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WO2002081689A1 - Regulation de la sous-unite ocnc2 humaine du canal a porte nucleotidique cyclique - Google Patents

Regulation de la sous-unite ocnc2 humaine du canal a porte nucleotidique cyclique Download PDF

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WO2002081689A1
WO2002081689A1 PCT/EP2002/001726 EP0201726W WO02081689A1 WO 2002081689 A1 WO2002081689 A1 WO 2002081689A1 EP 0201726 W EP0201726 W EP 0201726W WO 02081689 A1 WO02081689 A1 WO 02081689A1
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ocnc2
subunit
gated channel
polypeptide
nucleotide
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Zhimin Zhu
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Bayer AG
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Bayer AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention relates to the regulation of human cyclic-nucleotide-gated channel OCNC2 subunit.
  • Ion channels are integral membrane proteins, typically comprising multiple subunits, which form selective and highly regulated pores in cellular membranes. Each of these pores controls the influx and efflux of a given ion ⁇ e.g., sodium, potassium, calcium, or chloride) across the plasma membrane or the membranes of intracellular compartments.
  • ion e.g., sodium, potassium, calcium, or chloride
  • Many important physiological processes depend on the control of ion gradients by ion channels. Such processes include synaptic transmission, secretion, fertilization, muscle contraction, and regulation of intracellular and extracellular ion concentrations and pH.
  • Ion channels open in response to various stimuli. For example, there are ligand-gated channels, second messenger-gated channels, voltage- gated channels, and shear- or stress-gated channels.
  • Certain channels allow ions to leak across membranes without a specific stimulus.
  • the gating properties characteristic of a given channel include the period of time it is open, the frequency of opening, the strength of stimulus required for activation, and the refractory period. These characteristics can vary depending on the subunit composition of the channel, association of the channel with accessory proteins, and phosphorylation or other post-translational modification of channel polypeptides. See, e.g., U.S. Patent 6,071,720. Cyclic nucleotide-gated channels
  • Cyclic nucleotide-gated channels are present in a variety of cell types and take part in the regulation of a wide range of physiological phenomena. They are especially well known for their roles in visual and olfactory signal transduction, but are also involved in neuronal development and plasticity, renal transport of sodium and calcium, and cardiac pacemaker function.
  • the expression of cyclic nucleotide-gated channels has been found in retinal rods and cones, olfactory neuroepithelium, pineal gland, heart, aorta, kidney, and testis. D.E. McCoy et al., Kidney Int. 4:1125-33 (1995). Cyclic nucleotide-gated channels are found in invertebrates ⁇ e.g., C. elegans and Drosophil ⁇ ) as well as vertebrates, including humans.
  • cyclic nucleotide-activated channels are considered ligand-gated channels, they are structurally homologous to voltage-gated channels. Cyclic nucleotide-gated channels form heteromultimers comprising homologous alpha and beta subunits.
  • alpha and beta subunits contain a central pore, a structural unit consisting of six transmembrane segments, and a cyclic nucleotide binding domain.
  • a family of genes has been identified which encode the alpha and beta subunits. M. Biel et al., Naunyn. Schmied. Arch. Pharmacol. 353:1-10 (1995). These channels are typically activated
  • calmodulin ⁇ i.e., gated) by cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP).
  • cAMP cyclic adenosine monophosphate
  • cGMP cyclic guanosine monophosphate
  • binding of calmodulin provides further activation by modulating the sensitivity of the channels to cychc nucleotides.
  • Cychc nucleotide-gated channels are typically found in the plasma membrane of cells, where they permit the entry of sodium and calcium ions.
  • cyclic nucleotide-gated channels link light-driven signal transduction events, which modulate cGMP levels, to neuronal signals.
  • the channels are thought to be involved in the initial stages of odor adaptation.
  • cardiac pacemaker cells they are thought to produce the 1(f) current, which is also activated by hyperpolarization.
  • cyclic nucleotide-gated channels provide a variety of functional linkages between stimuli involving G protein-coupled receptors, calcium signaling mechanisms, and nitric oxide and responses involving ion transport or electrical excitability.
  • One embodiment of the invention is a cyclic nucleotide-gated channel OCNC2 subunit polypeptide comprising an amino acid sequence selected &om the group consisting of: amino acid sequences which are at least about 94% identical to the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence shown in SEQ ID NO: 2; amino acid sequences which are at least about 94% identical to the amino acid sequence shown in SEQ ID NO: 5 and; the amino acid sequence shown in SEQ ID NO:5;
  • Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a cyclic nucleotide-gated channel OCNC2 subunit polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 94%> identical to the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence shown in SEQ ED NO: 2; amino acid sequences which are at least about 94% identical to the amino acid sequence shown in SEQ ID NO: 5 and; the amino acid sequence shown in SEQ ID NO:5;
  • Binding between the test compound and the cyclic nucleotide-gated channel OCNC2 subunit polypeptide is detected.
  • a test compound which binds to the cyclic nucleotide-gated channel OCNC2 subunit polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the activity of the cyclic nucleotide-gated channel OCNC2 subunit.
  • Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a polynucleotide encoding a cyclic nucleotide-gated channel OCNC2 subunit polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence shown in SEQ ID NO: 1; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ED NO: 4 and; the nucleotide sequence shown in SEQ ID NO: 4;
  • a test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the amount of the cyclic nucleotide-gated channel OCNC2 subunit through interacting with the cyclic nucleotide-gated channel OCNC2 subunit mRNA.
  • Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation.
  • a test compound is contacted with a cyclic nucleotide-gated channel OCNC2 subunit polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 94% identical to the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence shown in SEQ ID NO: 2; amino acid sequences which are at least about 94% identical to the amino acid sequence shown in SEQ ID NO: 5 and; the amino acid sequence shown in SEQ ID NO:5;
  • a cyclic nucleotide-gated channel OCNC2 subunit activity of the polypeptide is detected.
  • a test compound which increases cyclic nucleotide-gated channel OCNC2 subunit activity of the polypeptide relative to cyclic nucleotide-gated channel OCNC2 subunit activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation.
  • a test compound which decreases cyclic nucleotide-gated channel OCNC2 subunit activity of the polypeptide relative to cyclic nucleotide-gated channel OCNC2 subunit activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • a test compound is contacted with a cyclic nucleotide-gated channel OCNC2 subunit product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1 ; the nucleotide sequence shown in SEQ ID NO: 1; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 4 and; the nucleotide sequence shown in SEQ ID NO: 4;
  • Binding of the test compound to the cyclic nucleotide-gated channel OCNC2 subunit product is detected.
  • a test compound which binds to the cyclic nucleotide-gated channel OCNC2 subunit product is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • Still another embodiment of the invention is a method of reducing extracellular matrix degradation.
  • a cell is contacted with a reagent which specifically binds to a polynucleotide encoding a cyclic nucleotide-gated channel OCNC2 subunit polypeptide or the.
  • the polynucleotide comprises a nucleotide sequence selected from the group consisting of: nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence shown in SEQ ID NO: 1; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 4 and; the nucleotide sequence shown in SEQ ID NO: 4;
  • Cyclic nucleotide-gated channel OCNC2 subunit activity in the cell is thereby decreased.
  • the invention thus provides a human cyclic-nucleotide-gated channel OCNC2 subunit that can be used to identify test compounds that may act, for example, as activators or inhibitors of the channel.
  • Human cyclic-nucleotide-gated channel OCNC2 subunit and fragments thereof also are useful in raising specific antibodies that can block the channel and effectively reduce its activity.
  • Fig. 1 shows the DNA-sequence encoding a cyclic nucleotide- gated channel OCNC2 subunit Polypeptide (SEQ ID NO:l).
  • Fig. 2 shows the amino acid sequence deduced from the DNA- sequence of Fig.l (SEQ ID NO:2).
  • Fig. 3 shows the amino acid sequence of a protein identified by swiss
  • Fig. 4 shows the DNA-sequence encoding a cyclic nucleotide- gated channel OCNC2 subunit Polypeptide (SEQ ID NO: 1
  • Fig. 5 shows the amino acid sequence deduced from the DNA- sequence of Fig. 4 (SEQ ID NO:5).
  • Fig. 6 shows the BLASTP - alignment of 341_protein (SEQ ID NO:2) against swiss
  • Fig. 7 shows the HMMPFAM - alignment of 341_protein (SEQ ID NO:2) against ⁇ fam
  • Fig. 8 shows the HMMPFAM - alignment of 341_protein (SEQ ID NO:2) against ⁇ fam
  • Fig. 9 shows the Prosite and BLOCKS search results.
  • Fig. 10 shows the Genewise output.
  • Fig. 11 shows the TMHMM result.
  • Fig. 12 shows the BLASTP - alignment of 341 jprotein against trembl
  • Fig. 13 shows the BLASTP - alignment of LBRI_341_v2j_rotein against swiss
  • Fig 14 shows the Expression of human cyclic-nucleotide-gated channel OCNC2 subunit (LBRI#341) in cardiovascular tissues.
  • Fig 15 shows the Expression of human cyclic-nucleotide-gated channel OCNC2 subunit (LBRI#341) in CNS tissues.
  • Fig 16 shows the Expression of human cyclic-nucleotide-gated channel OCNC2 subunit (LBRI#341) in various human organs.
  • the invention relates to an isolated polynucleotide from the group consisting of:
  • a novel cyclic- nucleotide-gated channel OCNC2 subunit particularly a human cyclic-nucleotide- gated channel OCNC2 subunit, can be used in therapeutic methods to treat a cardiovascular disorder or CNS disorder.
  • OCNC2 subunit comprises the amino acid sequence shown in SEQ ID NO:2.
  • a coding sequence for human cyclic-nucleotide-gated channel OCNC2 subunit is shown in SEQ ID NO:l. This sequence is located on chromosome 11.
  • Identification of the protein comprising SEQ ID NO:2 as a human ortholog of rat olfactory cyclic-nucleotide-gated channel OCNC2 subunit is based on its high degree of identity to swiss
  • PFAM search reveals that this protein contains transmembrane region for cyclic nucleotide gated channel and cyclic nucleotide-binding domain.
  • Prosite and BLOCK searches further indicate that this protem is a cyclic-nucleotide-gated channel.
  • Human cyclic-nucleotide-gated channel OCNC2 subunit of the invention is expected to be useful for the same purposes as previously identified cyclic nucleotide-gated ion channels. Human cyclic-nucleotide-gated channel OCNC2 subunit is believed to be useful in therapeutic methods to treat disorders such as cardiovascular disorders.
  • Human cyclic-nucleotide-gated channel OCNC2 subunit also can be used to screen for human cyclic-nucleotide-gated channel OCNC2 subunit activators and inhibitors.
  • Human cyclic-nucleotide-gated channel OCNC2 subunit polypeptides according to the invention comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225,
  • a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide of the invention therefore can be a portion of a cyclic-nucleotide-gated channel OCNC2 subunit protein, a full-length cyclic- nucleotide-gated channel OCNC2 subunit protein, or a fusion protein comprising all or a portion of a cyclic-nucleotide-gated channel OCNC2 subunit protein.
  • cyclic-nucleotide-gated channel OCNC2 subunit polypeptide variants have amino acid sequences which are at least about 94, 96, 96, 98, or 99% identical to the amino acid sequence shown in SEQ ID NO:2 or a fragment thereof. Percent identity between a putative cyclic-nucleotide- gated channel OCNC2 subunit polypeptide variant and an amino acid sequence of SEQ ID NO:2 is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes).
  • Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions.
  • Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can readily be determined by assaying for binding to a ligand or by conducting a functional assay, as described for example, in the Functional Assays section, below.
  • Fusion proteins are useful for generating antibodies against cyclic-nucleotide-gated channel OCNC2 subunit polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide. Protein affinity chromatography or library-based assays for protein- protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment comprises at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400, 425, 450, 475, 500, 525, 550, or 575 contiguous amino acids of SEQ ID NO:2 or of a biologically active variant, such as those described above.
  • the first polypeptide segment also can comprise full-length cyclic-nucleotide-gated channel OCNC2 subunit protein.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include ⁇ -galactosidase, ⁇ - glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-
  • G tags and thioredoxin (Trx) tags.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSN) BP16 protein fusions.
  • MBP maltose binding protein
  • S-tag S-tag
  • GAL4 DNA binding domain fusions GAL4 DNA binding domain fusions
  • HSN herpes simplex virus
  • a fusion protein also can be engineered to contain a cleavage site located between the cyclic-nucleotide-gated channel OC ⁇ C2 subunit polypeptide-encoding sequence and the heterologous protein sequence, so that the cyclic-nucleotide-gated channel
  • OCNC2 subunit polypeptide can be cleaved and purified away from the heterologous moiety.
  • a fusion protein can be synthesized chemically, as is known in the art.
  • a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology.
  • Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO:l in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art.
  • Many kits for constructing fusion proteins are available from companies such as Promega Corporation
  • Species homologs of human cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can be obtained using cyclic-nucleotide-gated channel OCNC2 subunit polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of cyclic-nucleotide-gated channel OCNC2 subunit polypeptide, and expressing the cDNAs as is known in the art.
  • Polynucleotides described below
  • a cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide.
  • a coding sequence for human cyclic-nucleotide-gated channel OCNC2 subunit is shown in SEQ ID NO: 1.
  • nucleotide sequences encoding human cyclic-nucleotide-gated channel OCNC2 subunit polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 9$, or 99% identical to the nucleotide sequence shown in SEQ ID NO:l or its complement also are cyclic- nucleotide-gated channel OCNC2 subunit polynucleotides.
  • Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2.
  • cDNA Complementary DNA
  • species homologs, and variants of cyclic- nucleotide-gated channel OCNC2 subunit polynucleotides that encode biologically, active cyclic-nucleotide-gated channel OCNC2 subunit polypeptides also are cyclic- nucleotide-gated channel OCNC2 subunit polynucleotides.
  • Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ ID NO:l or its complement also are cyclic-nucleotide-gated channel OCNC2 subunit polynucleotides. These fragments can be used, for example, as hybridization probes or as antisense oligonucleotides.
  • Variants and homologs of the cyclic-nucleotide-gated channel OCNC2 subunit polynucleotides described above also are cyclic-nucleotide-gated channel OCNC2 subunit polynucleotides.
  • homologous cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known cyclic-nucleotide-gated channel OCNC2 subunit polynucleotides under stringent conditions, as is known in the art.
  • homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25%o basepair mismatches, even more preferably 5-15% basepair mismatches.
  • Species homologs of the cyclic-nucleotide-gated channel OCNC2 subunit polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast.
  • Human variants of cyclic-nucleotide-gated channel OCNC2 subunit polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T m of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al, J. Mol Biol 81, 123 (1973).
  • Variants of human cyclic-nucleotide-gated channel OCNC2 subunit polynucleotides or cyclic-nucleotide-gated channel OCNC2 subunit polynucleotides of other species can therefore be identified by hybridizing a putative homologous cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO:l or the complement thereof to form a test hybrid.
  • the melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.
  • Nucleotide sequences which hybridize to cyclic-nucleotide-gated channel OCNC2 subunit polynucleotides or their complements following stringent hybridization and/or wash conditions also are cyclic-nucleotide-gated channel OCNC2 subunit polynucleotides.
  • Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.
  • T m of a hybrid between a cyclic- nucleotide-gated channel OCNC2 subunit polynucleotide having a nucleotide sequence shown in SEQ ID NO:l or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl Acad. Sci. U.S.A.
  • Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% formamide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65 °C.
  • Highly stringent wash conditions include, for example, 0.2X SSC at 65 °C.
  • a cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated cyclic- nucleotide-gated channel OCNC2 subunit polynucleotides.
  • restriction enzymes and probes can be used to isolate polynucleotide fragments, which comprise cyclic-nucleotide-gated channel OCNC2 subunit nucleotide sequences.
  • Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.
  • Human cyclic-nucleotide-gated channel OCNC2 subunit cDNA molecules can be made with standard molecular biology techniques, using cyclic-nucleotide-gated channel OCNC2 subunit mRNA as a template. Human cyclic-nucleotide-gated channel OCNC2 subunit cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.
  • cyclic- nucleotide-gated channel OCNC2 subunit polynucleotides can be synthesized using synthetic chemistry techniques to synthesize cyclic- nucleotide-gated channel OCNC2 subunit polynucleotides.
  • the degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide having, for example, an amino acid sequence shown in SEQ ID NO:2 or a biologically active variant thereof.
  • PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate R A polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al, Nucleic Acids Res. 16, 8186, 1988).
  • Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72 °C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al, PCR Methods Applic. 1, 111-119, 1991).
  • multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) that are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity can be converted to electrical signal using appropriate software ⁇ e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample.
  • Human cyclic-nucleotide-gated channel OCNC2 subunit polypeptides can be obtained, for example, by purification from human cells, by expression of cyclic- nucleotide-gated channel OCNC2 subunit polynucleotides, or by direct chemical synthesis.
  • Human cyclic-nucleotide-gated channel OCNC2 subunit polypeptides can be purified from any cell that expresses the polypeptide, including host cells that have been transfected with cyclic-nucleotide-gated channel OCNC2 subunit expression constructs.
  • a purified cyclic-nucleotide-gated channel OCNC2 subunit polypeptide is separated from other compounds that normally associate with the cyclic- nucleotide-gated channel OCNC2 subunit polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art.
  • Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography,. and preparative gel electrophoresis.
  • a preparation of purified cyclic-nucleotide-gated channel OCNC2 subunit polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.
  • the polynucleotide can be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding cyclic-nucleotide-gated channel OCNC2 subunit polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors ⁇ e.g., baculo virus), plant cell systems transformed with virus expression vectors ⁇ e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors ⁇ e.g., Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors ⁇ e.g., baculo virus
  • control elements or regulatory sequences are those non-translated regions of the vector ⁇ enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, can be used.
  • inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used.
  • the baculovirus polyhedrin promoter can be used in insect cells.
  • Promoters or enhancers derived from the genomes of plant cells can be cloned into the vector.
  • promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected depending upon the use intended for the cyclic-nucleotide-gated channel OCNC2 subunit polypeptide.
  • vectors which direct high level expression of fusion proteins that are readily purified can be used.
  • Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUES CRIPT (Stratagene).
  • BLUES CRIPT a sequence encoding the cyclic-nucleotide-gated channel
  • OCNC2 subunit polypeptide can be ligated into the vector in frame with sequences for the ammo-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced.
  • pBST vectors Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989
  • pGEX vectors Promega, Madison, Wis.
  • GST GST
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
  • Lf plant expression vectors are used, the expression of sequences encoding cyclic- nucleotide-gated channel OCNC2 subunit polypeptides can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Taka atsu, EMBO J. 6, 307-311, 1987).
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al, EMBO J. 3, 1671-1680, 1984; Broglie et al, Science 224,
  • constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews ⁇ e.g., Hobbs or Murray, inMcGRAW HELL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).
  • An insect system also can be used to express a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • Sequences encoding cyclic-nucleotide-gated channel OCNC2 subunit polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
  • cyclic-nucleotide-gated channel OCNC2 subunit polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which cyclic- nucleotide-gated channel OCNC2 subunit polypeptides can be expressed (Engelhard et al, Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).
  • Mammalian Expression Systems A number of viral-based expression systems can be used to express cyclic- nucleotide-gated channel OCNC2 subunit polypeptides in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding cyclic- nucleotide-gated channel OCNC2 subunit polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence.
  • Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus that is capable of expressing a cyclic- nucleotide-gated channel OCNC2 subunit polypeptide in infected host cells (Logan & Shenk, Proc. Nail. Acad. Sci. 81, 3655-3659, 1984).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • 6M to 10M are constructed and delivered to cells via conventional delivery methods ⁇ e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding cyclic-nucleotide-gated channel OCNC2 subunit polypeptides.
  • Such signals include the ATG initiation codon and adjacent sequences.
  • sequences encoding a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert.
  • Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic.
  • the efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al, Results Probl Cell Differ. 20, 125- 162, 1994).
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed cyclic-nucleotide-gated channel OCNC2 subunit polypeptide in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, hpidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture
  • Stable expression is preferred for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express cyclic-nucleotide-gated channel OCNC2 subunit polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced cyclic-nucleotide- gated channel OCNC2 subunit sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • herpes simplex virus thymidine kinase include, but are not limited to, the herpes simplex virus thymidine kinase
  • dhfr confers resistance to methotrexate (Wigler et al, Proc. Natl Acad. Sci. 77, 3567-70, 1980)
  • npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al, J. Mol.
  • trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl Acad. Sci. 85, 8047-51, 1988).
  • Visible markers such as anthocyanins, ⁇ -glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, Methods Mol. Biol 55, 121-131, 1995).
  • marker gene expression suggests that the cyclic-nucleotide- gated channel OCNC2 subunit polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a cyclic- nucleotide-gated channel OCNC2 subunit polypeptide is inserted within a marker gene sequence, transformed cells containing sequences that encode a cyclic- nucleotide-gated channel OCNC2 subunit polypeptide can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the cyclic- nucleotide-gated channel OCNC2 subunit polynucleotide.
  • host cells which contain a cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide and which express a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques that include membrane, solution, or chip-based technologies for the detection and or quantification of nucleic acid or protein.
  • the presence of a polynucleotide sequence encoding a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide.
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide to detect transformants that contain a cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide.
  • a variety of protocols for detecting and measuring the expression of a cyclic- nucleotide-gated channel OCNC2 subunit polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can be used, or a competitive binding assay can be employed.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding cyclic-nucleotide-gated channel OCNC2 subunit polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding a cyclic- nucleotide-gated channel OCNC2 subunit polypeptide can be cloned into a vector for the production of an mRNA probe.
  • RNA probes are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode cyclic-nucleotide- gated channel OCNC2 subunit polypeptides can be designed to contain signal sequences which direct secretion of soluble cyclic-nucleotide-gated channel OCNC2 subunit polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound cyclic-nucleotide-gated channel
  • purification facilitating domains include, but are not hrnited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension affinity purification system (Immunex Corp., Seattle, Wash.).
  • cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the cyclic-nucleotide-gated channel OCNC2 subunit polypeptide also can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing a cyclic- nucleotide-gated channel OCNC2 subunit polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by DVIAC (immobilized metal ion affinity chromatography, as described in Porath et al, Prot. Exp.
  • enterokinase cleavage site provides a means for purifying the cyclic-nucleotide-gated channel OCNC2 subunit polypeptide from the fusion protein.
  • Vectors that contain fusion proteins are disclosed in Kroll et al, DNA Cell Biol. 12, 441-453, 1993.
  • Sequences encoding a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl
  • a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • fragments of cyclic-nucleotide-gated channel OCNC2 subunit polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, NT., 1983).
  • the composition of a synthetic cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra).
  • any portion of the amino acid sequence of the cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein. Production of Altered Polypeptides
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life that is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter cyclic-nucleotide-gated channel OCNC2 subunit polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic ohgonucleotides can be used to engineer the nucleotide sequences.
  • site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • Antibody as used herein includes intact i ⁇ imunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding an epitope of a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide.
  • Fab fragment antigen binding protein
  • F(ab') 2 fragment antigen binding protein
  • Fv fragment antigen binding protein
  • An antibody which specifically binds to an epitope of a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen.
  • an antibody which specifically binds to a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to cyclic- nucleotide-gated channel OCNC2 subunit polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide from solution.
  • Human cyclic-nucleotide-gated channel OCNC2 subunit polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response.
  • a carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • various adjuvants can be used to increase the immunological response.
  • Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels ⁇ e.g., aluminum hydroxide), and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).
  • surface active substances e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG ⁇ bacilli Calmette-Gueri ⁇ and Corynebacterium parvum are especially useful.
  • Monoclonal antibodies that specifically bind to a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al, Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al, Nature 314, 452-454, 1985).
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues.
  • rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • humanized antibodies can be produced using recombinant methods, as described in GB2188638B.
  • Antibodies that specifically bind to a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies that specifically bind to cyclic-nucleotide-gated channel OCNC2 subunit polypeptides.
  • Antibodies with related specificity, but of distinct idiotypic composition can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl Acad. Sci. 88, 11120-23, 1991).
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al, 1996, Eur. J.
  • Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tefravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Noss, 1994, J Biol Chem. 269, 199-206.
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated- nucleotide synthesis, cloned into an expression construct using standard recombinant D ⁇ A methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology (Nerhaar et al, 1995, Int. J. Cancer 61, 497-501; ⁇ icholls et al, 1993, J Immunol. Meth. 165, 81- 91).
  • Antibodies which specifically bind to cyclic-nucleotide-gated channel OC ⁇ C2 subumt polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).
  • antibodies can be constructed and used therapeutically in methods of the invention.
  • chimeric antibodies can be constructed as disclosed in WO 93/03151.
  • Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.
  • Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of cyclic-nucleotide-gated channel OCNC2 subunit gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol 26, 1-72, 1994; Uhlmann et al, Chem. Rev. 90, 543-583, 1990.
  • Modifications of cyclic-nucleotide-gated channel OCNC2 subunit gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of the cyclic-nucleotide-gated channel OCNC2 subunit gene.
  • Ohgonucleotides derived from the transcription initiation site e.g., between positions -10 and +10 from the start site, are preferred.
  • inhibition can be achieved using "triple helix" base-pairing methodology..
  • Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g.,
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent cyclic-nucleotide-gated channel OCNC2 subumt nucleotides, can provide sufficient targeting specificity for cyclic-nucleotide-gated channel OCNC2 subunit mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non- complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular cyclic-nucleotide- gated channel OCNC2 subunit polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol 10,
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin.
  • Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673).
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art ⁇ see Haseloff et al. Nature 334, 585-591, 1988).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).
  • Specific ribozyme cleavage sites within a cychc-nucleotide-gated channel OCNC2 subunit RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC.
  • RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable.
  • Suitability of candidate cyclic-nucleotide-gated channel OCNC2 subunit RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target.
  • the hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated fransfection, elecfroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease cyclic-nucleotide-gated channel OCNC2 subunit expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • ribozymes can be engineered so that ribozyme expression will occur in response to factors that induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells. Differentially Expressed Genes
  • genes whose products interact with human cyclic-nucleotide-gated channel OCNC2 subunit may represent genes that are differentially expressed in disorders including, but not limited to, cardiovascular disorders. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human cyclic-nucleotide-gated channel OCNC2 subunit gene or gene product may itself be tested for differential-expression.
  • the degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques.
  • standard characterization techniques such as differential display techniques.
  • Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.
  • RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utihzed for the purification of such RNA samples. See, for example, Ausubel et al, ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Patent 4,843,155.
  • Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc. Natl Acad. Sci. U A. 85, 208-12, 1988), subrractive hybridization (Hedrick et al, Nature 308, 149-53; Lee et al., Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), and, preferably, differential display (Liang & Pardee, Science 257, 967-71, 1992; U.S. Patent 5,262,311).
  • the differential expression information may itself suggest relevant methods for the treatment of disorders involving the human cyclic-nucleotide-gated channel OCNC2 subunit.
  • treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human cyclic-nucleotide- gated channel OCNC2 subunit.
  • the differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human cyclic-nucleotide-gated channel OCNC2 subunit gene or gene product are up-regulated or down-regulated.
  • the invention provides assays for screening test compounds that bind to or modulate the activity of a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide or a cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide.
  • a test compound preferably binds to a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide or polynucleotide. More preferably, a test compound decreases or increases a functional activity of the protein by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
  • Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity.
  • the compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not hmited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.
  • High Throughput Screening Test compounds can be screened for the ability to bind to cyclic-nucleotide-gated channel OCNC2 subunit polypeptides or polynucleotides or to affect cyclic- nucleotide-gated channel OCNC2 subunit activity or cyclic-nucleotide-gated channel OCNC2 subunit gene expression using high throughput screening.
  • high throughput screening many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
  • the most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 ⁇ l.
  • many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.
  • free format assays or assays that have no physical barrier between samples, can be used.
  • an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl Acad. Sci. U.S.A. 19, 1614-18 (1994).
  • the cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose.
  • the combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
  • Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UN-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.
  • test samples are placed in a porous matrix.
  • One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • the test compound is preferably a small molecule that binds to the cyclic-nucleotide-gated channel OCNC2 subunit polypeptide, such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • either the test compound or the cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • Detection of a test compound that is bound to the cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can then be accomplished, for example, by direct counting of radioe mission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • OCNC2 subunit polypeptide can be determined without labeling either of the interactants.
  • a microphysiometer can be used to detect binding of a test compound with a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • a microphysiometer is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide (McConnell et al, Science 257, 1906-1912, 1992).
  • BIA Bimolecular Interaction Analysis
  • a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232,
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • polynucleotide encoding a cyclic-nucleotide-gated channel OCNC2 subumt polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence that encodes an unidentified protein (“prey” or "sample” can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey” proteins are able to interact in vivo to form an protein-dependent complex, the
  • DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein that interacts with the cyclic-nucleotide-gated channel OCNC2 subunit polypeptide.
  • a reporter gene e.g., LacZ
  • either the cyclic-nucleotide-gated channel OCNC2 subunit polypeptide (or polynucleotide) or the test compound can be bound to a solid support.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • any method known in the art can be used to attach the polypeptide (or polynucleotide) or te.st compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • the cyclic-nucleotide-gated channel OCNC2 subunit polypeptide is a fusion protein comprising a domain that allows the cyclic-nucleotide-gated channel OCNC2 subunit polypeptide to be bound to a solid support.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
  • a cychc- nucleotide-gated channel OCNC2 subunit polypeptide or polynucleotide
  • a test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated cyclic-nucleotide-gated channel OCNC2 subunit polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies which specifically bind to a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • GST-immobilized complexes include immunodetection of complexes using antibodies which specifically bind to the cychc-nucleotide-gated channel OCNC2 subunit polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the cyclic-nucleotide-gated channel OCNC2 subunit polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Screening for test compounds which bind to a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide or polynucleotide can be used in a cell-based assay system. A cyclic- nucleotide-gated channel OCNC2 subunit polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide or polynucleotide is determined as described above.
  • Test compounds can be tested for the ability to increase or decrease a biological effect of a cyclic nucleotide-gated channel-like polypeptide. Such biological effects can be determined for example using the functional assays described in the specific examples, below. Functional assays can be carried out after contacting either a purified cyclic nucleotide-gated channel-like polypeptide, a cell membrane preparation, or an intact cell with a test compound.
  • a test compound which decreases a functional activity of a cyclic nucleotide-gated channel by at least about 10, preferably about.50, more preferably about 75, 90, or 100% is identified as a potential agent for decreasing cyclic nucleotide-gated channel activity.
  • preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing cyclic nucleotide-gated channel-like activity.
  • the activity of cyclic nucleotide-gated channels in cells can be determined, for example, by loading the cells with a calcium-sensitive fluorescent indicator.
  • Cyclic nucleotide-gated channels can be activated by addition of a membrane permeating cyclic nucleotide derivative, e.g., dibutyryl cGMP, to the extracellular medium at a concentration such as 1 mM which is sufficient to activate the channels.
  • Fluorescent calcium indicators such as Fluo-3 can be loaded into cells in 96-well plates or another container, and the activity of cychc nucleotide-gated channels in the presence or absence of various test compounds can be simply and rapidly determined. See, e.g., U.S. Patent 6,057,114.
  • the luminescent protem aequorin can also be used as an indicator for changes in intracellular calcium ion concentration.
  • Cellular assays can also be designed where the Na + or Ca 2+ flux through the target channel itself is measured through its effects on membrane potential. Ion channel currents result in changes of electrical membrane potential (V m ) which can be monitored directly using potentiometric fluorescent probes. These electrically charged indicators (e.g., the anionic oxonol dye DiBAC 4 (3)) redistribute between extra- and intracellular compartment in response to voltage changes. The equilibrium distribution is governed by the Nernst-equation. Thus, changes in membrane potential results in concomitant changes in cellular fluorescence. Again, changes in V m might be caused directly by the activity of the target ion channel or through amplification and/or prolongation of the signal by channels co-expressed in the same cell.
  • V m electrical membrane potential
  • Target channel activity can cause cellular Ca 2+ entry either directly or through activation of additional Ca 2+ channels.
  • the resulting intracellular Ca 2+ signals regulate a variety of cellular responses, e.g. secretion or gene transcription. Therefore, modulation of the target channel can be detected by monitoring secretion of a known hormone/transmitter from the target-expressing cell or through expression of a reporter gene (e.g. luciferase) controlled by a Ca 2+ -responsive promoter element (e.g. cyclic AMP/ Ca 2+ -responsive elements (CRE)).
  • a reporter gene e.g. luciferase
  • a Ca 2+ -responsive promoter element e.g. cyclic AMP/ Ca 2+ -responsive elements (CRE)
  • cyclic nucleotide-gated channels Another approach to determining the activity of cyclic nucleotide-gated channels involves the electrophysiological determination of the corresponding channel currents.
  • Cells which endogenously express a particular cyclic nucleotide-gated channel can be used to study the effects of various test compounds or cyclic nucleotide-gated channel-like polypeptides on endogenous ionic currents attributable to the activity of cyclic nucleotide-gated channels.
  • cells which do not express a particular cychc nucleotide-gated channel can be employed as hosts for the expression of that channel, whose activity can then be studied by electrophysiological or other means.
  • Cells preferred as host cells for the heterologous expression of cychc nucleotide-gated channels are preferably mammalian cells such as COS cells, mouse L cells, CHO cells (e.g., DG44 cells), human embryonic kidney cells (e.g., HEK293 cells), African green monkey cells and the like; amphibian cells, such as Xenopus l ⁇ evis oocytes; or cells of yeast such as S. cerevisi ⁇ e or P. p ⁇ storis. See, e.g., U.S. Patent 5,876,958.
  • Electrophysiological procedures for measuring the current across a cell membrane are well known.
  • a preferred method is the use of a voltage clamp as in the whole-cell patch clamp technique.
  • Currents from other channel types can be eliminated by established methods so as to isolate the ionic current flowing through cyclic nucleotide-gated channels. See, e.g., U.S. Patent 5,876,958.
  • a further activity of cyclic nucleotide-gated channels which can be assessed is their ability to bind various ligands, including test compounds or cyclic nucleotide-gated channel-like polypeptides.
  • the ability of a test compound to bind cyclic nucleotide- gated channel polypeptides or fragments thereof may be determined by any appropriate competitive binding analysis (e.g., Scatchard plots), wherein the binding capacity and/or affinity is determined in the presence and absence of one or more concentrations a compound having known affinity for the cyclic nucleotide-gated channel.
  • Binding assays can be performed using whole cells which express cyclic nucleotide-gated channels (either endogenously or heterologously), membranes prepared from such cells, or purified cyclic nucleotide-gated channel polypeptides.
  • test compounds that increase or decrease cyclic-nucleotide- gated channel OCNC2 subunit gene expression are identified.
  • a cychc-nucleotide- gated channel OCNC2 subunit polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test, compound can then be identified as a modulator of expression based on this comparison.
  • test compound when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
  • test compound when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of cyclic-nucleotide-gated channel OCNC2 subunit mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • the presence of polypeptide products of a cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a cyclic- nucleotide-gated channel OCNC2 subunit polypeptide.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell that expresses a cychc-nucleotide-gated channel OCNC2 subunit polynucleotide can be used in a cell-based assay system.
  • the cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.
  • compositions of the invention can comprise, for example, a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide, cyclic-nucleotide-gated channel OCNC2 subunit polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide, or mimetics, activators, or inhibitors of a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide activity.
  • compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • agent such as stabilizing compound
  • the compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers also can be used for delivery.
  • the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penevers appropriate to the particular barrier to be permeated are used in the formulation. Such peneflops are generally known in the art.
  • compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation can be a lyopbilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.
  • Cardiovascular diseases include the following disorders of the heart and the vascular system: congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular arrhythmias, hypertensive vascular diseases, and peripheral vascular diseases.
  • Heart failure is defined as a pathophysiologic state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failure, such as high-output and low-output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause.
  • MI Myocardial infarction
  • Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen.
  • This group of diseases includes stable angina, unstable angina, and asymptomatic ischemia.
  • Arrhythmias include all forms of atrial and ventricular tachyarrhythmias (atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexcitation syndrome, ventricular tachycardia, ventricular flutter, and ventricular fibrillation), as well as bradycardic forms of arrhythmias.
  • vascular diseases include primary as well as all kinds of secondary arterial hypertension (renal, endocrine, neurogenic, others).
  • the disclosed gene and its product may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications.
  • Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon, and venous disorders.
  • PAOD peripheral arterial occlusive disease
  • acute arterial thrombosis and embolism inflammatory vascular disorders
  • Raynaud's phenomenon Raynaud's phenomenon
  • venous disorders venous disorders.
  • This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a cyclic- nucleotide-gated channel OCNC2 subunit polypeptide binding molecule
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • a reagent which affects cyclic-nucleotide-gated channel OCNC2 subunit activity can be administered to a human cell, either in vitro or in vivo, to reduce cyclic- nucleotide-gated channel OCNC2 subunit activity.
  • the reagent preferably binds to an expression product of a human cyclic-nucleotide-gated channel OCNC2 subunit gene. If the expression product is a protein, the reagent is preferably an antibody.
  • an antibody can be added to a preparation of stem cells that have been removed from the body.
  • the cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.
  • the reagent is delivered using a hposome.
  • the hposome is stable in the animal into which it has been administered for at least about
  • a hposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human.
  • the lipid composition of the hposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.
  • a liposome useful in the. present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell.
  • the fransfection efficiency of a liposome is about 0.5 ⁇ g of DNA per 16 nmole of hposome delivered to about 10 cells, more preferably about 1.0 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, and even more preferably about 2.0 ⁇ g of DNA per 16 nmol of liposome delivered to about 10 6 cells.
  • a liposome is between about 100 and 500 run, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
  • Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art.
  • More preferred liposomes include liposomes having a polycationic lipid composition and/or - liposomes having a cholesterol backbone conjugated to polyethylene glycol.
  • a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.
  • a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Patent 5,705,151).
  • a reagent such as an antisense oligonucleotide or ribozyme
  • antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery.
  • Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al Trends in Biotechnol. 11, 202-05 (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al., J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci. USA. 87, 3655-59 (1990); Wu et al, J. Biol. Chem. 266, 338-42 (1991). Determination of a Therapeutically Effective Dose
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases cyclic-nucleotide-gated channel OCNC2 subunit activity relative to the cyclic-nucleotide-gated channel OCNC2 subunit activity which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LDso/EDso-
  • compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect.
  • Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation. Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • reagent is a single-chain antibody
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated
  • Effective in vivo dosages of an antibody are in the range of about 5 ⁇ g to about 50 ⁇ g/kg, about 50 ⁇ g to about 5 mg/kg, about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight, and about 200 to about 250 ⁇ g/kg of patient body weight.
  • effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA.
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of a cyclic-nucleotide-gated channel OCNC2 subunit gene or the activity of a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent.
  • the effectiveness of the mechanism chosen to decrease the level of expression of a cyclic-nucleotide-gated channel OCNC2 subunit gene or the activity of a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to cyclic-nucleotide-gated channel OCNC2 subunit-specific mRNA, quantitative RT-PCR, immunologic detection of a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide, or measurement of cyclic-nucleotide-gated channel OCNC2 subunit activity.
  • any of the pharmaceutical compositions of the invention can be administered in combination with ' other appropriate therapeutic agents.
  • Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • Human cyclic-nucleotide-gated channel OCNC2 subunit also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the polypeptide. For example, differences can be determined between the cDNA or genomic sequence encoding cyclic-nucleotide-gated channel OCNC2 subunit in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.
  • Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method.
  • cloned DNA segments can be employed as probes to detect specific DNA segments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.
  • DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures ⁇ see, e.g., Myers et al, Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl.
  • the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA.
  • direct methods such as gel-electrophoresis and DNA sequencing
  • mutations can also be detected by in situ analysis. Altered levels of cyclic-nucleotide-gated channel OCNC2 subunit also can be detected in various tissues.
  • Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.
  • the polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-cyclic nucleotide-gated channel OCNC2 subunit polypeptide obtained is fransfected into human embryonic kidney 293 cells. These cells are incubated with a divalent cation-free solution containing 140 mM sodium chloride, 0.5 potassium chloride, lOmM Hepes, and 1.0 mM EGTA (pH 7.4). Measurements with Ca 2+-free solutions are performed under symmetrical ionic conditions.
  • the Ca2+-containing solution is comprised of 140 mM sodium cloride,
  • Cyclic nucleotides are applied by a multibarrelled perfusion pipette that allowed solution changes within 100ms. The membrane potential is held at 0 mV and stopped to ⁇ 105 mV. Capacitative transients and leak currents are substrated using currents recorded under the superfusion with solution without an agonist. Data are digitized at
  • Pichia pastoris expression vector pPICZB (Invifrogen, San Diego, CA) is used to produce large quantities of recombinant human cyclic-nucleotide-gated channel
  • OCNC2 subunit polypeptides in yeast The cychc-nucleotide-gated channel OCNC2 subunit-encoding DNA sequence is derived from SEQ ID NO:l.
  • the DNA sequence is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag and a termination codon.
  • at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pPICZ B with the corresponding restriction enzymes the modified DNA sequence is ligated into pPICZB.
  • This expression vector is designed for inducible expression in Pichia pastoris, driven by a yeast promoter.
  • the resulting pPICZ/md-His6 vector is used to transform the yeast.
  • the yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea.
  • the bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invifrogen, San Diego, CA) according to manufacturer's instructions.
  • enterokinase enterokinase (Invifrogen, San Diego, CA) according to manufacturer's instructions.
  • Purified human cyclic- nucleotide-gated channel OCNC2 subunit polypeptide is obtained.
  • OCNC2 subunit polypeptides comprising a glutathione-S-fransferase protein and absorbed onto glutathione-derivatized wells of
  • 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution.
  • Human cyclic-nucleotide-gated channel OCNC2 subunit polypeptides comprise the amino acid sequence shown in SEQ ID NO:2.
  • the test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.
  • the buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide is detected by fluorescence measurements of the contents of the wells.
  • a test compound that increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a cyclic-nucleotide-gated channel OCNC2 subunit polypeptide.
  • test compound is administered to a culture of human cells fransfected with a cyclic-nucleotide-gated channel OCNC2 subunit expression construct and incubated at 37 °C for 10 to 45 minutes.
  • a culture of the same type of cells that have not been fransfected is incubated for the same time without the test compound to provide a negative control.
  • RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18,
  • Northern blots are prepared using 20 to 30 ⁇ g total RNA and hybridized with a 32 P -labeled cyclic-nucleotide-gated channel OCNC2 subunit- specific probe at 65 ° C in Express-hyb (CLONTECH).
  • the probe comprises at least 11 contiguous nucleotides selected &om the complement of SEQ ID NO:l.
  • a test compound that decreases the cychc-nucleotide-gated channel OCNC2 subunit- specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of cyclic-nucleotide-gated channel OCNC2 subunit gene expression.
  • HEK-293 cells Human embryonic kidney cells (HEK-293 cells) are fransfected using the calcium phosphate method ⁇ see, e.g., R.S. Dhallan et al., Nature 347:184-87 (1990) with 10 micro grams of an expression plasmid for a cyclic-nucleotide-gated channel OCNC2 subunit, 10 micrograms carrier DNA (e.g., pBluescript), and 1 microgram of a simian virus 40 expression plasmid (RSV-TAg).
  • R.S. Dhallan et al. a cyclic-nucleotide-gated channel OCNC2 subunit
  • 10 micrograms carrier DNA e.g., pBluescript
  • RSV-TAg simian virus 40 expression plasmid
  • Activity of a cyclic-nucleotide-gated channel OCNC2 subunit is determined using the patch clamp technique.
  • Excised inside-out patches are prepared using a pipette containing 140 mM NaCl, 5 mM KC1, 1 mM Na 2 EGTA, and 10 mM Hepes-NaOH (pH 7.6).
  • the cells are maintained in Ringer's solution containing 140 mM NaCl, 5 mM KC1, 10 mM Hepes-NaOH (pH 7.6), 2 mM CaCl 2 , and 1 mM MgCl 2 .
  • Single channel currents are recorded after exchanging the bath solution with the same solution as in the pipette.
  • the pipette solution is clamped at +60 mV and cGMP in the range of 10 micromolar to 1 mM is added to the bath solution to activate the channels.
  • Channel activity is determined first in the absence of test compounds (confrol activity). Then, a test compound is added to the bath solution and or the pipette solution, and the channel activity is again determined and compared to control activity to dete ⁇ nine the effects of the test compound.
  • Quantitative expression profiling is performed by the form of quantitative PCR analysis called "kinetic analysis” firstly described in Higuchi et al, BioTechnology 10, 413-17, 1992, and Higuchi et al, BioTechnology
  • the probe is cleaved by the 5 '-3' endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et al, Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al, Genome Res. 6, 986-94, 1996, and Gibson et al, Genome Res. 6, 995-1001, 1996).
  • the amplification of an endogenous control can be performed to standardize the amount of sample RNA added to a reaction.
  • the confrol of choice is the 18S ribosomal RNA. Because reporter dyes with differing emission spectra are available, the target and the endogenous confrol can be independently quantified in the same tube if probes labeled with different dyes are used.
  • RNA extraction and cDNA preparation Total RNA from the tissues listed above are used for expression quantification. RNAs labeled “from autopsy” were extracted from autoptic tissues with the TRIzol reagent (Life Technologies, MD) according to the manufacturer's protocol.
  • RNA Fifty ⁇ g of each RNA were treated with DNase I for 1 hour at 37 C in the following reaction mix: 0.2 U/ ⁇ l RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/ ⁇ l RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; lOmM MgCl 2 ; 50 mM NaCl; and 1 mM DTT.
  • RNA is extracted once with 1 volume of phenoh.chloro- formtisoamyl alcohol (24:24:1) and once with chloroform, and precipitated with 1/10 volume of 3 M NaAcetate, pH5.2, and 2 volumes of ethanol.
  • RNA from the autoptic tissues Fifty ⁇ g of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, TX). After resuspension and specfrophotometric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is 200ng/ ⁇ L. Reverse transcription is carried out with 2.5 ⁇ M of random hexamer primers.
  • Total cDNA content is normalized with the simultaneous quantification (multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA).
  • PDAR Pre-Developed TaqMan Assay Reagents
  • the assay reaction mix is as follows: IX final TaqMan Universal PCR Master Mix (from 2X stock) (PE Applied Biosystems, CA); IX PDAR confrol - 18S RNA (from 20X stock); 300 nM forward primer; 900 nM reverse primer; 200 nM probe; 10 ng cDNA; and water to 25 ⁇ l.
  • the experiment is performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, CA). At. the end of the run, fluorescence data acquired during PCR are processed as described in the ABI Prism 7700 user's manual in order to achieve better background subtraction as well as signal linearity with the starting target quantity.

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Abstract

Cette invention concerne des réactifs qui régulant la sous-unité OCNC2 humaine du canal à porte nucléotidique cyclique ainsi que des réactifs se liant à des produits géniques de la sous-unité OCNC2 humaine du canal à porte nucléotidique cyclique, lesquels produits peuvent jouer un rôle dans la prévention, l'amélioration ou la correction de dysfonctionnements ou de maladies, notamment, mais pas exclusivement des troubles cardio-vasculaires ou du système nerveux central.
PCT/EP2002/001726 2001-02-20 2002-02-19 Regulation de la sous-unite ocnc2 humaine du canal a porte nucleotidique cyclique Ceased WO2002081689A1 (fr)

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WO2004058052A3 (fr) * 2002-12-20 2006-10-05 Applera Corp Polymorphismes genetiques associes a l'infarctus du myocarde, techniques de detection et utilisations de ceux-ci

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WO2001081578A2 (fr) * 2000-04-26 2001-11-01 Curagen Corporation Nouvelles proteines et acides nucleiques codant ces dernieres
WO2002002633A2 (fr) * 2000-06-29 2002-01-10 Incyte Genomics, Inc. Transporteurs et canaux ioniques
WO2002014467A2 (fr) * 2000-08-17 2002-02-21 Icagen, Incorporated Cng2b: nouveau canal ionique humain dependant de nucleotides cycliques

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WO2001081578A2 (fr) * 2000-04-26 2001-11-01 Curagen Corporation Nouvelles proteines et acides nucleiques codant ces dernieres
WO2002002633A2 (fr) * 2000-06-29 2002-01-10 Incyte Genomics, Inc. Transporteurs et canaux ioniques
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BRADLEY J ET AL: "Heteromeric olfactory cyclic nucleotide-gated channels: A subunit that confers increased sensitivity to cAMP", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 91, September 1994 (1994-09-01), pages 8890 - 8894, XP002197064 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004058052A3 (fr) * 2002-12-20 2006-10-05 Applera Corp Polymorphismes genetiques associes a l'infarctus du myocarde, techniques de detection et utilisations de ceux-ci

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