JP2002538768A - Ion channels, especially vanilloid receptor-like (VR-L) receptors - Google Patents

Abstract

  (57) [Summary] The invention provides (a) activatable by noxious heat; (b) obtainable from human T lymphocytes; (c) isolated, non-selective cation channels that are not capsaicin-sensitive. Provided are proteins, particularly VR-L proteins (SEQ ID NO: 2) and / or variants thereof (eg, homologs or fragments). Also provided are nucleic acids that encode them (eg, SEQ ID NO: 1), or those that have utility in search, amplification or down regulation. Methods for providing the above proteins and nucleic acids are disclosed as well as vectors, host cells and organisms, and antibodies utilizing them. Further disclosed are methods of affecting electrophysiological and pharmacological properties (eg, by therapy) based on the manipulation of proteins or nucleic acids as described above, and substances having ion channel modulating activity, Other analgesics or other substances that act on nociception, immunomodulators or neuromodulators).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field The present invention is an ion channel, in particular, especially that there is responsive to noxious heat, and to nucleic acids encoding them. It also produces these,
And to methods and materials for use.

Prior art Various proteinaceous ion channels have been characterized in the literature. Certain H ⁺ -dependent channels, further activated by noxious heat and pepper, have been cloned in Caterina et al. (1997) Nature, 389: 816-824.
VR1 "channel (or" vanilloid receptor-1 "), which appears to play a role in pain pathways.

[0003] Ion channels have received widespread attention, particularly for their usefulness in studying and manipulating the neurological and physiological processes that they mediate in vivo. In particular, the novel channels can be used for screening for agonists or antagonists (eg, analgesics that act on nociception) that can exert a desired physiological effect. Similarly, proteins or nucleic acids based on the channels themselves can be used directly to manipulate these processes.
Similarly, proteins or nucleic acids based on the channels themselves can be used directly to manipulate these processes.

[0004] It will therefore be appreciated that the provision of new channels, particularly those having one or more new activities, compared to those generally available, would provide a contribution to the art.

DISCLOSURE OF THE INVENTION The present inventors have cloned a novel ion channel derived from cultured Jurkat cells (human leukemia T lymphocyte cell line). The protein functions as a non-selective ion channel and has been shown to be thermosensory.

[0006] The ion channel has structural similarity to VR1 and
Share 7% identity (based on the number of identical amino acids divided by the total number of amino acids in the compared regions).

The ion channels that form the basis of the present invention are referred to herein as VR-like or VR-
L.

[0008] Like VR1, VR-L is the protein "t" for topological organization.
rp "family members. The Trp protein is a Drosophila transient receptor potential that exhibits photoreceptor deficiency.
tial, or Trp), is the first six transmembrane monomer identified in the mutant. Both VR1 and VR-L have characteristic N-terminal ankyrin repeats, and considerable sequence similarity is not limited to six transmembrane domains, their flanking sequences, and one of the putative pore regions. It is also present in the loop between transmembrane domains 5 and 6, which are believed to form a part. One trp gene is known as capacitive calcium influx, vaguely defined as Ca ²⁺ influx from the extracellular space following inositol 1,4,5-triphosphate-induced mobilization of an internal store. Provide a molecular basis for the phenomenon to be performed. It is known that trp plays an important role in the photoreduction of Drosophila, and a mediator of hyperalgesia, bradykinin, is obtained via activation of Gq heterotrimeric G protein obtained in a heterologous expression system. It is clear that trp-3 can be gated. C. A study by the Bargmann group in Elegans (Colb
ert et al. 1997, J Neurosci 17 (21: 8259-8269; Bargmann et al. 1998 An
nu Rev Neurosci 21: 279-308) is a trp-related protein (OSM-9)
Also play a role in mechanosensory transmission.

[0009] VR1 itself is a capsaicin-dependent channel. Such channels, which are present in sensory nerves, cause pain caused by acids and possibly other inflammatory mediators (eg, bradykinin, prostaglandins E2 and 5-HT) with tissue damage and ischemic symptoms. It is believed to mediate. VR1
Heterologous expression induces capsaicin-sensitive cation channels that are transiently activated by rapid extracellular acidification or capsaicin. The biophysical and pharmacological properties of the VR1 channel closely match those of the capsaicin-dependent cation channel described in sensory nerves (Helliwell et al.
l, 1998 Neurosci Lett 10; 250 (3): 177-180).

[0010] Like VR1, VR-L is expressed in sensory nerves and appears to play a role in mediating pain and inflammation (nociception) associated with tissue damage. Other endogenous ligands may activate the receptor in vivo, either directly or through a G protein that binds to the receptor by similarity to other TRP channels described above. Therefore, this novel channel is a particularly analgesic or anti-inflammatory drug target.

However, unlike VR1, VR-L does not appear to be capsaicin sensitive under the conditions used in the experiments below. Further, for example, V that is not present in Jurkat cells
For R1, it seems to have a different distribution. The structural and functional information about VR-L made available by the inventor suggests that it has additional utility for VR1.

[0012] Biro et al (1998) Blood 91: 1332-1340 is a system in which cells of a certain immune system (mast cell line) are capsaicin and RTX (a plant toxin having a similar action to capsaicin).
Reported calcium uptake in response to Biro et al. Further showed that, in contrast to sensory nerves, capsaicin did not kill mast cells or even induce their degranulation. However, the putative receptor important for the demonstrated activity was not characterized.

[0013] The presence of VR-L in a subset of cells of the immune system suggests an important role as a receptor involved in regulating the immune response. Accordingly, such channels have further utility in screening for factors that modulate the immune response.

[0014] VR-L based materials can be used in gene therapy for both sensory nerves (eg, pain) and diseases associated with white blood cells (eg, autoimmune disease, leukemia).

[0015] The nucleic acid sequence of VR-L and the encoded protein are shown in FIG. 3A. The nucleic acid sequence is referred to as SEQ ID NO: 1, while the encoded protein is referred to as SEQ ID NO: 2.

Certain aspects of the present invention will be immediately discussed in more detail.

Accordingly, in a first aspect of the present invention, there is provided a non-selective cation channel protein that can be activated by noxious heat.

The channel proteins of the present invention may be obtained from cells of the immune system, preferably human cells.

Preferably, said channel protein is not capsaicin-sensitive under the conditions described in the experiments below. The channel may not be activated by low pH, i.e. it may not be acid sensitive, which means that the cation permeability of the channel may be low at low pH (increased acidity or reduced alkalinity), e.g. pH, especially
Means little increase by pH 4.6.

The protein or polypeptide of the present invention can be produced recombinantly, isolated,
It may be provided in a concentrated or cell-free form. They may be heterogeneous and present in cells, which translates to them not occurring naturally there but being introduced by human intervention.

By “non-selective”, the activated channel can include one or more cations,
Preferably, it means that it is permeable to at least Na ⁺ , K ⁺ , and Ca ²⁺ , and preferably has broad and comparable permeability to each.

It can be activated by noxious heat, ie, a level of heat that can typically stimulate a pain response, for example, at about 45 ° C. or higher, 50 ° C. or 55 ° C., or higher.

The various activities of the channel can be assessed by one of ordinary skill in the art without undue burden, for example, one or more membrane preparations that expressed or otherwise introduced the protein (eg, Use voltage-clamping techniques similar to those described herein to analyze eukaryotic cells). Some commonly available methods are described in “Ionic Channels of Excitable Membranes” Hille, B. -Pub 1992, Sinaue
discussed in r.

[0024] Preferably, said protein is one of the VR-L proteins described herein.
Or it may have multiple electrophysiological and pharmacological characteristics. Preferably, said protein comprises the amino acid sequence of SEQ ID NO: 2.

In a further aspect of the invention there is disclosed a VR-L variant protein, which has ion channel activity and at least about 60% or 70% or 80% homology, most preferably at least about 90%, 95%, 96%, 97%
, 98% or 99% sequence identity to SEQ ID NO: 2. As noted above, the prior art VR1 channel has about 47% identity (56% similarity) to SEQ ID NO: 2.

[0026] Ion channel activity can be tested as described above, with the appropriate stimuli required.

Similarity (or identity or homology) is determined by Altschul et al. (1990) J. Mol. Bio
l. 215: The 403-10 TBLASTN program (which is standard in the industry, or this may be preferred), part of the eighth edition of the Wisconsin package, September 1994 (Genetics Computer Group, 575 Science D
rive, Madison, Wisconsin, USA, Wisconsin 53711), as defined and determined by the standard program Bestfit. B
estFit creates an optimal alignment of segments of the best similarity between two sequences. An optimal alignment is found by using the Smith and Waterman local homology algorithm to insert gaps to maximize the number of matches.

The parameters are preferably set using a default matrix as follows:

Gapopen (penalty of the first residue in the gap): in the case of protein -12 / in the case of DNA -16 Gapext (penalty of the additional residue in the gap): in the case of protein
2 / for DNA-4 KTUP word length: 2 for protein / 6 for DNA.

The homology may span the full length of the related sequences set forth herein, or may span a portion thereof, preferably about 20, 25, 30, 33, 40, 50. ,
67, 133, 167, 200, 300, 500, 600, 700 or more amino acids spanning the same or more contiguous sequences, or compared to SEQ ID NO: 2.

Thus, a variant polypeptide according to the present invention has the sequence shown in SEQ ID NO: 2
One amino acid or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10
, 15, 20, 30, 40 or 50 changes, or about 50, 60, 70, 8
May include zero or more than 90 changes. In addition to one or more changes in the indicated amino acids, the variant polypeptides may include additional amino acids at the C-terminal and / or N-terminal. Alternatively, it may represent an active (as an ion channel) fragment of the protein, for example a pore-forming fragment.

A protein or polypeptide of the present invention can be prepared by expressing the nucleic acid encoding it in a suitable host cell, as described in detail below.

In a further aspect of the present invention, there is provided a nucleic acid encoding the above-described protein of the present invention.

[0034] Nucleic acids according to the present invention may include cDNA, RNA, genomic DNA and modified nucleic acids or nucleic acid analogs (eg, peptide nucleic acids). Where the DNA sequence is described, for example, with respect to the figures, unless otherwise indicated, RNA equivalents having a substituted U instead of a T when it occurs are included.

The nucleic acids according to the present invention are isolated from their natural environment in substantially pure or homologous form, or in a form that is free or substantially free of other nucleic acids of the original species, and And / or may be provided in a purified form. As used herein, the phrase "isolated" encompasses all of these possibilities.

The nucleic acid molecule may be wholly or partially synthesized. In particular, they may be recombinant, in which nucleic acid sequences that are not found together in nature (do not work in contact) have been ligated or otherwise artificially combined. Alternatively, they may be synthesized directly, for example using an automatic synthesizer.

Thus, in one aspect of this aspect of the invention, a nucleic acid encoding VR-L, for example, a nucleic acid encoding SEQ ID NO: 2 (this nucleic acid sequence may be as set forth in SEQ ID NO: 1) Good) offer. In another aspect, there is a disclosure of a sequence that is degenerately equivalent to that sequence.

[0038] In another aspect, there is a disclosure of a nucleic acid encoding a VR-1 variant as described above. In general, this has, in equivalent terms as described above for the variant protein, homology or identity with the sequence encoding VR-L, but with an amino acid change here. Corresponds to a codon or individual nucleotide change.

Variants may include unique portions or fragments (which may be produced) corresponding to portions of the provided sequences. The above fragment
It may encode a particularly functional part of said polypeptide.

At the same time, the fragment may have utility in searching or amplifying what the sequence provides or is closely related to.

Suitable fragment lengths and conditions for the above method are discussed in further detail below.

[0042] For any of these embodiments, nucleic acids extended at the 3 'or 5' end are also included.

Preferred mutant nucleic acids, in addition to those encoding the VR-L mutant, are poly- or oligo-, having a sequence complementary to a unique portion of SEQ ID NO: 1 under low stringency conditions. Those capable of hybridizing with nucleotides, more preferably those capable of hybridizing with one or more of the above-mentioned poly- or oligonucleotides under highly stringent conditions.

The expressions “low stringency conditions” and “high stringency conditions” are:
As will be appreciated by those skilled in the art, US 5,202,257, Col 9-Col.
This is simply exemplified as described in FIG. For example, screening may be performed at a temperature of about 37 ° C. or less, a formamide concentration of about 50% or less, and mild low salt (eg, normal salt-citrate buffer (“SSC”) = 0.15 M sodium chloride;
. 15M sodium citrate; pH 7) concentration.

Alternatively, a temperature of about 50 ° C. or less and a high salt concentration (eg, “SSPE
"= 0.180 mM sodium chloride; 9 mM disodium hydrogen phosphate; 9 mM sodium dihydrogen phosphate; 1 mM sodium EDTA; pH 7.4). Preferably, the screening is performed at about 37 ° C., about 20%. Performed at a formamide concentration and a salt concentration of about 5 × SSC, or at a temperature of about 50 ° C. and a salt concentration of about 2 × SSPE, These conditions require complete homology for the identification of stable hybrids. Without doing so, it would allow the identification of sequences having a substantial degree of homology (similarity, identity) with the probe sequence.

One general formula for calculating the stringent conditions necessary to achieve hybridization between molecules of homology of the described sequences is (Sambrook et al.
al., 1989): T _m = 81.5 ° C. + 16.6 Log [Na +] + 0.41 (% G +
C) -0.63 (% formamide) -600 / number of base pairs in duplex.

For example, conditions suitable for detecting sequences that are about 80-90% identical are at 42 ° C. and 0.25
Overnight hybridization in M Na ₂ HPO ₄ , pH 7.2, 6.5% SDS, 10% dextran sulfate, and final wash in 0.1 × SSC, 0.1% SDS at 55 ° C. including. Suitable conditions for detection of sequences that are about 90% or more identical are 65 ° C., 0.25 M Na ₂ HPO ₄ , pH 7.2, 6.5% SDS.
Overnight hybridization in 10% dextran sulfate and at 60 ° C.
Includes final wash in 0.1 × SSC, 0.1% SDS.

[0048] Naturally occurring sequence variants may include alleles (which may include polymorphisms or mutations at one or more bases) or pseudoalleles. Also included within the scope of the invention are isogenic or other homologous genes from other species having similarity to SEQ ID NO: 1.

The phrase “variant” nucleic acid as used herein encompasses all of these possibilities.

Thus, in an aspect of the present invention, there is provided a method of identifying and / or cloning a nucleic acid according to the present invention (ie, encoding an ion channel as described above), which comprises SEQ ID NO: 1 or Use that unique fragment or region.

“Unique” means based on regions not present in VR1 or other trp proteins. The sequence information for the above regions can be used directly for identification of cloned variants or, optionally, from different species, in a database (eg, expressi
It can be used to identify the corresponding region in on sequence tags (or sequence tagged sites).

For example, using the information disclosed herein, the V of both rats and mice,
The RL homolog was identified. A part of the encoded amino acid sequence is shown in FIG.

Thus, in a further aspect, a variant according to the invention provides: (a) a preparation of nucleic acids from, for example, cells of the immune system, or sensory nerves; (b) SEQ ID NO: 1 or a unique thereof. Providing a nucleic acid molecule having a nucleotide sequence set forth in a fragment or complementary thereto, and (c) conditions for hybridization of said nucleic acid molecule to any of said genes or homologues in said preparation. Contacting the nucleic acid in the preparation with the nucleic acid molecule and hybridizing it with the nucleic acid molecule
Identifying whether the gene or homolog is present.

The search may optionally be performed by means of a so-called “nucleic acid chip” (see Marshall & Hodgson (1998) Nature Biotechnology 16: 27-31 for a review).

The nucleic acids of the invention may be derived, for example, from genomic DNA, DRG cDNA, or from an mRNA template, eg, by using the polymerase chain reaction, or by using reverse transcription (RT) followed by the polymerase chain reaction. Amplification can also be performed from template DNA from cells using a specific DNA amplification reaction with specific primers targeted to amplify the required sequence from RNA, e.g., SEQ ID NO: 1 ("" PCR Protocols; A Guide to Methods and Applications
", Eds. Innis et al, Academic Press, New York, 1990).

Thus, methods involving the use of PCR in obtaining nucleic acids according to the present invention include: (a) providing a preparation of nucleic acid, eg, from a DRG, or other suitable tissue or organ; Providing a primer pair of nucleic acid molecules useful for PCR (ie, suitable), wherein one primer has the sequence shown in SEQ ID NO: 1 or a unique fragment thereof or has a sequence complementary thereto; Contacting said nucleic acid with said primers under conditions for performing PCR, (d) performing PCR and determining the presence or absence of the amplified PCR product. The presence of the amplified PCR product may indicate the identity of the mutant.

In all cases, if necessary, the clones or fragments identified in the above studies can be expanded. For example, if you think they are incomplete, the source of the original DNA (eg, clone library, mRNA preparation, etc.)
To isolate the lost portion, it may be revisited using a sequence, probe or primer based on a portion thereof already obtained, for example to identify other clones containing overlapping sequences.

[0058] Specific fragments or oligonucleotides for use in probing or PCR can be readily selected and prepared by one of skill in the art in light of the present disclosure. Typically, they are about 10-30 or less in length (about 18,21 or 24
) Nucleotides. Generally, specific primers are at least 14 nucleotides in length. For optimal specificity and cost effectiveness, primers 16 to 24 nucleotides in length may be preferred. If necessary, the search can be performed using all restriction fragments of the genes disclosed herein, which can be hundreds (eg, 500 or more) or even thousands of nucleotides in length. .

The oligonucleotides and their complements (except any oligonucleotides consisting solely of the disclosed regions in the form of EST clones or other parts whose function is not defined) Form part of the present invention.

Artificial variants (derivatives) can be prepared by those skilled in the art based on the sequences provided herein, eg, by site-directed or random mutagenesis, or by direct synthesis. Preferably, the mutant nucleic acid is directly or indirectly (eg, via one or more amplification or replication steps) from an original nucleic acid having all or a portion of the sequence set forth in SEQ ID NO: 1. Generate.

Accordingly, in a further aspect of the present invention, there is disclosed a method of producing a derivative nucleic acid comprising modifying the coding sequence of SEQ ID NO: 1.

Sequence changes to produce derivatives may lead to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide, of one or more nucleotides of one or more nucleotides of the nucleic acid. May be due to addition, insertion, deletion or substitution.

The alterations are characterized by the following features: restriction endonuclease sequence; codon usage; other sites requiring post-translational modification; cleavage sites in the encoded polypeptide; encoded for post-translational modification. It may be desirable for a number of reasons, including introducing or removing motifs in the polypeptide. A leader or other targeting sequence (eg, a membrane or Golgi locating sequence) can be added to the expressed protein to determine its location after expression. All of these can assist in effectively cloning and expressing the recombinant active polypeptide.

Another desired mutation is the activity (eg, specificity) of the encoded polypeptide.
Alternatively, it may be random (eg, chemical) or site-directed mutagenesis to alter stability. For the method, for example, the vectorized D
NA is exposed to a mutagenic material such as hydroxylamine, or
In the case of M, a PCR reaction is performed on the DNA using mutagenic primers, thereby encoding a protein that differs in sequence to SEQ ID NO: 2 at several, each predictable or predetermined site. DNA
Is manufactured.

The particular target region of the sequence can determine any of the following: specificity of activation of the ion channel (eg, whether a given stimulus, eg, pH, elicits a response) or ions Channel specificity (eg, specific permeability to different cations).

All of these properties can be easily tested, for example using suitable voltage-clamping techniques.

Preferably, said amino acid sequence differs from SEQ ID NO: 2 only by conservative substitutions. The expression "conservative substitution" when used in reference to an amino acid relates to the substitution of an amino acid by an amino acid having the same class of physicochemical properties. Thus, if the amino acid of SEQ ID NO: 2 has a group that characterizes hydrophobicity, a conservative substitution will replace it with another amino acid that also has a group that characterizes hydrophobicity; The above characterizing groups are those that include hydrophilic, cationic, anionic or thiol or thioether. Such substitutions are known to those skilled in the art (see, eg, US Pat. No. 5,380,712), and it is only intended that the resulting protein have ion channel activity.

[0068] Variants with non-conservative substitutions are also included. As is known to those of skill in the art, substitutions for regions of the peptide that are not important for determining its conformation may not significantly affect its activity because they do not significantly alter the three-dimensional structure of the peptide. It is. In areas important to determine the conformation or activity of the peptide, such changes may confer advantageous properties on the polypeptide.

[0069] In an aspect of the invention, the nucleic acids of the invention are in the form of recombinant and preferably replicable constructs, such as vectors.

A “vector” may be self-propagating or activatable or may not be, either double- or single-stranded, linear or circular, in particular, Or a plasmid, cosmid, phage or other vector, which transforms a prokaryotic or eukaryotic host, or integrates extrachromosomally, by integration into the genome of the cell. (Eg, a self-replicating plasmid with an origin of replication).

Specifically included are shuttle vectors, which are actinomycetes and related species,
A DNA vehicle that can be naturally or artificially replicated in two different host organisms, which can be selected from bacteria and eukaryotes (eg, mammalian, yeast or fungal cells).

A vector comprising a nucleic acid according to the present invention may include a promoter or other regulatory sequences, particularly if the vector is to be used to introduce the nucleic acid into a cell for recombination into the genome. It does not need to be included.

Preferably, the nucleic acid in said vector is under the control of an operably linked, suitable promoter or other regulatory element for transcription in a host cell, eg, a microorganism, eg, a bacterial cell. is there. The vector may be a bifunctional expression vector that functions in multiple hosts. In the case of genomic VR-L DNA (isolatable as described above), this may include its own promoter or other regulatory elements, and in the case of cDNA, it may be a promoter suitable for expression in a host cell. Or it may be under the control of other regulatory factors.

“Promoter” refers to a nucleotide sequence capable of initiating transcription of DNA operably linked downstream (3 ′ direction on the sense strand of double-stranded DNA).

“Operably linked” means linked as part of the same nucleic acid molecule, properly positioned and oriented for transcription that can be initiated from the promoter. The DNA operably linked to the promoter is the "
Under the regulation of transcription initiation ".

Thus, this aspect of the present invention provides a gene construct, preferably a replicable vector, which comprises a nucleotide sequence provided by the present invention, such as V
Comprising a promoter operably linked to the RL gene (see SEQ ID NO: 1) or a variant thereof.

In general, those skilled in the art will be able to correctly construct vectors and design protocols for expression of recombinant genes. Suitable vectors can be selected or constructed to contain appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other appropriate sequences. For further details see, for example, Molecular Cloning:
a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Ha
See rbor Laboratory Press.

For the manipulation of nucleic acids, for example, the preparation of nucleic acid constructs, mutagenesis (see discussion above for variants), sequencing, introduction of DNA into cells and gene expression, and Many known techniques and protocols for the analysis of proteins are described in Current Protocols in Molecular Biology, Second Edition, Ausube
l et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. And Ausubel et al. Are incorporated herein by reference.

In one embodiment of this aspect of the invention, there is provided a gene construct, preferably a replicable vector, which is operably linked to a nucleotide sequence provided by the invention, such as SEQ ID NO: 1. Comprising a possible promoter.

The term “inducible” when applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on" or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters result in low or undetectable levels of expression (or no expression) without appropriate stimulation.
Other inducible promoters provide for detectable constitutive expression without stimulation. Whatever the level of expression in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus.

Particularly preferred are vectors suitable for the expression of mammalian DNA, such as the HSV or vaccinia vectors, which will occur to those skilled in the art.
Or a pcDNA3 shuttle vector, such as the lambda expression system (Str
, which is capable of expressing heterologous proteins in both bacteria and eukaryotic cells, such as COS cells. Suitable bacterial vectors will include lambda-Zap vectors, such as the lambda-Zap-II vector available from Stratagene Cloning Systems. A bacterial clone containing a plasmid capable of gene expression can be obtained by excising pBluescript from the lambda-Zap-II construct in the presence of a filamentous fungal helper phage available from Stratagene. A typical protocol is provided in the following example in the insert of the Stratagene kit.

A further aspect of the invention provides a cell comprising, or more preferably, transformed (or transfected with) a nucleic acid of the invention. Such a cell is a host cell, preferably a eukaryotic cell, such as COS, CHO or HEK29.
3 cells or oocytes, preferably Xenopus oocytes, especially CO
S cells are provided by transformation with the DNA of the present invention, which can be integrated by recombinant DNA technology into a vector or directly into the cell's genomic DN.
A, for example by electroporation or other such DNA incorporation techniques.

The cells may express the VR-L protein, as described above, or may be capable of having it expressed. Thus, the cell is, in some respects,
It can mimic the electrophysiological and pharmacological properties of native VR-L expressing cells.

It is also possible to produce cells having the receptor protein of the present invention by direct injection of the RNA of the present invention into the cells where it begins to be translated.

The foregoing considerations generally relate to the use of the nucleic acids of the invention for the production of functional polypeptides, thereby increasing the VR-L or related ion channel activity of said cells. .

[0086] However, the information disclosed herein can also be used to reduce the activity of VR-L or a variant thereof, in cells where it is desired to do so.

For example, down-regulation of target gene expression can be achieved using antisense technology.

When using antisense genes or partial gene sequences to down regulate gene expression, the nucleotide sequence is placed under the control of a “reverse orientation” promoter so that transcription is Produces RNA complementary to normal mRNA, transcribed from the "sense" strand of the gene. For example, Rothstein et al, 1987;
Smith et al, (1988) Nature 334, 724-726; Zhang et al, (1992) The Plant
Cell 4, 1575-1588, English et al., (1996) The Plant Cell 8, 179-188. Antisense technology is also described in Bourque, (1995), Plant Science 105, 125-14.
9 and Flavell, (1994) PNAS USA 91, 3490-3496.

“Complementary to” means that A can base pair with T (and U); G can base pair with C by base pairing.

A “complement” of a reference nucleic acid consists of a nucleotide sequence that is the counterpart of the entire nucleotide sequence of the reference nucleic acid.

An alternative to antisense is to use a copy of all or a portion of the target gene inserted in sense, in the same orientation as the target gene, to achieve reduced target gene expression by co-suppression.

Further choices for down-regulating gene expression include the use of ribozymes, for example, hammerhead ribozymes that can catalyze the site-specific cleavage of RNA, eg, mRNA (eg, Jaeger (1997) “The new w
orld of ribozymes ”Curr Opin Struct Biol 7: 324-335 or Gibson & Shill
itoe (1997) “Ribozymes: their functions and Strategies form their use”
Mol Biotechnol 7: 242-251.).

[0093] Methods for generating regulatory sequences will not be difficult for one of skill in the art in light of the present disclosure. Antisense or sense or ribozyme-based regulation can be performed using the vectors described above, and can be regulated by itself using an inducible promoter in a suitable construct. For example, integration of this DNA into mammalian cells can be readily achieved using a vector such as HSV, vaccinia or adenovirus (Principl
es of Gene Manipulation (1994) 5th Edit.Old and Primrose 5th Edition, B
lackwell Scientific Publications).

It is not necessary to use the complete sequence corresponding to the coding sequence described above (in the case of antisense, in the reverse orientation). For example, fragments of sufficient length may be used. It is a routine matter for one skilled in the art to screen fragments of different sizes and from different parts of the coding sequence to optimize the level of antisense inhibition. It may be advantageous to include the ATG codon of the initiation methionine, and possibly one or more nucleotides upstream of the initiation codon. A further possibility is to target a conserved sequence of the gene, for example a sequence characteristic of one or more genes, for example a regulatory sequence.

[0095] The sequences utilized may be about 500 nucleotides or less, perhaps about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100 nucleotides. Oligonucleotides of very short length, 14-2
Although it may be possible to use three nucleotides, longer fragments,
And generally much longer than about 500 nucleotides, preferably, for example, about 600 nucleotides or more, about 700 nucleotides or more, about 800 nucleotides or more, about 1000 nucleotides or more.

It may be preferred that there is complete sequence identity with the sequence used for down-regulating the expression of the target sequence and with said target sequence, but the overall complementarity or similarity of the sequences is not essential . One or more nucleotides may differ in the sequence used from the target gene. Thus, according to the present invention, the sequence utilized for down-regulation of gene expression may be a wild-type sequence (eg, a gene) selected from those available, or a variant of such a sequence.

The sequence need not include an open reading frame or specifically specify an RNA that is believed to be translatable. Preferably, there will be sufficient homology between each antisense and sense RNA molecule in order to hybridize. There may be between about 5%, 10%, 15% or 20% between the sequence used and the target gene.
Even with% or more mismatches, there may be down regulation of gene expression. In effect, the homology should be sufficient to cause down-regulation of gene expression.

One aspect of this aspect of the invention is typically 10 to 30 bases in length, conveniently about 20 bases in length, optionally from degradation by, for example, thiolation. Utilizes a DNA oligonucleotide that is in a protected form, and
It is conveniently synthesized chemically and oriented to hybridize to a portion of the 5 'coding region of VR-L mRNA. Annealing with the oligomeric DNA results in mRNA that can be degraded by the activation of RNase H, or interferes with the translation of said mRNA into protein. Such oligomers of small size are described in the present VR-
Facilitates their direct contact into target cells expressing the L protein.

Another embodiment is a tissue specific inducible or constitutively active promoter,
In vivo, by inserting a factor upstream of the coding region, or a portion of the coding region, of the DNA of SEQ ID NO: 1 in an enhancer or site regulatory region or consequently a construct cloned into a vector. Generate antisense RNA. For use in mammals in therapy, the vector should be able to infect but not kill the target cell. A convenient vector for use in this embodiment that can target mammalian dorsal root ganglion cells is a herpes simplex virus (HSV) vaccinia or adenovirus-derived vector. Viral vectors for use in gene therapy are discussed in Vile (1997) Nature Biotechnology 15: 840-841. Non-viral gene therapy approaches are described in Sebestyen et al (1998) Nature Biotechnolog
y 16: 80-85. Various gene therapy transport systems (HSV VP22
Use of Fernandez & Baylay (1998) Nature Biotechnology 16:41
Discussed by 8-420 and references therein.

When antisense down-regulated DNA or RNA is provided in dorsal root ganglion cells, the number of VR-L channels on the surface of sensory cells is actually reduced, which is an indication of painful Strongly inhibits response. When DNA or RNA for which antisense is down-regulated is provided by the immune system, VRs on the surface of the nerve
It alters the function of leukocytes by actually reducing the number of L channels.

Accordingly, nucleotide sequences complementary to any of the encoding nucleic acids discussed with respect to the earlier aspects of the invention form part of the invention.

The present invention further provides a method of affecting the electrophysiological and pharmacological properties of a cell, said method resulting in or expression of said heterologous nucleic acid sequence in said cell. Comprising the step of allowing.

The invention further relates to down-regulation of gene expression, in particular genes encoding ion channels, more preferably cation channels, most preferably VR
Provided is the use of the nucleotide sequence of SEQ ID NO: 1, or its complement, or any variant thereof, for down-regulating the expression of -L.

In a further aspect of the invention, there is provided a nucleic acid (eg, antisense DNA) of the invention for use in gene therapy or for use in the manufacture of a medicament for use in gene therapy. Is done.

In a further aspect of the invention, the activity of VR-L or a variant thereof is altered by the use of the methods and materials described above, wherein the organism, preferably comprising a damaged cell, A human mammal is disclosed. Particularly preferred are rodents, for example, mouse organisms. Methods for producing "knock-out" mammals with impaired activity of specific genes are now known in the art-see, for example, Boerrigter et al (1995
) Nature 377: 657-659 or Gossen and Vick (1993) Trends Genet 9: 27-3
See 1.

In a further aspect of the present invention, the use of a protein, transformed or transfected cell, or transgenic organism, as described above, for identifying a substance having ion channel modulating activity is described. Provided.

The substances may serve, for example, as agonists, partial agonists or antagonists.

[0108] Thus, the protein or cell may comprise a protein (eg, one associated with a membrane, eg, a liposome) or a cell surface, wherein the substance and the VR-L (or mutant VR-L) in the membrane are combined with the substance. Is exposed to a solution of the substance so as to interact with and then measures the electrophysiological response of the cell or membrane to this interaction. This measurement can optionally be compared to the reference figures.

Typically, the response can be measured by the use of microelectrode technology that accompanies a measurement strategy, such as voltage clamping of the cells, whereby activation of ion channels is reduced by the It can be identified by an inward or outward current flow as detected using a microelectrode. Cations or ¹⁴ C or ³ H guanidine radiolabeled with ²² Na, ⁸⁶ Rb, ⁴⁵ Ca can be used to assess the influx of such ions; sodium, calcium or potassium ion sensitive dyes (eg, Fura-2 or indo) can be used to monitor the passage of ions through the ion channel of the receptor, or voltage sensitive dyes to monitor changes in potential, eg, in depolarization Can be used for

Agonists and partial agonists may be distinguished by their relative potency, as compared to other known agonists and stimuli in activating the receptor, or, in the case of partial agonists and antagonists, other Can be identified by their ability to prevent activation caused by known agonists.

The substance may optionally be in the form of a medicament (eg a pharmaceutically acceptable carrier), as an analgesic or other immunological or neuromodulatory agent and as such in a further aspect of the invention. Or excipients).

[0112] Purified VR-L protein, or a variant thereof, eg, recombinantly produced by expression from the nucleic acid encoding it, can be used to raise antibodies using standard techniques in the art. It can be used to produce.

Antibodies and other polypeptides comprising an antigen-binding fragment of an antibody can be used (among others) as antagonists or inhibitors, or for use in identifying homologs.

A method for producing an antibody comprises immunizing a mammal with said protein or a fragment thereof. Antibodies can be obtained from the immunized animal using any of a variety of techniques known in the art, and preferably screened using the binding of the antibody to the antigen of interest.

For example, Western blotting techniques or immunoprecipitation may be used (Armi
tage et al, 1992, Nature 357: 80-82). Antibodies can be polyclonal or monoclonal.

[0116] Antibodies can be modified in several ways. In fact, the phrase “antibody”
It should be construed to cover any specific binding substance that has a binding domain with the required specificity. Thus, the term encompasses antibody fragments, derivatives, functional equivalents and homologs of antibodies, and includes any polypeptide, comprising natural or synthetic, immunoglobulin binding domains. A chimeric molecule comprising an immunoglobulin binding domain fused to another polypeptide, or equivalent, is then included. The cloning and expression of the chimeric antibody is described in EP-A-01206.
94 and EP-A-0125023. Fragments of whole antibodies have been shown to serve the function of binding antigens. An example of a combined fragment is
(I) Fab fragment consisting of VL, VH, CL and CH1 domains; (ii)
A) an Fd fragment consisting of the V1 and VH domains of a single antibody; and (iv) a dAb fragment consisting of the VH domain (Ward, ES et al., Nature 341). 544-546 (1989
(Vi) an isolated CDR region; (vi) a F (ab ') 2 fragment, a bivalent fragment comprising two linked Fab fragments (vii) a single chain Fv molecule (scFv) (VH domain) And the VL domains are linked by a peptide linker that associates the two domains to form an antigen-binding site) (Bird et
al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883,
(Viii) bispecific single-chain Fv dimers (PCT / US92 / 09965) and (ix) "diabodies" of multivalent or multispecific fragments constructed by gene fusion ( WO94 / 13804; P Holliger e
Natl. Acad. Sci. USA 90 6444-6448, 1993).

The present invention will now be described only by way of illustration of the following non-limiting figures and examples. Further aspects of the invention, which fall within the scope of the claims provided herein, will be apparent to those skilled in the art from these perspectives.

[0118] adopted the approach of innovative "virtual cloning" for finding the cloning summary VR-L of Examples Example 1-VR-2. A Genbank dbEST tblastn search identified at least 30 human and mouse entries (Figure 6, Table 1), which showed considerable homology to different regions of the amino acid sequence of rat VR1. Duplicate human EST clones, even if gaps are inserted, are virtual transcripts (FIG. 1).
Build). The contig formed was composed of 86-7 of the amino acid sequence of rat VR1.
Shows sequences that were broadly similar to position 63.

Since the EST clone is obtained from a different cDNA library, and therefore, the contig may comprise ESTs from different tissues, and the “transcript” may not be the actual transcript. There was no way to know if there was. R, including overlapping primer sets (see FIGS. 1 and 7, Table 2)
A T-PCR based approach was therefore used to bridge the gap between the different contigs and to establish whether these clones actually form part of a single transcript. Total RNA from Jurkat cells (leukemia T cell line; from which EST clones were obtained) was used as a template for reverse transcription. Sequencing of the PCR product confirmed that the individual entries were identical transcripts, truncated at various times during reverse transcription.

As shown in FIG. 1, a tblastn search of the EST database did not yield any clones with apparent homology to the amino-terminal region of VR1.

A search of the Human Gene Index (HGI) THC database of the Genome Institute (TIGR) found a further 5 ′ sequence (THC176254) that is adjacent to other EST clones but different from that of VR1. Revealed that VR1
EST clones homologous to the further 5 'region of GenBank (GenBank
Accession number H20025, AA461295, H51393, AA236417)
This was performed using The putative initiation methionine was designated based on the identification of the stop codon of what should be the 5 'untranslated region of the transcript.

A human EST clone having an amino acid sequence substantially identical to most of the 3 ′ region of VR1 (GenBank Accession No. AA321554) is one of the transcripts formed by all other ESTs extracted. And that there are actually at least two subtypes of the vanilloid receptor (
And AA321554 can form part of a homolog of human VR-1). This EST-based primer, combined with primers from the further 5 'region of VR-L (see FIG. 1), results in failed PCR or either VR1 or VR-L Have some similarity to
It did not give a clear PCR product.

Suspected, further 3 ′ VR-L EST clone (GenBank accession number A
Subsequent THC database searches using A281348, W92895, N35179, T12251) revealed stop codons and 3 'untranslated sequences (VR1
Leading to the identification of the 3 'end of the kidney,
The 'sequence (THC161190) was extracted. The primer designed from this putative 3 'end further combined with the 5' primer to give a distinct product, which was later demonstrated by sequencing to be flanking the rest of the VR-L sequence. Was. Long PCR products can be trans-PCR (splicing by overlap extension) or ligation utilizing unique restriction sites in the region of overlap (FIGS. 2a and 2b).
) And both spliced together. FIG. 3b shows a comparison of the amino acid sequence of human VR-L with that of rat VR1.

To further demonstrate that these clones simply represent the human homolog of rat VR1, a mouse EST clone (GenBank accession number AA476107) was used.
)) Was used to amplify VR-L from rat dorsal root ganglia from which VR1 was isolated. Sequencing revealed a transcript similar to VR1 but different, confirming that there are at least two subtypes of the vanilloid receptor in the rat genome. FIG. 4 shows an alignment of the rat, mouse and human VR-L sequences in this region with the rat VR1.

Methods of RT-PCR and Subcloning Total RNA was isolated from cultured Jurkat cells (leukemia T lymphocytes) by Chomcyznsk.
i and Sacci's method (Analytical Biochemistry 162: 156-159, 1987). Reverse transcription was performed as follows. Total RNA (2 μg) and 50 ng of primer (random hexamer or gene-specific oligonucleotide) were heated to 70 ° C. for 10 minutes in a total volume of 9 μl to destroy possible secondary structure. The mixture was subsequently diluted with 1 × buffer [50 mM Tris-HCl (pH 8.3), 75 mM KCl,
3 mM MgCl ₂ ], 10 mM dithiothreitol and 125 mM of each dNTP (d
(ATP, dCTP, dGTP and dTTP) at 37 ° C. for 2 minutes. Superscript II reverse transcriptase (200 U) was added to a final volume of 20 μl and the mixture was incubated at 37 ° C. for 60 minutes. The reaction is 9
Stopped by incubation at 4 ° C. for 5 minutes.

For the first PCR amplification, the reaction was performed with 1 μl of the first strand cDNA, 40
pmol of each primer, 1 × buffer [50 mM KCl, 10 mM Tris-HCl
pH9.0,0.1% Triton X-100], 1.5mM MgCl 2,
Performed in a final volume of 20 μl using 0.2 mM dNTPs and 1 U of Taq polymerase. The amplification program used was one cycle of 94 ° C for 5 minutes, 53-57 ° C for 1 minute, 72 ° C for 1-2 minutes; 94 ° C for 1 minute, 53-57 ° C for 45 seconds, 72 ° C for 1-minute. Included 35 cycles of 2 minutes; and a final extension at 72 ° C. for 5 minutes. For cloning, 0.4 U of Gibco-BRL'S eLONGa was used.
se enzyme mix (mixture of Taq polymerase and proofreading Pyrococcus sp. GB-D thermostable DNA polymerase)
With 1 μl of first strand cDNA, 40 pmol of each primer, 1 × buffer [60
_{mM Tris-SO 4 (pH9.1)} , between 1.5～1.9MM, including _{18mM (NH 4) 2 SO 4} ] and 0.2 mM dNTPs with over MgSO _4, used in 20μl of reaction solution did. Cycling conditions are: 1 cycle at 94 ° C for 3 minutes, 55 to 57 ° C for 1 minute, 68 ° C for 45 seconds to 2 minutes, 94 ° C for 30 seconds, 55 to 57 ° C for 30 seconds, 68 ° C for 45 seconds to 35 cycles of 2 minutes; and a final extension of 5 minutes at 68 ° C. The PCR product was prepared using the vector pGem-T Easy (Promega).
) And sequenced using standard methods. eLONGa
For PCR products with blunt ends generated by s, incubation for 10 minutes with 1 U of Taq polymerase after the PCR reaction is
It was necessary to add the "A" overhang required for cloning to the pGem-T Easy vector.

The larger PCR product was spliced together by either trans-PCR (splicing by overlap extension) or conventional ligation involving a unique restriction site to obtain a full-length VR-L clone. (See FIGS. 2a and 2b). The full-length clone was then transferred to a mammalian expression vector (pGW-1) and checked for correct orientation. The resulting recombinant clone was subsequently used for transfection of COS-7 cells by electroporation.

Example 2 Expression of VR-L in COS-7 Cells A full-length VR-L clone (in pGemT easy) was excised with EcoRI and subcloned into the EcoRI site of pGW1. The orientation of the resulting construct was confirmed by restriction analysis and sequencing (see FIG. 2).

A shuttle vector containing VR-L, such as pTracer-CMV or pq
w1 was grown and purified from cultures of bacteria transformed with the recombinant plasmid. These vectors were used to transfect COS cells to express ion channels. 100mm Petri dish (80-90%
) Was trypsinized and suspended in 350 microliters of ice-cold HEBS buffer. 20-30 micrograms of the plasmid of interest were dissolved in 150 microliters of HEBS buffer, then mixed with the COS cell suspension in an electroporation cuvette and kept on ice for 5 minutes to cool . At the same time, an electroporator (Invitroge)
n) is set to 250 microfarads, and 330 V, 25 mA, and 25
Charged with W for 3 minutes. The cells were resuspended by flipping the cuvette and electroporation was applied.

After transfection by electroporation, COS cells were harvested for 35 days.
Seed at low density on a 2 mm Petri dish and 3 ml with 2 ml MEM / 10% FCS.
The cells were cultured at 7 ° C for 1-2 days.

Example 3 VR-L Electrophysiology Recordings of voltage clamps on whole cells were made 1-2 days after transfection.

Membrane currents were recorded using an Axopatch 200B amplifier. Current is 5kH
z low-pass filtered and digitized using a Digidata 1200 interface. Current capture and analysis was achieved using pClamp 6 software. Pipette KCl 120, NaCl 8, MgC
l ₂ 3, HEPES 40, and a BAPTA 10 (mM), pH7.35
Was filled with the intracellular solution. Recordings were made at room temperature (18-22 ° C).

The solution for extracellular recordings was: NaCl 146; KCl 5; glucose 10;
It was composed of MgCl ₂ 1 and CaCl ₂ 0.01 (mM). pH value 7.4
For extracellular solutions of ~ 6.5, 10 mM HEPES was used as buffer, while for solutions of pH 6.5-4.0, MES at a concentration of 10 mM was used.

Results VR-L forms a functional channel when transiently expressed in COS-7 cells. Despite the homology of VR-L to VR1, the channels do not appear to open at capsaicin or low pH.

However, exposure of COS-7 cells expressing VR-L to noxious heat did not elicit inward currents and significantly differentiated this channel in its function as compared to VR1.

FIG. 5 shows the response from a typical cell. Application of 10 μM capsaicin at a holding potential of −60 mV to the cells failed to induce a change in membrane current (1 A). Application of the bath of extracellular recording solution at room temperature resulted in an increase in current noise (1B), probably due to mechanical interference. A similar application of the extracellular solution at 55 ° C. elicited a large inward current (1C). These studies suggest that the channel is non-selective with respect to cation permeability. Thus, natural cells expressing the channel appear to respond to noxious thermal conditions. The overall effect appears to be excitatory, as the current appears to be inward at the resting membrane potential. Activation of the channel will result in depolarization, which will recruit voltage-gated sodium and calcium channels, further enhancing the excitatory effect.

Example 4 Screening of Ion Channel Modulators The compounds to be evaluated as agonists, partial agonists or antagonists of the VR-L channel can be applied in solution in the above system, and The current can be used to measure activation or blockage of the channel encoded by the transfected vector.

Alternatively, permanently or transiently transfected cells expressing VR-L are discussed in Example 3 with or without the addition of compounds capable of exhibiting agonist, partial agonist or antagonist activity. It is used to measure the increase in intracellular calcium in response to an applied stimulus.

Changes in intracellular calcium can be determined using Ca-45 uptake, eg, Wood et
al (1989) J Neurochem 53: 1203-1211, or Zeilhofer et al (1996) J
Neurophysiol 76 (5): 2834-40 or Hansen et al (1998) Am J Physiol 2
74 (6 pt 1): a calcium-sensitive dye described in C1552-62 (for example, Fura-
Measure using cells in a multi-well plate using the activation of II).

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1. VR-L "virtual transcript" based on duplication of human EST clones. Similar positions in VR1 for which primers were designed are indicated by numbers on the map. The primers and their orientation are indicated by the arrows. The vertical dash-dot line is adjacent to a gap not represented by any EST clone. The 5 'untranslated region (UTR) and the region around the stop codon were derived from the results of a query of the human genome index for a hypothetical human consensus sequence.

FIG. 2A. Generation of full-length sequences from PCR products by trans (PCR).

FIG. 2B. Use of unique restriction sites.

FIG. 3A. VR-L nucleotide and amino acid sequence. Note: Ankyrin-like repeat (ANK), predicted transmembrane helix (TM
) And possible hole loops are ( ^* ), ( ) And (＾).

FIG. 3A (cont. 1) VR-L nucleotide and amino acid sequence. Note: Ankyrin-like repeat (ANK), predicted transmembrane helix (TM
) And possible hole loops are ( ^* ), ( ) And (＾).

FIG. 3A (continued 2) VR-L nucleotide and amino acid sequence. Note: Ankyrin-like repeat (ANK), predicted transmembrane helix (TM
) And possible hole loops are ( ^* ), ( ) And (＾).

FIG. 3B. Alignment of the amino acid sequences of rat VR1 (upper) and human VR-L (lower).

FIG. 4: Mouse, rat and human (Jurkat cells) in the 5/6 transmembrane region
Alignment of multiple sequences of VR-L and rat VR1.

FIG. 5: Whole-cell voltage-clamp recording of COS-7 cells transfected with VR-L.

FIG. 6: EST clones showing homology to rat vanilloid receptor subtype 1 (VR1).

FIG. 6 (continued) Table 1: EST clones showing homology to rat vanilloid receptor subtype 1 (VR1).

FIG. 7: Table 2: Primers used for cloning the VR-L fragment.

[Procedural Amendment] Submission of translation of Article 34 Amendment

[Submission date] December 1, 2000 (2000.12.1)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0123

[Correction method] Change

[Contents of correction]

Suspected, further 3 ′ VR-L EST clone (GenBank accession number A
Subsequent THC database searches using A281348, W92895, N35179, T12251) revealed stop codons and 3 'untranslated sequences (VR1
Leading to the identification of the 3 'end of the kidney,
The 'sequence (THC161190) was extracted. The primer designed from this putative 3 'end further combined with the 5' primer to give a distinct product, which was later demonstrated by sequencing to be flanking the rest of the VR-L sequence. Was. Long PCR products can be trans-PCR (splicing by overlap extension) or ligation utilizing unique restriction sites in the region of overlap (FIGS. 2a and 2b).
) And both spliced together. FIG. 3b shows the amino acid sequence of rat VR1.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] FIG. 3B

[Correction method] Change

[Contents of correction]

FIG. 3B: Amino acid sequence of rat VR1.

[Procedure amendment 3]

[Document name to be amended] Drawing

[Correction target item name] FIG. 3A (continued 1)

[Correction method] Change

[Contents of correction]

[Procedure amendment 4]

[Document name to be amended] Drawing

[Correction target item name] FIG. 3B

[Correction method] Change

[Contents of correction]

FIG. 3B

[Procedure amendment 5]

[Document name to be amended] Drawing

[Correction target item name] Fig. 4

[Correction method] Change

[Contents of correction]

FIG. 4

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 29/00 A61P 37/02 4C086 37/02 C07K 14/705 4H045 C07K 14/705 16/28 16/28 C12Q 1/02 C12N 5/10 1/68 A C12Q 1/02 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 33/53 D 33/50 33/566 33/53 C12N 15/00 ZNAA 33/566 5/00 B (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Wood, John Nicholas United Kingdom, London W Sea 1E 6 Beatty, Gower Street, Med War Building, University College London Department of Anatomy and Developmental Biology (72) Inventor England, Stephen, United Kingdom, Kent Seatie 139E NJ, Sandwich, Ramsgate Road, Pfizer Limited F-term (reference) 2G045 AA29 AA40 CB01 CB17 DA36 DA77 FB02 FB03 4B024 AA01 AA11 BA63 CA04 DA02 EA04 FA15 GA14 GA18 HA01 HA14 HA15 HA17 4B063 QA01 QA18 QQ08 QQ42 QQ52 QR55 QR62 QR80 QS05 QS25 QS34 QS36 AXAAA4A65 AAXA4A0AA4A65 AA17 NA14 ZA082 ZB072 4C086 AA01 AA02 AA03 AA04 EA16 MA01 MA04 NA14 ZA08 ZB07 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA50 DA76 EA21 EA22 EA50 FA74

Claims (38)

[Claims] 1. An isolated non-selective cation channel protein that is (a) activatable by noxious heat, (b) available from human T lymphocytes, and (c) not capsaicin-sensitive. . 2. An isolated VR-L protein comprising amino acid SEQ ID NO: 2. (3) having ion channel activity; and (b) at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%.
An isolated VR-L variant protein comprising an amino acid sequence having%, 98% or 99% sequence identity to SEQ ID NO: 2. 4. An isolated nucleic acid comprising a nucleotide sequence encoding a protein according to any one of claims 1 to 3. 5. The nucleic acid of claim 4, wherein said nucleotide sequence comprises SEQ ID NO: 1 or a sequence degenerately equivalent thereto. 6. The nucleic acid of claim 4, wherein said nucleotide sequence encodes the VR-L variant of claim 3. 7. The nucleic acid according to claim 4, which is capable of hybridizing with a nucleic acid comprising a sequence complementary to SEQ ID NO: 1. 8. The nucleic acid according to claim 6, wherein said nucleotide sequence encodes an allelic variant of SEQ ID NO: 1. 9. The nucleic acid of claim 6, wherein said nucleotide sequence encodes a variant derived from one of non-human origin. 10. The nucleic acid of claim 9, wherein said nucleotide sequence encodes the amino acid sequence of FIG. 11. The method according to claim 6, wherein the nucleotide sequence is a derivative of SEQ ID NO: 1 by one or more additions, insertions, deletions or substitutions of one or more nucleotides therein. Nucleic acids. A nucleic acid which is a complement of the nucleic acid according to any one of claims 4 to 11. 13. Use as a probe consisting of a nucleotide sequence that is unique to at least 30 contiguous nucleotides of SEQ ID NO: 1, or a sequence that is degenerately equivalent to, or complementary to either of them. For nucleic acid molecules. 14. A primer comprising a nucleotide sequence specific to 10 to 30 contiguous nucleotides of SEQ ID NO: 1 or a sequence degenerately equivalent thereto or complementary to either of them. Nucleic acid molecules for use. 15. A method for identifying and / or cloning a nucleic acid according to any one of claims 4 to 12, wherein the method uses the probe or primer according to claim 13 or 14. 16. (a) providing a preparation of nucleic acid; (b) providing the probe of claim 13; (c) conditions for hybridizing the nucleic acid in the preparation with the probe. 16. The method of claim 15, comprising contacting underneath, and (d) identifying any nucleic acids that hybridize to the probe. 17. A method comprising: (a) providing a preparation of a nucleic acid; (b) at least one primer is the primer of claim 14.
Providing primer pairs of nucleic acid molecules suitable for PCR, (c) contacting the nucleic acid in the preparation with the primers under conditions for performing PCR, (d) performing PCR and amplifying 16. The method of claim 15, comprising determining the presence or absence of a PCR product. 18. A method for producing a derivative nucleic acid, comprising a step of modifying the nucleic acid of claim 5. 19. The method of claim 19, wherein the ion channel protein encoded by said derivative nucleic acid is modified for its activation or permeability specificity. A recombinant vector comprising the nucleic acid according to any one of claims 4 to 12. 21. The vector of claim 20, wherein said nucleic acid is operably linked to a promoter or other regulatory element for transcription of a host cell. 22. The vector according to claim 20, which is suitable for expressing the nucleic acid in a mammal. A cell comprising the heterologous nucleic acid according to any one of claims 4 to 12, or the vector according to any one of claims 20 to 22. 24. A cell transformed with the heterologous nucleic acid according to any one of claims 4 to 12, or the vector according to any one of claims 20 to 22. 25. The cell according to claim 23, wherein the cell is selected from COS, CHO or HEK293 cells or oocytes. A cell comprising the heterologous protein according to any one of claims 1 to 3. 27. A method for affecting the electrophysiological and / or pharmacological properties of a cell, wherein the heterologous nucleic acid according to any one of claims 4 to 12 is expressed in said cell. Providing or causing the same. 28. The method of claim 27, for increasing the VR-L ion channel activity of the cell. 29. The method of claim 27 for reducing VR-L ion channel activity of said cell. 30. All or a specific part of the nucleic acid according to claim 12, which reduces VR-L activity by an antisense mechanism, (ii) a method for reducing activity by co-suppression. 12. Use of any of the specific part of the nucleic acid according to any one of items 4 to 11 or (iii) use of a ribozyme specific to the nucleic acid according to any one of items 4 to 11. 30. The method of claim 29 comprising comprising. 31. The cell according to any one of claims 23 to 26, or the electrophysiological and / or pharmacological properties of the cell according to any one of claims 27 to 30.
A transgenic non-human mammal comprising cells that have been altered according to the methods of paragraphs. 32. A method for identifying a substance having an activity of regulating an ion channel, comprising: (i) the protein according to any one of claims 1 to 3, and (ii) the protein according to any one of claims 23 to 26. 31. The cell of claim 1, (iii) a cell whose electrophysiological and / or pharmacological properties have been altered according to the method of any one of claims 27-30, (iv) the cell of claim 31. A method comprising the use of any of a transgenic organism. 33. A solution of a substance that causes the heterologous protein according to any one of claims 1 to 3 to interact with a substance and the protein, wherein the substance interacts with a membrane or a cell surface. 33. The method of claim 32, comprising: (ii) measuring the electrophysiological response of the cell or membrane to this interaction. 34. An analgesic or other compound that acts on nociception; an immunomodulator;
34. The method of claim 32 or 33 for screening for a neuromodulator. 35. A method for affecting the electrophysiological and / or pharmacological properties of a cell, comprising modulating the activity of the protein according to any one of claims 1 to 3. Method. 36. A polypeptide comprising an antigen-binding site of an antibody capable of specifically binding to the protein of claim 2. 37. The method according to any one of claims 4 to 12, for use in gene therapy or for use in the preparation of a medicament for use in gene therapy.
The nucleic acid according to claim or the vector according to any one of claims 20 to 22. 38. The nucleic acid or vector according to claim 37, wherein the treatment is for a disease involving cells of the sensory nerve and / or the immune system.