA CTA TAG GGC-3′), 200 nM cCMR1 specific primer (dcmr1-1 for 5′-RACE PCR or dcmr1-3 for 3′-RACE PCR) and 1 μl of 50× AdvantageTM-HF2 DNA polymerase mixture (Clontech). The thermal cycler parameters for the RACE-PCR were: a) initial denaturing at 94° C. for 2 min; b) 5 cycles of: 94° C. for 30 sec, 72° C. for 3 min; c) 5 cycles of: 94° C. for 30 sec, 70° C. for 30 sec, 72° C. for 3 min; and d) 25 cycles of 94° C. for 5 sec, 68° C. for 30 sec, 72° C. for 3 min. After the reaction, the RACE-PCR products were purified, polished and directly subcloned into pPCRscript. Four independent clones from either 5′-RACE or 3′-RACE were picked and subjected to DNA sequencing analysis.
E. Sequence of Full-length cCMR1 cDNA:
The sequence of the full-length canine CMR1 cDNA was confirmed by synthesizing PCR primers based on the sequence of the 5′ and 3′ ends obtained by RACE-PCR. Full length cCMR1 cDNA was amplified from canine DRG double-stranded cDNAs prepared in B. by high-fidelity DNA polymerase with forward primer dcmr1-7, which had the following sequence: 5′-AAGCTTCAT ATG TCC TTC GAG GGG GCC AGG CTC AGC ATG AGG AA-3′ (SEQ ID NO: 8) and reverse primer dcmr1-8, which had the following sequence: 5′-CTCGAG CTA TTT GAT TTT ATT AGC GAT CTC TTT CAG AAG GCCC-3′ (SEQ ID NO: 9). The PCR was performed in a final volume of 50 μl containing 5 μl of above dog DRG double-stranded cDNAs, 5 μl of 10× reaction buffer, 200 μM dNTP, 200 nM forward primer dcmr1-7, 200 nM reverse primer dcmr1-8, and 1 μl of 50× AdvantageTM-HF2 DNA polymerase mixture (Clontech, CA). The PCR reaction parameters were: 1 cycle: initial denaturing at 94° C. for 2 min; 35 cycles: a) denaturing at 94° C. for 30 sec, b) annealing and extension at 70° C. for 5 min. After PCR, the 3.4 kb PCR fragment was purified and subcloned into pPCRscript following the same cloning protocol as in C. Four independent clones were picked and subjected to DNA sequencing analysis. The clone NQC562 was used for further subcloning and studying. The sequence results revealed that the nucleic acid sequence of cCMR1 cDNA (nucleotides 69-3380 of SEQ ID NO: 1) was 86.2%, 86.6%, and 90.9% identical to the cDNA sequences of mouse (Accession number: AY095352), rat (Accession number: AY072788) and human (Accession number: NM—024080) CMR1, respectively.
F. Sequence Analysis
5′- and 3′-RACE-PCR allowed for the determination of the 68 nucleotide sequence of the 5′ untranslated region; 3′-RACE-PCR allowed for the determination of the 431 bp of the 3′ untranslated region including the 37-mer poly(A+) tail. No in-frame stop codon was identified.
The predicted cCMR1 open reading frame consists of a 3315 bp sequence that is predicted to encode a polypeptide of 1104 amino acids (SEQ ID NO: 2) having a calculated molecular mass of 127.6 kDa (see Table 1). A Kyte-Doolitle hydrophilicity analysis (not shown) of primary sequence predicts the presence of eight putative hydrophobic domains clustered near the carboxyl terminus. A high probability of coiled-coil domain located at the very carboxyl terminal from residue 1070 to the end, which may be implicated in oligomerization of the channel, was identified. Further, the primary sequence analysis with GCG SeqWeb revealed that cCMR1 contained multiple N-glycosylation sites located at residues 15, 256, 317, 812, 934, 1050 and 1072, respectively. cCMR1 also contains one putative PKA (protein kinase A) phosphorylation sites at residue 92, three tyrosine phosporylation sites at residues 30, 228 and 288, and 17 PKC (protein kinase C) phosphorylation sites.
The cCMR1 amino acid sequence was aligned with the human (GenBank Acc. No.: NP—076985), rat (GenBank Acc. No.: NP—599198), and mouse (GenBank Acc. No.: AAM23261) sequences which revealed a 95.1%, 94.1%, and 93.9% identity, respectively, using the Gap program from Seqweb version 2 of Accelrys. The Gap program uses the algorithm of Needleman and Wunsch (J. Mol. Biol., 48:443 (1970)) to find the alignment of two complete sequences. It maximizes the number matches and minimizes the number of gaps.
EXAMPLE 2
Recombinant Expression of CMR1
A. Cloning of cCMR1 into a Mammalian Expression Vector
For expression of cCMR 1 in mammalian cell lines, the full-length cDNA of cCMR1 was subcloned into pcDNA3.1 by performing a three-way ligation. First, the full-length cCMR1 clone NQC562 was digested with HindIII and NcoI to yield a 0.8 kb 5′ fragment. Next, in an independent restriction reaction, NQC562 was digested with NcoI and SalI to yield a 2.5 kb 3′ fragment. The 0.8 kb 5′ and 2.5 kb 3′ cCMR1 fragments were purified and ligated with pcDNA3.1 that was predigested with HindIII and SalI, creating vector pcDNA3.1-cCMR1.
For in vitro translational analysis, full-length cCMR1 cDNA was subcloned into pAGA4 vectors (modified from pGEM3 of Promega, Sanford 1991 and Qin, et al 1997). Briefly, 0.8 kb N-terminal fragment was obtained by digestion of NQC562 with NdeI and NcoI and a 2.5 kb C-terminal fragment was obtained by digestion of NQC562 with NcoI and XhoI. The two purified fragments were ligated together with vector pAGA4 predigested with NdeI and SalI, creating construct cCMR1/pAGA4. All the final constructs were confirmed by DNA sequencing.
B. In Vitro Translation of cCMR1
In vitro translation of the canine CMR1 was done with TnTTM T7 Quick Coupled Transcription/Translation System (Promega), according to the vendor-recommended protocol. Briefly, 1 μl of 0.1 μg/μl cCMR1/pAGA4 was added to 9 μl of TNT Quick Master Mix with 0.2 μl of [35S]-methionine (1000 Ci/mmol at 10 mCi/ml). The reaction mixture was incubated at 30° C. for 90 min. The reaction was stopped by adding an equal volume of 2×SDS/PAGE loading buffer, and then, the samples were subjected to 4-20% gradient SDS-PAGE analysis. After electrophoresis, the gel was stained with Commassie Blue R250, dried and exposed to X-ray film. The in vitro translated cCMR1 migrated to an approximate molecular weight of 135 kDa as predicted by faithful translation of the amino acid sequences from the corresponding nucleic acid sequences.
The in vitro translated cCMR1 protein was also analyzed by Western blot. 5 μl of in vitro translated cCMR1 protein was subjected to 4-20% gradient SDS-PAGE. The proteins on the gel were then transferred to nitrocellulose. The blot was then blocked with 5% dry milk in TTBS (0.5% Tween 20, 100 mM Tris-HCl, and 0.9% NaCl at pH=7.5) at room temperature for 1 hour and then incubated with anti-cCMR1 polyclonal antibody (1:500) at 4° C. overnight. The next day, the blot was washed three times with 100 ml TTBS, and incubated with goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (Pierce) at room temperature for 1 hour. The blot was washed three times with 100 ml TTBS and visualized with ECL-Plus luminescent reagents (Amersham-Pharamacial Biotech) according to the manufacturer's instructions.
The pcDNA3.1-cCMR1 construct was transfected into HEK293 (human embryonic kidney cells (ATCC CRL-1573) using the GeneJammerTM kit (Stratagene, CA), according to manufacturer's protocol. Stable cell clones were selected by growth in the presence of G418. Single G418 resistant clones were isolated and purified. Clones containing the cCMR1 cDNA were analyzed using a calcium influx assay.
EXAMPLE 3
Calcium Influx Functional Assay of cCMR1
FLIPR assay was performed to study the properties of cCMR1 channels within a population of cells.
To demonstrate functionality of the cCMR1 expressed in recombinant cells, CMR1/HEK293 stably transfected cells were seeded in a 384-well plate at a concentration of 6.7×105 cells/well and incubated overnight at 37° C. The following day, the cells were loaded with buffer and calcium dye (Molecular Devices, Sunnyvale, Calif.) in a final volume of 40 μl and incubated for 30 minutes at room temperature. The fluorescence intensity was measured by FLIPR before and after the addition of menthol or icilin, which were added to the cells at a concentration of 100 μM or 10 μM each, respectively. The results are shown in FIG. 1.
EXAMPLE 4
CMR1 Functional Assay with Reduced Ca++ Loading Concentrations
CMR1 opens in response to agonists, such as menthol or icilin, and also to mildly cold temperatures (15° C. to 25° C.). Therefore, at room temperature (22-24° C.) CMR1 could be active and induce Ca2+ influx. However, Ca2+ influx will also induce Ca2+-dependent inactivation, resulting in negative feedback regulation of CMR1. In this case, CMR1 will be inactivated after activation by room temperature and will not be reactivated until the temperature is increased above about 25° C. Therefore, under normal test conditions (room temperature and in the presence of buffer containing 2 mM Ca 2), CMR1 from certain species, such as rat CMR1, is not responsive to any agonist. To prepare a system wherein CMR1 would be used to screen antagonist at room temperature, a Ca2+ influx assay was developed by removing Ca2+ from the dye loading buffer and then challenging the CMR1-continaing system with 4 mM Ca2+. Under this condition, although CMR1 is active (at room temperature), no calcium will enter the cell through the channel and inactivation will not occur. Under these assay conditions CMR1 is constitutively active and primed to permit Ca2+ influx as soon as Ca2+ is added into the extracellular solution.
Human Embryonic Kidney cells (HEK293) transfected with rat CMR1 were seeded in a 384-well plate (6.7×105 cells/well). The following day, the culture media was removed and the cells were rinsed with complete Hank's buffer. Cells were then loaded with buffers and calcium dye (Mol. Dev.) in a final volume of 40 μl and incubated for 30 minutes at room temperature. The plates were then transferred to a FLIPR apparatus wherein compounds tested for antagonist activity were added to a final concentration of 4.2 μM at time zero. Calcium was added to a final concentration of 4 mM at about time 10 second, and fluorescence intensity was measure by FLIPR. A representative result is shown in FIG. 2 wherein no test compound was added at time zero.
EXAMPLE 5
A Screening Assay for a Desensitizer or Inactivator of a CMR1 Channel
Generally, upon prolonged exposure of an ion channel to an activating stimulus (e.g., an agonist) or in response to a direct desensitizing or inactivating stimulus, the channel may assume alternate conformations that are variably less activatable in response to an activating stimulus. These less activatable or inactivatable conformations may be referred to functionally as being desensitized or inactivated, and compounds that produce these states as being desensitizers or inactivators, respectively. Such conformations may be induced or stabilized by or in the presence of these so-called desensitizers or inactivators, and may arise by the preferential action of the desensitizer or inactivator upon an open or upon a closed channel. In addition, such conformations may be reversible, across variable time courses and conditions, or may be irreversible, pending de novo synthesis of nascent channels. Compounds that are identified as desensitizers or inactivators, either being reversible or irreversible, may be useful in the treatment of certain conditions, including pain conditions, in which decreased CMR1 activity would be therapeutic.
Therefore, in another embodiment, the invention provides a method of identifying reversible and irreversible desensitizers or inactivators of CMR1 activity. The first method is designed to identify compounds that induce and/or stabilize the channel in a desensitized or inactivated state from a closed state and comprises the steps of: (a) providing a recombinant cell comprising a nucleic acid encoding a cCMR1 protein, (b) contacting the recombinant cell at a temperature above the threshold for activation (typically above about 28° C.) with a test compound for varying lengths of time, (c) extensively washing out the test compound and (d) at varying time points, determining the extent to which the test compound diminishes CMR1 activity in response to a subsequent exposure to a CMR1-activating stimulus. The second method is designed to identify compounds that induce and/or stabilize the channel in a desensitized or inactivated state from an open state and comprises EITHER the steps of: (a) providing a recombinant cell comprising a nucleic acid encoding a cCMR1 protein, (b) contacting the recombinant cell at a temperature above the threshold for activation (typically above about 28° C.) with a CMR1 agonist, (c) contacting the recombinant cell with a test compound for varying lengths of time, (d) extensively washing out the test compound and agonist and (e) at varying time points, determining the extent to which the test compound diminishes CMR1 activity in response to a subsequent exposure to a CMR1-activating stimulus OR the steps of: (a) providing a recombinant cell comprising a nucleic acid encoding a cCMR1 protein, (b) incubating the recombinant cell at a temperature below the threshold for activation (typically below about 28° C.), (c) contacting the recombinant cell with a test compound for varying lengths of time, (d) extensively washing out the test compound and (e) at varying time points, determining the extent to which the test compound diminishes CMR1 activity in response to a subsequent exposure to a CMR1-activating stimulus.
EXAMPLE 6
Activation of cCMR1 by Mustard Oil
Mustard oil is a nature product that elicits pain and inflammation when applied to the skin. Recently, TRPA1, a novel member of TRP family, has been proposed as one of the cellular and molecular targets for the pungent action of mustard oils (Jordt, et al. 2004, Nature, 427: 260-265). We demonstrated that Mustard oil also activates cCMR1.
HEK293 cells stably transfected with cCMR1 were seeded in a 384 will plate at a concentration of 6.7×105 cells/well and incubated overnight at 37° C./5% CO2. The following day the cells were loaded with calcium dye and incubated for 30 minutes at room temperature. The calcium-mediated fluorescence intensity was measured by FLIPR before and after the compound was administered to the cells. As shown in FIG. 3, cCMR1 is not only sensitive to cooling compounds such as 100 nM icilin (solid line), but also is activated by the pungent compound, 1 mM mustard oil (dash line).
EXAMPLE 7
Whole-cell Patch Clamp Studies
Patch clamp experiments were performed to study the properties of cCMR1 channels expressed in a single cell.
HEK293 cells stably transfected with canine cCMR1 were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 1 mg/ml G418. Cells were maintained at 37° C. and in 5% CO2.
Unless otherwise indicated, the standard extracellular solution used for recording contained (in mM): NaCl, 132; EGTA, 1; KCl, 5.4; MgCl2, 0.8; HEPES, 10; glucose, 10; pH=7.4. In experiments where the extracellular solution contained Ca2+, the extracellular solution used was one of the following (in mM), depending on the Ca2+ concentration used: (1) NaCl, 132; CaCl2, 0.1 or 1.8; KCl, 5.4; MgCl2, 0.8; HEPES, 10; glucose, 10; pH=7.4; (2) NaCl, 116; CaCl2, 10; KCl, 5.4; MgCl2, 0.8; HEPES, 10; glucose, 10; pH=7.4. The intracellular solution used to fill recording pipettes contained (in mM): CsCl, 145; EGTA, 5; HEPES, 10; glucose, 5; pH=7.4.
Recordings were performed using the conventional whole-cell patch clamp technique, 1-2 days after plating cells onto glass coverslips at densities appropriate for single cell recording. Currents were amplified by a patch clamp amplifier and filtered at 2 kHz (Axopatch 200B, Axon Instruments). Menthol (100 μM) or icilin (1 μM) was applied to the cell at 0.5 ml/min via a gravity-fed perfusion system. Recordings involving agonist stimulations were performed at 22° C.
In experiments where temperatures were varied, temperature ramps were generated by heating/cooling the perfusate in a dual in-line heater/cooler (Model SC-20, Warner Instruments, Hamden, Conn.) controlled by a bipolar temperature controller (Model CL-100, Warner Instruments). The temperature in the vicinity of the recorded cell was measured with a custom-made miniature thermo-microprobe connected to a monitoring thermometer (Model TH-8, Physitemp, Clifton, N.J.), and sampled using Digidata 1322A and pClamp 9.0 (Axon Instruments, Union City, Calif.), as were the currents concurrently measured in the whole-cell patch clamp mode. Two voltage protocols were used in these studies. The first involved a 600 ms voltage ramp from −100 mV to +60 mV at a sampling rate of 10 kHz. This voltage pulse was repeated once every 5 seconds. The cell was held at −100 mV between voltage pulses. In the second protocol, the cell was held at −80 mV and the current was continuously sampled (at 100 Hz) at this holding potential.
FIG. 4 illustrates that cCMR1 is strongly outwardly rectifying and non-selective to cations. Whole-cell patch clamp recording of cCMR1 was performed using the voltage ramp protocol described above. Upon application of 100 μM menthol, a cooling agent, there was a large increase of the whole-cell current amplitude (solid line) compared to control (dashed line) at both hyperpolarized and depolarized membrane potentials. This increase was much more pronounced at depolarized potentials than at hyperpolarized potentials. Hence, the channel is strongly outwardly rectifying. In addition, the menthol-activated current had a reversal potential near 0 mV, indicating the relatively unselective (at least to the cations used in these experiments) nature of the channel. Qualitatively similar results have also been obtained for another cooling agent, icilin.
The temperature sensitivity of cCMR1 is illustrated in FIG. 5. As the temperature of the solution perfusing the cCMR1-expressing cell was lowered, the current passing through the cell at +60 mV was significantly increased with an activation threshold of  ̃17° C. The cCMR1 channel was not open at room temperature, but was activated by cool temperatures below about 17° C.
FIG. 6 demonstrates that extracellular Ca2+ desensitizes the cCMR1 channel. Menthol at 100 μM activated a non-desensitizing current in the absence of extracellular Ca2+ (−80 mV; gray trace). In contrast, desensitization readily occurred in the presence of 1.8 mM extracellular Ca2+ under otherwise identical recording conditions (black trace; normalized to the Ca2+-free trace for display clarity).
Extracellular Ca2+ decreased the current amplitude of cCMR1 when the channel was activated by menthol, for example at 1 mM. This apparent inhibition by extracellular Ca2+ was concentration dependent (FIG. 7). The higher concentration of extracellular Ca2+, the stronger the inhibition of the current amplitude. The dashed line in FIG. 7 is a logistic function representing the best fit to the data. An IC50 value of 1.6 mM extracellular Ca2+ was derived from the best fit analyses. In addition, the apparent inhibition by extracellular Ca2+ was voltage-dependent (FIG. 8). Extracellular Ca2+ (10 mM) strongly inhibited the current amplitude at hyperpolarized potentials. The inhibition was lessened at more depolarized potentials.

## Claims

1. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide capable of detecting and transducing cold stimuli and having at least 98% sequence identity to SEQ ID NO: 2.

2. The isolated nucleic acid molecule of claim 1 that encodes a polypeptide of SEQ ID NO: 2.

3. The isolated nucleic acid molecule of claim 1 comprising nucleotide 69 to 3380 of SEQ ID NO: 1.

4. An expression vector comprising the isolated nucleic acid sequence of claim 1.

5. A recombinant host cell comprising the isolated nucleic acid sequence of claim 1.

6. A method for expressing a polypeptide having at least 98% sequence identity to SEQ ID NO: 2 comprising the steps of:
(a) introducing an expression vector capable of encoding a polypeptide having at least 98% sequence identity to SEQ ID NO: 2 into a cell; and
(b) culturing the cell under conditions that allow expression of the polypeptide from the expression vector.