GB2375349A - Chloride intracellular channel-related proteins - Google Patents

Abstract

An isolated chloride intracellular channel-related polypeptide comprising: <SL> <LI>(i) the amino acid sequence of SEQ ID NO: 2 or <LI>(ii) a variant thereof which is capable of modulating chloride ion efflux across a membrane in response to a change in extracellular chloride ion concentration or <LI>(iii) a fragment of (i) or (ii) which is capable of modulating chloride ion efflux across a membrane in response to a change in extracellular chloride ion concentration. </SL> The polypeptide may be used in an assay to identify substances which may be used in the treatment of asthma, bronchitis, COPD, cystic fibrosis, respiratory distress syndrome, diabetes, obesity, arthritis, constipation, Crohn's disease, bowel disease, amenorrhea and urinary incontinence.

Description

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POLYPEPTIDE Field of the Invention The present invention relates to chloride intracellular channel-related polypeptides.

Background of the Invention Ion channels are involved in a wide variety of neurological and other disorders in man. Members of the chloride intracellular channels (CLIC) family, including human chloride intracellular channel 4 (CLIC4), rabbit chloride intracellular channel-related protein (parchorin) and the chloride channel from bovine kidney (p64), play a role in water-secreting tissues through the regulation of chloride transport.

Rabbit parchorin is a 120-kDa phosphoprotein that, in association with stimulation of HCI secretion, translocates from cytosol to the apical membrane of gastric parietal cells. Pachorin is highly enriched in tissues that secrete water, such as parietal cell, choroid plexus, salivary duct, lacrimal gland, kidney, airway epithelia, and chorioretinal epithelia. When expressed as a fusion protein with green fluorescent protein (GFP) in the LLC-PK1 kidney cell line, GFP-parchorin, unlike other CLIC family members, exists mainly in the cytosol. However, when Cl-efflux from the cell is elicited, GFP-parchorin translocates to the plasma membrane. It is generally thought that parchorin plays a critical role in water-secreting cells, possibly through the regulation of chloride ion transport.

Summary of the Invention

A novel chloride intracellular channel, referred to herein as HIPHUM 182, is I now provided. HIPHUM182 is shown to be primarily expressed in urinary bladder, thymus, thyroid, lung, parotid gland, stomach, ovary, tonsil, trachea and rectum. The novel chloride intracellular channel is a screening target for the identification and development of novel pharmaceutical agents, including modulators of chloride intracellular channel activity. These agents may be used in the treatment and/or prophylaxis of disorders such as asthma, bronchitis, chronic obstructive pulmonary

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disease (COPD), cystic fibrosis, respiratory distress syndrome, diabetes mellitus type II, hypoglycemia, obesity, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, constipation, Crohn's disease, diarrhea, hyperthyroidism, hypothyroidism, inflammatory bowel disease, irritable bowel syndrome, amenorrhea and urinary incontinence.

Accordingly, the present invention provides an isolated chloride intracellular channel-related polypeptide comprising: (i) the amino acid sequence of SEQ ID NO: 2; (ii) a variant thereof which is capable of modulating chloride ion efflux across a membrane in response to a change in extracellular chloride ion concentration; or (iii) a fragment of (i) or (ii) which is capable of modulating chloride ion efflux across a membrane in response to a change in extracellular chloride ion concentration.

According to another aspect of the invention there is provided a polynucleotide encoding a polypeptide of the invention which polynucleotide includes a sequence comprising: (a) the nucleic acid sequence of SEQ ID NO: 1 and/or a sequence complementary thereto; (b) a sequence which hybridises under stringent conditions to a sequence as defined in (a) ; (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b); or (d) a sequence having at least 80% identity to a sequence as defined in (a), (b) or (c).

The invention also provides: an expression vector which comprises a polynucleotide of the invention and which is capable of expressing a polypeptide of the invention; a host cell comprising an expression vector of the invention; a method of producing a polypeptide of the invention which method comprises maintaining a host cell of the invention under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide ;

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an antibody specific for a polypeptide of the invention; a method for identification of a substance that modulates intracellular chloride channel activity and/or expression, which method comprises contacting a polypeptide, polynucleotide, expression vector or host cell of the invention with a test substance and determining the effect of the test substance on the activity and/or expression of the said polypeptide or the polypeptide encoded by the said polynucleotide, thereby to determine whether the test substance modulates chloride intracellular channel activity and/or expression; a compound which stimulates or modulates chloride intracellular channel activity and which is identifiable by the method referred to above ; a method of treating a subject having a disorder that is responsive to chloride intracellular channel stimulation or modulation, which method comprises administering to said subject an effective amount of substance of the invention; and use of a substance that stimulates or modulates chloride intracellular channel activity in the manufacture of a medicament for the treatment or prophylaxis of a disorder that is responsive to stimulation or modulation of chloride intracellular channel activity.

Preferably the disorder is selected from asthma, bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, respiratory distress syndrome, diabetes mellitus type II, hypoglycemia, obesity, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, constipation, Crohn's disease, diarrhea, hyperthyroidism, hypothyroidism, inflammatory bowel disease, irritable bowel syndrome, amenorrhea and urinary incontinence.

Brief Description of the Sequences SEQ ID NO: 1 shows the nucleotide and amino acid sequences of human protein HIPHUM 182.

SEQ ID NO: 2 shows the amino acid sequence alone of HIPHUM 182.

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Detailed Description of the Invention Throughout the present specification and the accompanying claims the words "comprise"and"include"and variations such as"comprises","comprising", "includes"and"including"are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

The present invention relates to a human chloride intracellular channel-related polypeptide, referred to herein as HIPHUM182, and variants thereof. Sequence information for HIPHUM182 is provided in SEQ ID NO: 1 (nucleotide and amino acid) and in SEQ ID NO: 2. A polypeptide of the invention thus consists essentially of the amino acid sequence of SEQ ID NO: 2 or of a variant of that sequence, or of a fragment of either thereof.

Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 50%, e. g. more than 80%, 90%, 95% or 99%, by weight of the polypeptide in the preparation is a polypeptide of the invention.

Routine methods, can be employed to purify and/or synthesise the proteins according to the invention. Such methods are well understood by persons skilled in the art, and include techniques such as those disclosed in Sambrook et al, Molecular Cloning : a Laboratory Manual, 2"Edition, CSH Laboratory Press, 1989, the disclosure of which is included herein in its entirety by way of reference.

The term"variant"refers to a polypeptide which has a same essential character or basic biological functionality as HIPHUM182. The essential character of HIPHUM182 can be defined as follows: HIPHUM 182 is a chloride intracellular channel. Preferably the polypeptide is capable of modulating chloride ion efflux across a membrane in response to a change in extracellular chloride ion concentration. Preferably a variant polypeptide is one which potentiates chloride ion efflux across the plasma membrane in response to a decrease in extracellular chloride ion concentration. HIPHUM182 may exist in a soluble form in the resting state and

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may associate with the cell membrane in the secreting state. Preferably a variant polypeptide may be translocated to the cell membrane in response to a stimulus such as a change in extrallular chloride ion concentration.

HIPHUM182 contains a single consensus N-glycosylation site (at amino acids 487 to 490 in SEQ ID NO: 2), a consensus glycosaminoglycan attachment site (at amino acid positions 299 to 302 in SEQ ID NO : 2), a consensus cAMP and cGMP-dependent protein kinase site (290-293 in SEQ ID NO : 2), three consensus protein kinase C-phosphorylation sites (472-474,575-577 and 617-619 in SEQ ID NO : 2) and seven consensus casein kinase II phosphorylation sites (83-86,322-325, 489-492,495-498, 575-578,610-613 and 628-631 in SEQ ID NO : 2).

A polypeptide having the same essential character as HIPHUM182 may be identified by monitoring for a function of the chloride intracellular channel selected from chloride ion channel activity, translocation of the polypeptide between the cytoplasm and cell membrane, or modulation of the polypeptide by a kinase.

In another aspect of the invention, a variant is one which does not show the same activity as HIPHUM182 but is one which inhibits a basic function of HIPHUM182. For example, a variant polypeptide is one which inhibits chloride ion channel activity of HIPHUM 182, for example a variant polypeptide may be capable of binding to extracellular chloride ions but may be incapable of transporting chloride ions across the cell membrane.

Typically, polypeptides with more than about 80% identity preferably at least 90% or at least 95% and particularly preferably at least 96% at least 97% or at least 99% identity, with the amino acid sequences of SEQ ID NO: 2, are considered as variants of the proteins. Such variants may include allelic variants and the deletion, modification or addition of single amino acids or groups of amino acids within the protein sequence, as long as the peptide maintains a basic biological functionality of HIPHUM182.

Amino acid substitutions may be made, for example from 1,2 or 3 to 10,20 or 30 substitutions. The modified polypeptide generally retains activity as a chloride intracellular channel-related. Conservative substitutions may be made, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for

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eacn other.

ALIPHATIC Non-polar GAP ILV Polar-uncharged CSTM NQ Polar-charged DE KR AROMATIC H F W Y

Shorter polypeptide sequences are within the scope of the invention. For example, a peptide of at least 20 amino acids or up to 50,60, 70,80, 100,150, 200, 300,400 or 500 amino acids in length is considered to fall within the scope of the invention as long as it demonstrates a basic biological functionality ofHIPHUM182.

In particular, but not exclusively, this aspect of the invention encompasses the situation when the protein is a fragment of the complete protein sequence and may represent a chloride ion channel pore forming region. Such fragments can be used to construct chimeric receptors preferably with another chloride ion channel polypeptide, more preferably with another chloride intracellular channel polypeptide.

Such fragments of HIPHUM 182 or a variant thereof can also be used to raise antiHIPHUM182 antibodies. In this embodiment the fragment may comprise an epitope of the HIPHUM182 polypeptide and may otherwise not demonstrate the functional properties of HIPHUM 182 Polypeptides of the invention may be chemically modified, e. g. posttranslationally modified. For example, they may be glycosylated or comprise modified amino acid residues. They may also be modified by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote insertion into the cell membrane. Such modified polypeptides fall within the scope

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of the term"polypeptide"of the invention.

The invention also includes nucleotide sequences that encode for HIPHUM182 or variant thereof as well as nucleotide sequences which are complementary thereto. The nucleotide sequence may be RNA or DNA including genomic DNA, synthetic DNA or cDNA. Preferably the nucleotide sequence is a DNA sequence and most preferably, a cDNA sequence. Nucleotide sequence information is provided in SEQ ID NO: 1. Such nucleotides can be isolated from human cells or synthesised according to methods well known in the art, as described by way of example in Sam brook et ai, 1989.

Typically a polynucleotide of the invention comprises a contiguous sequence of nucleotides which is capable of hybridizing under selective conditions to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1.

A polynucleotide of the invention can hydridize to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1 at a level significantly above background. Background hybridization may occur, for example, because of other cDNAs present in a cDNA library. The signal level generated by the interaction between a polynucleotide of the invention and the coding sequence or complement of the coding sequence of SEQ ID NO: 1 is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ ID NO: 1. The intensity of interaction may be measured, for example, by radiolabelling the probe, e. g. with 32p. Selective hybridisation may typically be achieved using conditions of medium to high stringency. However, such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al, 1989). For example, if high stringency is required suitable conditions include from 0.1 to 0.2 x SSC at 60 C up to 65 OC. If lower stringency is required suitable conditions include 2 x SSC at 60 C.

The coding sequence of SEQ ID NO : 1 may be modified by nucleotide substitutions, for example from 1,2 or 3 to 10,25, 50 or 100 substitutions. The polynucleotide of SEQ ID NO: 1 may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends.

A polynucleotide may include one or more introns, for example may comprise genomic DNA. Additional sequences such as signal sequences which may assist in

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insertion of the polypeptide in a cell membrane may also be included. The modified polynucleotide generally encodes a polypeptide which has HIPHUM182 activity.

Alternatively, a polynucleotide encodes a chloride channel forming portion of a polypeptide or a polypeptide which inhibits HIPHUM182 activity. Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the Table above.

A nucleotide sequence which is capable of selectively hybridizing to the complement of the DNA coding sequence of SEQ ID NO: 1 will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the coding sequence of SEQ ID NO: 1 over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, more preferably at least 100 contiguous nucleotides or most preferably over the full length of SEQ ID NO : 1.

For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul (1993) J. Mol. Evol.

36: 290-300; Altschul et al (1990) J Mol. Biol. 215: 403-10.

Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http ://www. ncbi. nlm. nih. gov/).

This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy

some positive-valued threshold score T when aligned with a word of the same length p z : l in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, 1990). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value ; the cumulative score goes to zero or below, due to the accumulation of one or

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more negative-scoring residue alignments: or the end of either sequence is reached.

The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad.

Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e. g. , Karlin and Altschul (1993) Proc. Natl. Acad. Sci.

USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N) ), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0. 001.

Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i. e. higher sequence identity over longer lengths) being preferred. Thus, for example a polynucleotide which has at least 90% sequence identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 95% sequence identity over 40 nucleotides.

The nucleotides according to the invention have utility in production of the proteins according to the invention, which may take place in vitro, in vivo or ex vivo.

The nucleotides may be involved in recombinant protein synthesis or indeed as therapeutic agents in their own right, utilised in gene therapy techniques. Nucleotides complementary to those encoding HIPHUM 182, or antisense sequences, may also be used in gene therapy.

Polynucleotides of the invention may be used as a primer, e. g. a PCR primer, a primer for an alternative amplification reaction, a probe e. g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.

Such primers, probes and other fragments will preferably be at least 10,

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preferably at least 15 or at least 20. for example at least 25, at least 30 or at least 40 nucleotides in length. They will typically be up to 40,50, 60,70, 100 or 150 nucleotides in length. Probes and fragments can be longer than 150 nucleotides in length, for example up to 200,300, 400,500, 600,700, 1000,1500 or 2000 nucleotides in length, or even up to a few nucleotides, such as five or ten nucleotides, short of the coding sequence of SEQ ID NO: 1.

The present invention also includes expression vectors that comprise nucleotide sequences encoding the proteins or variants thereof of the invention. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al. 1989.

Polynucleotides according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense polynucleotides may also be produced by synthetic means. Such antisense polynucleotides may be used as test compounds in the assays of the invention or may be useful in a method of treatment of the human or animal body by therapy.

Preferably, a polynucleotide of the invention or for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i. e. the vector is an expression vector. The term"operably linked"refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.

A regulatory sequence, such as a promoter,"operably linked"to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

The vectors may be for example, plasmid, virus or phage vectors provided with a origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain

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one or more selectable marker genes, for example an ampicillin resistence gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also be adapted to be used in vivo, for example in a method of gene therapy.

Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. For example, yeast

promoters include S. cerevisiae GAL4 and ADH promoters, S. pombe nmtl and adh promoter. Mammalian promoters include the metallothionein promoter which can be induced in response to heavy metals such as cadmium. Viral promoters such as the SV40 large T antigen promoter or adenovirus promoters may also be used. All these promoters are readily available in the art.

Mammalian promoters, such as -actin promoters, may be used. Tissuespecific promoters are especially preferred. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR). Viral promoters are readily available in the art.

The vector may further include sequences flanking the polynucleotide giving rise to polynucleotides which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombination. In particular, a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for delivering the polynucleotides of the invention to a mammalian cell. Other examples of suitable viral vectors include herpes simplex viral vectors and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses. Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the polynucleotide into the host genome. Replication-defective adenovirus vectors

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by contrast remain episomal and therefore allow transient expression.

The invention also includes cells that have been modified to express the HIPHUM182 polypeptide or a variant thereof. Such cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells. using for example a baculovirus expression system, lower eukaryotic cells, such as yeast or prokaryotic cells such as bacterial cells. Particular examples of cells which may be modified by insertion of vectors encoding for a polypeptide according to the invention include mammalian HEK293T, CHO, HeLa, BHK, 3T3 and COS cells.

Preferably the cell line selected will be one which is not only stable, but also allows for mature glycosylation and cell surface expression of a polypeptide. Expression

may be achieved in transformed oocytes. A polypeptide of the invention may be expressed in cells of a transgenic non-human animal, preferably a mouse. A transgenic non-human animal expressing a polypeptide of the invention is included within the scope of the invention. A polypeptide of the invention may also be expressed in Xenopus laevis oocytes, in particular for use in an assay of the invention. A polypeptide of the invention may be purified from any suitable cell type from any species for reconstitution into lipid bilayers or vesicles.

According to another aspect, the present invention also relates to antibodies, specific for a polypeptide of the invention. Such antibodies are for example useful in purification, isolation or screening methods involving immunoprecipitation techniques or, indeed, as therapeutic agents in their own right.

Antibodies may be raised against specific epitopes of the polypeptides according to the invention. Such antibodies may be used to block the chloride channel formed by the polypeptide. An antibody, or other compound,"specifically binds"to a protein when it binds with preferential or high affinity to the protein for which it is specific but does substantially bind not bind or binds with only low affinity to other proteins. A variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of an antibody are well known in the art (see for example Maddox et al, J. Exp. Med. 158, 1211-1226,1993). Such immunoassays typically involve the formation of complexes between the specific protein and its antibody and the measurement of complex formation.

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Antibodies of the invention may be antibodies to human polypeptides or fragments thereof. For the purposes of this invention, the term"antibody", unless specified to the contrary, includes fragments which bind a polypeptide of the

invention. Such fragments include Fv, F (ab') and F (ab') 2 fragments, as well as single I I fragments, as well as single chain antibodies. Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDR-grafted antibodies or humanised antibodies.

Antibodies may be used in a method for detecting polypeptides of the invention in a biological sample, which method comprises: I providing an antibody of the invention ; II incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and III determining whether antibody-antigen complex comprising said antibody is formed.

A sample may be for example a tissue extract, blood, serum and saliva.

Antibodies of the invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions, etc.

Antibodies may be linked to a revealing label and thus may be suitable for use in methods of in vivo HIPHUM182 imaging.

Antibodies of the invention can be produced by any suitable method. Means for preparing and characterising antibodies are well known in the art, see for example Harlow and Lane (1988)"Antibodies : A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, an antibody may be produced by raising antibody in a host animal against the whole polypeptide or a fragment thereof, for example an antigenic epitope thereof, herein after the "immunogen".

A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the animal's serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified.

A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by

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fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256,495-497).

An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

For the production of both monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat or mouse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier.

The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified.

An important aspect of the present invention is the use of polypeptides according to the invention in screening methods. The screening methods may be used to identify substances that bind to chloride intracellular channel-relateds and in particular which bind to HIPHUM182. Screening methods may also be used to identify agonists or antagonists which may modulate chloride intracellular channelrelated activity, inhibitors or activators ofHIPHUM182 activity, and/or agents which up-regulate or down-regulate HIPHUM182 expression.

Any suitable format may be used for the assay. In general terms such screening methods may involve contacting a polypeptide of the invention with a test substance and monitoring for binding of the test substance to the polypeptide or measuring receptor activity. A polypeptide of the invention may be incubated with a test substance. Modulation of chloride intracellular channel-related activity may be determined. In a preferred aspect, the assay is a cell-based assay. Preferably the assay may be carried out in a single well of a microtitre plate. Assay formats which allow high throughput screening are preferred.

Modulator activity can be determined by contacting cells expressing a polypeptide of the invention with a substance under investigation and by monitoring an effect mediated by the polypeptide. The cells expressing the polypeptide may be

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in vitro or in vivo. The polypeptide of the invention may be naturally or recombinantly expressed. Preferably, the assay is carried out in vitro using cells expressing recombinant polypeptide. Preferably, control experiments are carried out on cells which do not express the polypeptide of the invention to establish whether the observed responses are the result of activation of the polypeptide.

The binding of a test substance to a polypeptide of the invention can be determined directly. For example, a radiolabelled test substance can be incubated with the polypeptide of the invention and binding of the test substance to the polypeptide can be monitored. Typically, the radiolabelled test substance can be incubated with cell membranes containing the polypeptide until equilibrium is reached. The membranes can then be separated from a non-bound test substance and dissolved in scintillation fluid to allow the radioactive content to be determined by scintillation counting. Non-specific binding of the test substance may also be determined by carrying out a competitive binding assay.

Assays may be carried out using cells expressing HIPHUM182, and incubating such cells with the test substance. The results of the assay are compared to the results obtained using the same assay in the absence of the test substance. Cells expressing HIPHUM182 constitutively may be provided for use in assays for HIPHUM182 function. Additional test substances may be introduced in any assay to look for inhibitors or enhancers of HIP HUM 182-mediated activity, preferably chloride ion channel activity.

In preferred aspects, a host cell is provided expressing the polypeptide and containing a chloride, bromide or iodide sensitive photoprotein. Such photoproteins increase or decrease light emission on the influx or efflux of chloride ions and can be detected using an imaging system. A reporter gene assay using such photoproteins can be used to assay for modulation of chloride channel activity. For example, reporter assays that link the intracellular chloride, bromide or iodide ion concentration to a signalling cascade that causes up-or down-regulation of expression or activity of a detectable reporter protein such as luciferase. The assay enables determination of whether a test substance modulates the HIPHUM 182 regulated flow of chloride, bromide or iodide ions through chloride channels in target cells.

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The ability of a test substance to modulate the HIPHUM 182 regulated flow of chloride ions through chloride ion channels may also be determined using fluorescence based assays using a Fluorometric Imaging Plate Reader (FLIPR) and membrane voltage sensitive dyes, such as DiBac, or chloride sensitive dyes such as 6-methyoxy-N-[3-sulfopropyl]quinolinium (SPQ) (Molecular Probes), N- (4aminobutyl)-6-methoxyquinolinium chloride (ABQ), N- (ethoxycarbonylmethyl)-6- methoxyquinolinium bromide (MQAE) or N- (3-sulfopropyl) acridinium (SPA).

Alternatively bromide or iodide sensitive dyes may be used. FRET/BRET based membrane voltage sensitive dyes with VIPR may also be used.

Assays may also be carried out by measuring the influx or efflux of radioactive chloride ions in cells expressing a polypeptide of the invention.

Electrophysiological recordings of cell membrane currents or membrane potentials from cells expressing a polypeptide of the invention may also be used to assay for modulatory activity of a test substance.

The structural conformation of the channel on activation or inhibition may also be assayed. The location of the polypeptide within the cell may also be monitored to determine whether a substance promotes or inhibits translocation of the polypeptide from the cytosol to the plasma membrane.

Assays may also be carried out to identify substances which modify HIPHUM182 expression, for example substances which up-or down-regulate expression. Such assays may be carried out for example by using antibodies for HIPHUM182 to monitor levels of HIPHTJM 1 82 expression. Other assays which can be used to monitor the effect of a test substance on HIPHUM182 expression include using a reporter gene construct driven by the HIPHUM 182 regulatory sequences as the promoter sequence and monitoring for expression of the reporter polypeptide.

Further possible assays could utilise membrane fractions from overexpression of HIPHUM182 polypeptide either in X laevis oocytes or cell lines such as HEK293, CHO, COS7, BHK, 3T3 and HeLa cells.

Additional control experiments may be carried out.

Suitable test substances which can be tested in the above assays include combinatorial libraries, defined chemical entities and compounds, peptide and peptide mimetics, oligonucleotides and natural product libraries, such as display (e. g.

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phage display libraries) and antibody products.

Typically, organic molecules will be screened, preferably small organic molecules which have a molecular weight of from 50 to 2500 daltons. Candidate products can be biomolecules including, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

Test substances may be used in an initial screen of, for example, 10 substances per reaction, and the substances of these batches which show inhibition or

activation tested individually. Test substances may be used at a concentration of from 10M to 1000uM, preferably from 1/lM to 100uM, more preferably from IlM to 10/lM. Preferably, the activity of a test substance is compared to the activity shown by a known activator or inhibitor. A test substance which acts as an inhibitor may produce a 50% inhibition of activity of the channel. Alternatively a test substance which acts as an activator may produce 50% of the maximal activity produced using a known activator.

Another aspect of the present invention is the use of polynucleotides encoding the HIPHUM 182 polypeptides of the invention to identify mutations in HIPHUM182 genes which may be implicated in human disorders. Identification of such mutations may be used to assist in diagnosis or susceptibility to such disorders and in assessing the physiology of such disorders. Polynucleotides may also be used in hybridisation studies to monitor for up-or down-regulation of HIPHUM 182 expression. Polynucleotides such as SEQ ID NO: 1 or fragments thereof may be used to identify allelic variants, genomic DNA and species variants.

The present invention provides a method for detecting variation in the expressed products encoded by HIPHUM182 genes. This may comprise determining the level of HIPHUM 182 expressed in cells or determining specific alterations in the expressed product. Sequences of interest for diagnostic purposes include, but are not limited to, the conserved portions as identified by sequence similarity and

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conservation of intron/exon structure. The diagnosis may be performed in conjunction with kindred studies to determine whether a mutation of interest cosegregates with disease phenotype in a family.

Diagnostic procedures may be performed on polynucleotides isolated from an individual or alternatively, may be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Appropriate procedures are described in, for example, Nuovo, G. J., 1992,"PCR In Situ Hybridization: Protocols And Applications", Raven Press, NY). Such analysis techniques include, DNA or RNA blotting analyses, single stranded conformational polymorphism analyses, in situ hybridization assays, and polymerase chain reaction analyses. Such analyses may reveal both quantitative aspects of the expression pattern of a HIPHUM182, and qualitative aspects ofHIPHUM182 expression and/or composition.

Alternative diagnostic methods for the detection of HIPHUM 182 nucleic acid molecules may involve their amplification, e. g. by PCR (the experimental embodiment set forth in U. S. Patent No. 4,683, 202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA 88: 189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. 15 USA 86: 1173- 1177), Q-Beta Replicase (Lizardi et aI., 1988, Bio/Technology 6 : 1197) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

Particularly suitable diagnostic methods are chip-based DNA technologies such as those described by Hacia et at., 1996, Nature Genetics 14: 441-447 and Shoemaker et al., 1996, Nature Genetics 4 : 450-456. Briefly, these techniques involve quantitative methods for analyzing large numbers of nucleic acid sequence targets rapidly and accurately. By tagging with oligonucleotides or using fixed probe

arrays, one can employ chip technology to segregate target molecules as high density t : l Z=l L > arrays and screen these molecules on the basis of hybridization.

Following detection, the results seen in a given patient may be compared with

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a statistically significant reference group of normal patients and patients that have HIPHUM 182 related pathologies. In this way, it is possible to correlate the amount or kind of HIPHUM182 encoded product detected with various clinical states or predisposition to clinical states.

Another aspect of the present invention is the use of the substances that have been identified by screening techniques referred to above in the treatment of disease states, which are responsive to regulation of chloride intracellular channel-related activity. The treatment may be therapeutic or prophylactic. The condition of a patient suffering from such a disease state can thus be improved.

In particular, such substances may be used in the treatment of asthma, bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, respiratory distress syndrome, diabetes mellitus type II, hypoglycemia, obesity, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, constipation, Crohn's disease, diarrhea, hyperthyroidism, hypothyroidism, inflammatory bowel disease, irritable bowel syndrome, amenorrhea and urinary incontinence.

Additional disease states that may be treated include lymphocytic interstitial pneumonitis, pleural effusion and empysema, pleurisy, pneumonia, pulmonary edema, pulmonary hypertension, respiratory acidosis, respiratory alkalosis, tuberculosis, fibromyomas, leiomyomas, myomas, uterine fibroids, large cell lung cancer, small cell lung cancer, squamous cell carcinoma, cardiac tumors, breast cancer, periodic paralyses, malignant hyperthermia, cardiac arrhythmias, cardiac tamponade, cardiogenic shock, coarctation of the aorta, congestive heart failure, dilated cardiomyopathy, dyslipidaemia, hypovolemic shock and periodic paralyses.

Substances that act as inhibitors of HIPHUM182 activity may be used in the treatment of disease states in which HIPHUM182 expression is up-regulated such as lung tumour, lung asthma, lung COPD or breast tumour. Substances that act as activators of HIPHUM182 activity may be used in the treatment of disease states in which expression of HIPHUM182 is down-regulated.

Substances identified according to the screening methods outlined above may be formulated with standard pharmaceutically acceptable carriers and/or excipients as is routine in the pharmaceutical art. For example, a suitable substance may be dissolved in physiological saline or water for injections. The exact nature of a

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formulation will depend upon several factors including the particular substance to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania, 17"'Ed. 1985, the disclosure of which is included herein of its entirety by way of reference.

The substances may be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, topical or other appropriate administration routes.

A therapeutically effective amount of a modulator is administered to a patient. The dose of a modulator may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific modulator, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.

Nucleic acid encoding HIPHUM 182 or a variant thereof which inhibits or enhances HIPHUM182 activity or antisense nucleic acid may be administered to the mammal. Nucleic acid, such as RNA or DNA, and preferably, DNA, is provided in the form of a vector, such as the polynucleotides described above, which may be expressed in the cells of the mammal.

Nucleic acid administered to the mammal for gene therapy may encode functional HIPHUM 182 or a variant thereof with an impaired function such as a dominant negative mutant that disrupts the function of the whole chloride ion channel.

Nucleic acid encoding HIPHUM 182 or a variant thereof which inhibits HIPHUM182 activity may be administered to the mammal. In particular, a nucleic acid encoding a polypeptide with HIPHUM182 activity may be administered to a subject suffering from a condition in which HIPHUM182 expression is downregulated. A nucleic acid encoding a variant of HIPHUM 182 that inhibits

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HIPHUM182 activity may be administered to a patient suffering from a condition in which HIPHUM182 expression is up-regulated such as lung tumour, lung asthma, lung COPD or breast tumour. Nucleic acid, such as RNA or DNA, and preferably, DNA, is provided in the form of a vector, such as the polynucleotides described above, which may be expressed in the cells of the mammal.

Nucleic acid encoding the polypeptide may be administered by any available technique. For example, the nucleic acid may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the nucleic acid may be delivered directly across the skin using a nucleic acid delivery device such as particle-mediated gene delivery. The nucleic acid may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, intravaginal or intrarectal administration.

Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use oftransfection agents.

Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam. The dosage of the nucleic acid to be administered can be altered. Typically the nucleic acid is administered in the range of 1 pg to 1mg, preferably to Ipg to log nucleic acid for particle mediated gene delivery and lOg to 1mg for other routes.

The following Examples illustrate the invention.

Example 1: Characterisation of the sequence A chloride intracellular channel-related polypeptide, designated as HIPHUM182 has been identified. The nucleotide and amino acid sequences of the receptor have been determined. These are set out below in SEQ ID NOs: 1 and 2.

Suitable primers and probes were designed and used to analyse tissue expression.

HIPHUM182 was found to be primarily expressed in urinary bladder. thymus, thyroid, lung, parotid gland, stomach, ovary, tonsil, trachea and rectum.

The chromosomal localization was also mapped. Human HIPHUM182 has been mapped to 21 q22. 1.

Example 2: Screening for substances which exhibit protein modulating activity

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Mammalian cells, such as HEK293, CHO, COS, BHK, 3T3 or HeLa cells, or Xenopus oocytes over-expressing a polypeptide of the invention together with one or more appropriate chloride ion channels subunit are generated for use in the assay. 96 and 384 well plate, high throughput screens (HTS) are employed using fluorescence based chloride indicator molecules or voltage sensitive indicator molecules.

Secondary screening involves electrophysiological assays utilising two electrodes, voltage clamp or patch clamp technology. Tertiary screens involve the study of modulators in rat and mouse models of disease relevant to the target.

A brief screening assay protocol based on a chloride binding fluorescent dye is as follows. Changes in intracellular chloride concentration are measured in cells expressing a polypeptide of the invention using the chloride sensitive dye, 6methoxy-N-[3-sulfopropyl] quinolinium (SPQ). Cells expressing the channel protein are grown on glass coverslips and incubated with 25mM SPQ (Molecular Probes) in loading buffer (lOlmM NaCl, 5mM KC1, 2mM CaCI, 2mM MgCl,, 5mM HEPES, pH7.4, 29mM sodium gluconate) diluted 1: 1 with water, for 4 minutes at room temperature. Cells are then washed with loading buffer before transfer to the perfusion chamber of a Quanticell 700 imager, maintained at 37 C. Fluorescence is excited at 355nm and detected at 450nm with an interference filter (435 20nm) and the time course of SBQ fluorescence is monitored. A field with several cells is selected for measurement, the initial loss of fluorescence due to passive loss of the dye from cells is monitored for 10 minutes and then the perfusate is switched to chloride-free buffer (lOImM sodium gluconate, 5mM potassium gluconate, 2mM calcium acetate, 2mM MgS04, 50mM mannitol, 5mM HEPES, pH 7.4). The efflux of Cl- is observed as an increase in fluorescence. Agents that activate or inhibit Cl' efflux mediated by the novel protein can be identified and assayed by their inclusion in the perfused buffer at the appropriate concentration.

Channel activity is monitored in the presence and absence of a test substance and modulation of channel activity by the test substance is compared in the presence and absence of the test substance to determine whether the test substance is an agonist or antagonist of the chloride intracellular channel-related.

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A typical electropysiology protocol using electrophysiology in oocytes expressing HIPHUM182 is as follows. HIPHUM182 is expressed in Xenopus laevis oocytes either : i) by injection of plasmid DNA that allows the expression of the ion channel cDNA or gene by virtue of an upstream promoter (for example the CMV promoter), or preferably ii) by injection of in vitro transcribed, m'G (5') pp (5') GTP-capped, complementary RNA synthesised from the ion channel cDNA by virtue of an upstream Sp6, T3 or T7 promoter and Sp6, T3 or T7 RNA polymerase.

Typically, 20-50ng of plasmid DNA or cRNA is injected per oocyte and whole-cell currents are recorded using two-microelectrode voltage-clamp (Geneclamp amplifier, Axon instruments Inc.) 1 to 7 days post-injection. Typical microelectrodes have a resistance of 0.5 to 2M.

Chloride currents elicited by decreasing the extracellular chloride concentration by perfusion of chloride-free buffer over the cell are recorded. Voltageprotocols can be generated using pCLAMP8 software (Axon Instruments) and a P/N leak subtraction protocol is used throughout (to remove artefacts generated by nonspecific'leak'current across the membrane). In these experiments the effects of a test compound on current mediated by the channel is studied by inclusion of the compound in the extracellular buffer which is superfused across the oocyte.

A typical electrophysiology assay using mammalian cells expressing a polypeptide of the invention is as follows.

Cells are grown on a glass coverslip, placed into a recording chamber (0.5ml volume) and superfused with an extracellular recording solution at 2 ml min-'. Drugs are applied either via addition to the bath perfusate, or alternatively using a rapid perfusion system which consists of a series of reservoirs connected to a small microfil tube that is placed in close proximity to the voltage-clamped cell. Wholecell currents are recorded using an Axopatch 200B amplifier (Axon Instruments) or other voltage-clamp amplifier (e. g. HEKA), using standard electrophysiological methods (Hamill et al., (1981) Pflugers Arch. 391 : 85-100). Patch pipettes are fabricated from 1. 5mm outside diameter borosilicate capillary glass (Clark Electromedical) using a micropipette puller (Sutter model P97), and fire polished

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(Narishige Microforge) to give final tip resistances of 2-4MQ. A silver/silver chloride pellet is used as the bath reference electrode and the potential difference between this and the recording electrode will be adjusted for zero current flow before seal formation. Cells are visualised using a Diaphot200 inverted microscope (Nikon) with modulation contrast optics at a final magnification ofx400. High resistance seals (1-1 OGQ) between pipette and neuronal cell membranes are achieved by gentle suction, and the'whole cell'configuration attained by applying further suction.

Cells are patch-clamped in an extracellular buffer containing 140mM NaCl, 4.7mM KC1, 1. 2mM MgCI,, ImM CaCI,, 1 ImM glucose, 5mM HEPES (pH 7.4 at 25 C) using microelectrode pipettes containing 120mM CsF, 15mM NaCl, lOmM Cs-EGTA (ethylene glycol-bis (-aminomethyl ester) N, N, N', N-tetra acetic acid, Cs salt), 10mM HEPES (pH7.25 at 25C). Patch electrodes should have resistances of 2 to 6MQ when filled with the pipette-filling solution. Channels are activated by decreasing the extracellular chloride concentration by perfusion of chloride-free buffer over the cell (140mM sodium gluconate, 4.7mM potassium gluconate, 1. 2mM magnesium gluconate, 1mM calcium gluconate, 1 ImM glucose, 5mM HEPES (pH7.4 at 25 C) and the resulting currents recorded.

Voltage command protocols are generated, and current records stored, via a digidata 1200 analog/digital interface (Axon Instruments) controlled by microcomputer (Hewlett Packard Kayak XA) using pCLAMP8 Clampex software (Axon Instruments). Signals are prefiltered at 5kHz bandwidth and sampled at 20kHz. Capacitance transients and series resistance errors are compensated for (80- 85%) using the amplifier circuitry, and linear leakage currents subtracted using an on-line'P-4'procedure provided by the commercial software package.

Data are analysed using pCLAMP8/Clampfit (Axon Instruments), ORIGIN (MicroCal) and DAISI data handling and graphical presentation software packages.

Results can be presented as either arithmetic mean s. e mean or geometric mean with 95% confidence limits. Statistical comparisons are made using paired or unpaired Student's t-test and considered of significance when P < 0. 05.

Channel activity is monitored in the presence and absence of a test substance and modulation of channel activity by the test substance is compared in the presence

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and absence of the test substance to determine whether the test substance is an activator or inhibitor of the chloride intracellular channel.

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SEQUENCE LISTING < 110 > GLAXO GROUP LIMITED < 120 > POLYPEPTIDE < 130 > QG1045 (P80916) < 160 > 2 < 170 > PatentIn version 3.0 < 210 > 1 < 211 > 2661 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1).. (2088) < 220 > < 221 > Unsure < 222 > (2345).. (2345) < 223 > A, C, G or T < 220 > < 221 > Unsure < 222 > (2517).. (2517) < 223 > A, C. G or T < 220 > < 221 > Unsure < 222 > (2559).. (2559) < 223 > A, C, G or T < 220 > < 221 > Unsure < 222 > (2628).. (2628) < 223 > A, C, G or T < 400 > 1

atg gcc gag gcc gcg gag ccg gag ggg gtt gcc ccg ggt ccc cag ggg 48 Met Ala Glu Ala Ala Glu Pro Glu Gly Val Ala Pro Gly Pro Gln Gly 1 5 10 15 ccg ccg gag gtc ccc gcg cct ctg get gag aga ccc gga gag cca gga 96 Pro Pro Glu Val Pro Ala Pro Leu Ala Glu Arg Pro Gly Glu Pro Gly 20 25 30 gcc gcg ggc ggg gag gca gaa ggg ccg gag ggg age gag ggc gca gag 144 Ala Ala Gly Gly Glu Ala Glu Gly Pro Glu Gly Ser Glu Gly Ala Glu 35 40 45 gag gcg ccg agg ggc gcc gcc get gtg aag gag gca gga ggc ggc ggg 192 Glu Ala Pro Arg Gly Ala Ala Ala Val Lys Glu Ala Gly Gly Gly Gly 50 55 60

<Desc/Clms Page number 27>

cca gac agg ggc ccg gag gcc gag gcg egg ggc acg agg ggg gcg cac 240 Pro Asp Arg Gly Pro Glu Ala Glu Ala Arg Gly Thr Arg Gly Ala His 65 70 75 80 ggc gag act gag gcc gag gag gga gcc ccg gag ggt gcc gag gtg ccc 288 Gly Glu Thr Glu Ala Glu Glu Gly Ala Pro Glu Gly Ala Glu Val Pro 85 90 95 caa gga ggg gag gag aca age ggc gcg cag cag gtg gag ggg gcg age 336 Gin Gly Gly Glu Glu Thr Ser Gly Ala Gin Gin Val Glu Gly Ala Ser 100 105 110 ccg gga cgc ggc gcg cag ggc gag ccc cgc ggg gag get cag agg gag 384 Pro Gly Arg Gly Ala Gln Gly Glu Pro Arg Gly Glu Ala Gin Arg Glu 115 120 125 ccc gag gac tct gcg gcc ccc gag agg cag gag gag gcg gag cag agg 432 Pro Glu Asp Ser Ala Ala Pro Glu Arg Gln Glu Glu Ala Glu Gln Arg 130 135 140 cct gag gtc ccg gaa ggt age gcg tec ggg gag gcg ggg gac age gta 480 Pro Glu Val Pro Glu Gly Ser Ala Ser Gly Glu Ala Gly Asp Ser Val 145 150 155 160 gac gcg gag ggc ccg ctg ggg gac aac ata gaa gcg gag ggc ccg gcg 528 Asp Ala Glu Gly Pro Leu Gly Asp Asn He Glu Ala Glu Gly Pro Ala 165 170 175 ggc gac age gta gag gcg gag ggc egg gtg ggg gac age gta gac gcg 576 Gly Asp Ser Val Glu Ala Glu Gly Arg Val Gly Asp Ser Val Asp Ala 180 185 190 gaa ggt ccg gcg ggg gac age gta gac gcg gag ggc ccg ctg ggg gac 624 Glu Gly Pro Ala Gly Asp Ser Val Asp Ala Glu Gly Pro Leu Gly Asp 195 200 205 aac ata caa gcc gag ggc ccg gcg ggg gac age gta gac gcg gag ggc 672 Asn He Gln Ala Glu Gly Pro Ala Gly Asp Ser Val Asp Ala Glu Gly 210 215 220 egg gtg ggg gac age gta gac gcg gaa ggt ccg gcg ggg gac age gta 720 Arg Val Gly Asp Ser Val Asp Ala Glu Gly Pro Ala Gly Asp Ser Val 225 230 235 240 gac gcg gag ggc egg gtg ggg gac age gta gag gcg ggg gac ccg gcg 768 Asp Ala Glu Gly Arg Val Gly Asp Ser Val Glu Ala Gly Asp Pro Ala 245 250 255 ggg gac ggc gta gaa gcg ggg gtc ccg gcg ggg gac age gta gaa gcc 816 Gly Asp Gly Val Glu Ala Gly Val Pro Ala Gly Asp Ser Val Glu Ala 260 265 270 gaa ggc ccg gcg ggg gac age atg gac gcc gag ggt ccg gca gga agg 864 Glu Gly Pro Ala Gly Asp Ser Met Asp Ala Glu Gly Pro Ala Gly Arg ' ?'7c' ? sn ooc

<Desc/Clms Page number 28>

o gcg cgc egg gtc teg ggt gag ccg cag caa teg ggg gac ggc age etc 912 Ala Arg Arg Val Ser Gly Glu Pro Gln Gln Ser Gly Asp Gly Ser Leu 290 295 300 teg ccc cag gcc gag gca att gag gtc gca gcc ggg gag agt gcg ggg 960 Ser Pro Gln Ala Glu Ala Ile Glu Val Ala Ala Gly Glu Ser Ala Gly 305 310 315 320 cgc age ccc ggt gag etc gcc tgg gac gca gcg gag gag gcg gag gtc 1008 Arg Ser Pro Gly Glu Leu Ala Trp Asp Ala Ala Glu Glu Ala Glu Val 325 330 335 ccg ggg gta aag ggg tec gaa gaa gcg gcc ccc ggg gac gca agg gca 1056 Pro Gly Val Lys Gly Ser Glu Glu Ala Ala Pro Gly Asp Ala Arg Ala 340 345 350 gac get ggc gag gac agg gta ggg gat ggg cca cag cag gag ccg ggg 1104 Asp Ala Gly Glu Asp Arg Val Gly Asp Gly Pro Gln Gln Glu Pro Gly 355 360 365 gag gac gaa gag aga cga gag egg age ccg gag ggg cca agg gag gag 1152 Glu Asp Glu Glu Arg Arg Glu Arg Ser Pro Glu Gly Pro Arg Glu Glu 370 375 380 gaa gca gcg ggg ggc gaa gag gaa tec ccc gac age age cca cat ggg 1200 Glu Ala Ala Gly Gly Glu Glu Glu Ser Pro Asp Ser Ser Pro His Gly 385 390 395 400 gag gcc tec agg ggc gcc gcg gag cct gag gcc cag etc age aac cac 1248 Glu Ala Ser Arg Gly Ala Ala Glu Pro Glu Ala Gln Leu Ser Asn His 405 410 415 ctg gcc gag gag ggc ccc gcc gag ggt age ggc gag gcc gcg cgc gtg 1296 Leu Ala Glu Glu Gly Pro Ala Glu Gly Ser Gly Glu Ala Ala Arg Val 420 425 430 aac ggc cgc egg gag gac gga gag gcg tec gag ccc egg gcc ctg ggg 1344 Asn Gly Arg Arg Glu Asp Gly Glu Ala Ser Glu Pro Arg Ala Leu Gly 435 440 445 cag gag cac gac ate ace etc ttc gtc aag get ggt tat gat ggt gag 1392 Gln Glu H1S Asp Ile Thr Leu Phe Val Lys Ala Gly Tyr Asp Gly Glu 450 455 460 agt ate gga aat tgc ccg ttt tct cag cgt etc ttt atg att etc tgg 1440 Ser Ile Gly Asn Cys Pro Phe Ser Gln Arg Leu Phe Met Ile Leu Trp 465 470 475 480 ctg aaa ggc gtt ata ttt aat gtg ace aca gtg gac ctg aaa agt tct 1488 Leu Lys Gly Val Ile Phe Asn Val Thr Thr Val Asp Leu Lys Ser Ser 485 490 495 caa gag ttg ctt etc tta aac tgg aaa ccc gca gac ctg cag aac ctg 1536 Gln Glu Leu Leu Leu Leu Asn Trp Lys Pro Ala Asp Leu Gln Asn Leu 500 505 510 get ccc gga aca aac cct cct ttc atg act ttt gat ggt gaa gtc aag 1584

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L. 7 Ala Pro Gly Thr Asn Pro Pro Phe Met Thr Phe Asp Gly Glu Val Lys 515 520 525 acg gat gtg aat aag ate gag gag ttc tta gag gag aaa tta get ccc 1632 Thr Asp Val Asn Lys Ile Glu Glu Phe Leu Glu Glu Lys Leu Ala Pro 530 535 540 ccg agg tat ccc aag ctg ggg ace caa cat ccc gaa let aat tec gca 1680 Pro Arg Tyr Pro Lys Leu Gly Thr Gln His Pro Glu Ser Asn Ser Ala 545 550 555 560 gga aat gac gtg ttt gcc aaa ttc tea gcg ttt ata aaa aac acg aag 1728 Gly Asn Asp Val Phe Ala Lys Phe Ser Ala Phe Ile Lys Asn Thr Lys 565 570 575 aag gat gca aat gag att cat gaa aag aac ctg ctg aag gcc ctg agg 1776 Lys Asp Ala Asn Glu Ile His Glu Lys Asn Leu Leu Lys Ala Leu Arg 580 585 590 aag ctg gat aat tac tta aat age cct ctg cct gat gaa ata gat gcc 1824 Lys Leu Asp Asn Tyr Leu Asn Ser Pro Leu Pro Asp Glu Ile Asp Ala 595 600 605 tac age acc gag gat gtc act gtt tct gga agg aag ttt ctg gat ggg 1872 Tyr Ser Thr Glu Asp Val Thr Val Ser Gly Arg Lys Phe Leu Asp Gly 610 615 620 gac gag ctg acg ctg get gac tgc aac etc tta ccc aag etc cat att 1920 Asp Glu Leu Thr Leu Ala Asp Cys Asn Leu Leu Pro Lys Leu His Ile 625 630 635 640 att aag att gtg gcc aag aag tac aga gat ttt gaa ttt cct tct gaa 1968 Ile Lys Ile Val Ala Lys Lys Tyr Arg Asp Phe Glu Phe Pro Ser Glu 645 650 655 atg act ggc ate tgg aga tac ttg aat aat get tat get aga gat gag 2016 Met Thr Gly Ile Trp Arg Tyr Leu Asn Asn Ala Tyr Ala Arg Asp Glu 660 665 670 ttc aca aat acg tgt cca get gat caa gag att gaa cac gca tat tea 2064 Phe Thr Asn Thr Cys Pro Ala Asp Gln Glu Ile Glu His Ala Tyr Ser 675 680 685 gat gtt gca aaa aga atg aaa tga agctgggctg ttttctgtct tatttctcag 2118 Asp Val Ala Lys Arg Met Lys 690 695 ttgagtgagc aaggatacga aaacagtgtg tttgaaaaca aattaggttt gggttcaatt 2178 ccttcaattt ttaaaaaact ggtctctgag agttttttaa atcattgaga gcctgttttt 2238 cttctctaaa acattagttt aattttcttc aaaatgaaaa tactgctttg taattacaaa 2298 atgagacaca cctatcttga tattttaaag caatatcaga gggtgtnaag aaggacattt 2358 taacaatcgc cttcaatttt actccactta attaccgaaa acttactgga gaacatgttc 2418

<Desc/Clms Page number 30>

caaatcttca gtatcttgtt ctctctctct ctctctctct ctctctctct ctatcacaca 2478 cacacacaca cacacacaca cacaatttna ttcatatatg gtattgcatt attttatttt 2538 aaagcactgg ngaggggacc tcttggtgat tcctggatga tcatacacag aggacttaca 2598 ccatacaaaa atattgggcn ccgcagtgcc agagaagatg cttgaggtta gattttaaga 2658 agg 2661 < 210 > 2 < 211 > 695 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Ala Glu Ala Ala Glu Pro Glu Gly Val Ala Pro Gly Pro Gln Gly 1 5 10 15 Pro Pro Glu Val Pro Ala Pro Leu Ala Glu Arg Pro Gly Glu Pro Gly 20 25 30 Ala Ala Gly Gly Glu Ala Glu Gly Pro Glu Gly Ser Glu Gly Ala Glu 35 40 45 Glu Ala Pro Arg Gly Ala Ala Ala Val Lys Glu Ala Gly Gly Gly Gly 50 55 60 Pro Asp Arg Gly Pro Glu Ala Glu Ala Arg Gly Thr Arg Gly Ala His 65 70 75 80 Gly Glu Thr Glu Ala Glu Glu Gly Ala Pro Glu Gly Ala Glu Val Pro 85 90 95 Gln Gly Gly Glu Glu Thr Ser Gly Ala Gln Gln Val Glu Gly Ala Ser 100 105 110 Pro Gly Arg Gly Ala Gln Gly Glu Pro Arg Gly Glu Ala Gln Arg Glu 115 120 125 Pro Glu Asp Ser Ala Ala Pro Glu Arg Gln Glu Glu Ala Glu Gln Arg 130 135 140 Pro Glu Val Pro Glu Gly Ser Ala Ser Gly Glu Ala Gly Asp Ser Val 145 1 iso 155 160 Asp Ala Glu Gly Pro Leu Gly Asp Asn Ile Glu Ala Glu Gly Pro Ala 165 170 175 Gly Asp Ser Val Glu Ala Glu Gly Arg Val Gly Asp Ser Val Asp Ala 180 185 190 Glu Gly Pro Ala Gly Asp Ser Val Asp Ala Glu Gly Pro Leu Gly Asp 195 200 205 Asn Ile Gln Ala Glu Gly Pro Ala Gly Asp Ser Val Asp Ala Glu Gly

<Desc/Clms Page number 31>

210 215 220 Arg Val Gly Asp Ser Val Asp Ala Glu Gly Pro Ala Gly Asp Ser Val 225 230 235 240 Asp Ala Glu Gly Arg Val Gly Asp Ser Val Glu Ala Gly Asp Pro Ala 245 250 255 Gly Asp Gly Val Glu Ala Gly Val Pro Ala Gly Asp Ser Val Glu Ala 260 265 270 Glu Gly Pro Ala Gly Asp Ser Met Asp Ala Glu Gly Pro Ala Gly Arg 275 280 285 Ala Arg Arg Val Ser Gly Glu Pro Gln Gln Ser Gly Asp Gly Ser Leu 290 295 300 Ser Pro Gln Ala Glu Ala Ile Glu Val Ala Ala Gly Glu Ser Ala Gly 305 310 315 320 Arg Ser Pro Gly Glu Leu Ala Trp Asp Ala Ala Glu Glu Ala Glu Val 325 330 335 Pro Gly Val Lys Gly Ser Glu Glu Ala Ala Pro Gly Asp Ala Arg Ala 340 345 350 Asp Ala Gly Glu Asp Arg Val Gly Asp Gly Pro Gln Gln Glu Pro Gly 355 360 365 Glu Asp Glu Glu Arg Arg Glu Arg Ser Pro Glu Gly Pro Arg Glu Glu 370 375 380 Glu Ala Ala Gly Gly Glu Glu Glu Ser Pro Asp Ser Ser Pro His Gly 385 390 395 400 Glu Ala Ser Arg Gly Ala Ala Glu Pro Glu Ala Gln Leu Ser Asn His 405 410 415 Leu Ala Glu Glu Gly Pro Ala Glu Gly Ser Gly Glu Ala Ala Arg Val 420 425 430 Asn Gly Arg Arg Glu Asp Gly Glu Ala Ser Glu Pro Arg Ala Leu Gly 435 440 445 Gln Glu His Asp Ile Thr Leu Phe Val Lys Ala Gly Tyr Asp Gly Glu 450 455 460 Ser Ile Gly Asn Cys Pro Phe Ser Gln Arg Leu Phe Met Ile Leu Trp 465 470 475 480 Leu Lys Gly Val Ile Phe Asn Val Thr Thr Val Asp Leu Lys Ser Ser 485 490 495 Gln Glu Leu Leu Leu Leu Asn Trp Lys Pro Ala Asp Leu Gln Asn Leu 500 505 510 Ala Pro Gly Thr Asn Pro Pro Phe Met Thr Phe Asp Gly Glu Val Lys

<Desc/Clms Page number 32>

515 520 525

Thr Asp Val Asn Lys Ile Glu Glu Phe Leu Glu Glu Lys Leu Ala Pro 530 535 540 Pro Arg Tyr Pro Lys Leu Gly Thr Gln His Pro Glu Ser Asn Ser Ala 545 550 555 560 Gly Asn Asp Val Phe Ala Lys Phe Ser Ala Phe Ile Lys Asn Thr Lys 565 570 575 Lys Asp Ala Asn Glu Ile His Glu Lys Asn Leu Leu Lys Ala Leu Arg 580 585 590 Lys Leu Asp Asn Tyr Leu Asn Ser Pro Leu Pro Asp Glu Ile Asp Ala 595 600 605 Tyr Ser Thr Glu Asp Val Thr Val Ser Gly Arg Lys Phe Leu Asp Gly 610 615 620 Asp Glu Leu Thr Leu Ala Asp Cys Asn Leu Leu Pro Lys Leu His Ile 625 630 635 640

Ile Lys Ile Val Ala Lys Lys Tyr Arg Asp Phe Glu Phe Pro Ser Glu 645 650 655 Met Thr Gly Ile Trp Arg Tyr Leu Asn Asn Ala Tyr Ala Arg Asp Glu 660 665 670 Phe Thr Asn Thr Cys Pro Ala Asp Gln Glu Ile Glu H1S Ala Tyr Ser 675 680 685 Asp Val Ala Lys Arg Met Lys 690 695

Claims (17)

1. CLAIMS 1. An isolated chloride intracellular channel-related polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2 or (ii) a variant thereof which is capable of modulating chloride ion efflux across a membrane in response to a change in extracellular chloride ion concentration or (iii) a fragment of (i) or (ii) which is capable of modulating chloride ion efflux across a membrane in response to a change in extracellular chloride ion concentration.
2. 2. A polypeptide according to claim 1 wherein the variant (ii) has at least 80% identity to the amino acid sequence of SEQ ID NO: 2.
3. 3. A polynucleotide encoding a polypeptide according to claim 1 or 2.
4. 4. A polynucleotide according to claim 3 which is a cDNA sequence.
5. 5. A polynucleotide encoding a chloride intracellular channel-related polypeptide which is capable of modulating chloride ion efflux across a membrane in response to a change in extracellular chloride ion concentration, which polynucleotide comprises: (a) the nucleic acid sequence of SEQ ID NO: 1 and/or a sequence complementary thereto; (b) a sequence which hybridises under stringent conditions to a sequence as defined in (a); (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b); or (d) a sequence having at least 80% identity to a sequence as defined in (a), (b) or (c).
6. 6. An expression vector comprising a polynucleotide according to any one of claims 3 to 5.
7. 7. A host cell comprising an expression vector according to claim 6.
8. 8. An antibody specific for a polypeptide according to claim 1 or 2.
9. 9. A method for the identification of a substance that modulates chloride intracellular channel activity and/or expression, which method comprises:

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   (i) contacting a test substance and a polypeptide according to claim 1 or 2, a polynucleotide according to any one of claims 3 to 5, an expression vector according to claim 6 or a host cell according to claim 7, and (ii) determining the effect of the test substance on the activity and/or expression of the said polypeptide or the polypeptide encoded by said polynucleotide, thereby to determine whether the test substance modulates chloride intracellular channel activity and/or expression.
10. 10. A method according to claim 9 wherein the polypeptide is expressed in a cell.
11. 11. A method according to claim 9 or 10 wherein step (ii) comprises monitoring any chloride ion channel activity.
12. 12. A substance which modulates chloride intracellular channel activity and which is identifiable by a method according to any one of claims 9 to 11.
13. 13. A method of treating a subject having a disorder that is responsive to chloride intracellular channel modulation, which method comprises administering to said subject an effective amount of a substance according to claim 12.
14. 14. A method according to claim 13 wherein the disorder is selected from asthma, bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, respiratory distress syndrome, diabetes mellitus type II, hypoglycemia, obesity, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, constipation, Crohn's disease, diarrhea, hyperthyroidism, hypothyroidism, inflammatory bowel disease, irritable bowel syndrome, amenorrhea and urinary incontinence.
15. 15. Use of a substance as defined in claim 12 in the manufacture of a medicament for treatment or prophylaxis of a disorder that is responsive to stimulation or modulation of chloride intracellular channel activity.
16. 16. A use according to claim 15 wherein the disorder is selected from asthma, bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, respiratory distress syndrome, diabetes mellitus type II, hypoglycemia, obesity, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, constipation, Crohn's disease, diarrhea, hyperthyroidism, hypothyroidism, inflammatory bowel disease, irritable bowel syndrome, amenorrhea and urinary incontinence.

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17. 17. A method of producing a polypeptide according to claim 1 or 2, which method comprises maintaining a host cell as defined in claim 7 under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide.