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Human kcnq5 potassium channel, methods and compositions thereof

Human kcnq5 potassium channel, methods and compositions thereof description/claims

This application is a divisional of patent application U.S. Ser. No. 10/948,493, filed Sep. 23, 2004, which is a divisional of non-provisional patent application U.S. Ser. No. 09/866,020, filed May 24, 2001, which claims benefit of provisional patent application U.S. Ser. No. 60/207,389, filed May 26, 2000.

The present invention relates generally to the KCNQ family of potassium channels. More specifically, the present invention provides a new potassium channel polypeptide, KCNQ5, which was cloned and isolated from human tissue. Human KCNQ5 is found to be expressed primarily in brain and skeletal muscle. The present invention further provides methods for modulating the KCNQ5 potassium channel and assays for detecting channel modulators, which have use in the treatment of a variety of neurological, neurophysiological and neuropsychological conditions, disorders and diseases.

Potassium channels are membrane-spanning proteins that generally act to hyperpolarize neurons. Physiological studies indicate that potassium currents are found in most cells and are associated with a wide range of functions, including the regulation of the electrical properties of excitable cells. Depending on the type of potassium channel, its functional activity can be controlled by transmembrane voltage, different ligands, protein phosphorylation, or other second messengers.

In the last decade, the cloning of potassium channels has resulted in the discovery of the molecular isolation and characterization of greater than 50 potassium channel genes and many of their associated regulatory subunits. More recently, a new family of potassium channel genes, the KCNQ gene family, has been described (N. A. Singh et al., 1998, Nature Genet., 18:25-29; C. Charlier et al., 1998, Nature Genet., 18:53-55; C. Biervert et al., 1998, Science, 279:403406). The KCNQ family of potassium channels are voltage dependent potassium channels. They contain the voltage sensor and pore signature sequences characteristic of a 6 transmembrane potassium channel gene and have a longer carboxy-terminus than other known voltage-dependent potassium channels.

A remarkable aspect about the KCNQ 1-4 gene family is that mutations in each channel are associated with a particular disease, including cardiac arrhythmias (KCNQ1), epilepsy (KCNQ2 and KCNQ3), and hearing loss (KCNQ4). The present invention provides a newly isolated, cloned and characterized member of the KCNQ family, called KCNQ5. Human KCNQ5 provides the art with an additional member of the KCNQ family of potassium channel proteins, isolated from a human source, for use in the methods and compositions described herein.

It is an object of the present invention to provide a novel isolated polynucleotide molecule encoding a novel potassium channel polypeptide referred to herein as KCNQ5, or hKCNQ5 (i.e., human KCNQ5). The present invention encompasses the amino acid sequence of the hKCNQ5 protein and the nucleic acid sequence encoding the hKCNQ5 protein. Also embraced by the present invention are variations in the hKCNQ5 nucleic acid sequence due to degeneracy in the genetic code.

The present invention provides for polynucleotide molecules which are at least about 70% identical to the polynucleotide sequence of the native hKCNQ5 sequence disclosed herein and set forth as SEQ ID NO:1. Preferably, the present invention provides: (a) a purified and isolated nucleic acid molecule encoding a KCNQ5 protein according to the present invention; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences having at least 70% sequence identity, preferably at least 80% to at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a); or (d) a fragment of (a) or (b) which will hybridize to (a) or (b) under low, moderately stringent and/or highly stringent conditions (described further herein), said fragment preferably comprising at least 15 nucleotides to 20 nucleotides (e.g., encoding at least 5 codons, more preferably at least about 7-15 codons), and more preferably encoding a functional or biologically active fragment of the hKCNQ5 polypeptide, such as the pore region (which spans about amino acid 191 to amino acid 209 of the KCNQ5 polypeptide sequence) or the S4 voltage sensor region (which spans about amino acid 265 to amino acid 285 of the KCNQ5 polypeptide sequence) (FIG. 5A).

It is another object of the present invention to provide an amino acid sequence which is at least about 70% to 80% identical to the hKCNQ5 polypeptide sequence disclosed herein and set forth as SEQ ID NO:2. Preferably, the present invention includes: (a) the amino acid sequence of the KCNQ5 protein of the present invention; and (b) amino acid sequences having at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to (a).

It is yet a further object of the present invention to provide a novel KCNQ5 polynucleotide molecule, associated vectors, host cells, and methods of use. Preferably, the polynucleotide molecule is a DNA molecule. In accordance with the present invention, polynucleotide sequences having approximately 70% or greater identity to the hKCNQ5 sequence are preferred, preferably under moderate or high stringency conditions. Also preferred are nucleotide sequences that about 80% or more identical to the KCNQ5 polynucleotide sequence of SEQ ID NO:1.

Yet another object of the present invention is to provide nucleic acids obtained by PCR with degenerate oligonucleotide primers. The ordinarily skilled practitioner in the art can devise such primers based on the hKCNQ5 nucleic acid sequence and/or the consensus sequence(s) described herein using techniques known and practiced in the art. For example, PCR techniques are described in White et al., 1989, Trends Genet. 5: 185-9.

Another object of the present invention is to provide expression vectors comprising a nucleic acid sequence coding for a KCNQ5 polypeptide, or fragment thereof, preferably a functional or biologically active fragment thereof; host cells containing such vectors; and polypeptides comprising the amino acid sequence of the KCNQ5 protein. Such polypeptides, or fragments thereof, may be isolated and purified employing conventional methodologies, following expression in the host cell. Preferably, the vector encodes a full-length KCNQ5 protein and the polypeptide is a full-length KCNQ5 protein. Preferred are frog (Xenopus) expression vectors, such as pSP64T or derivatives thereof (Melton et al., 1984, Nucl. Acids Res., 12: 7057-70); mammalian cell expression vectors, such as pcDNA3 (available from Invitrogen); or bacterial cell expression vectors, such as pET-30 (available from Novagen or Promega).

Yet another object of the present invention is to provide host cells transformed with the above-described vectors. Preferred are Xenopus oocytes, mammalian cells (e.g., HEK-293, CHO, L929), and bacterial cells (e.g., E. coli, especially BL21 (DE3), available from Novagen). Particularly preferred are HEK-293 cells deposited as ATCC Accession No. CRL-1573 (American Type Culture Collection, 10801 University Boulevard, Manassas Va. 20110-2209). The present invention also provides a method for producing a KCNQ5 polypeptide having the amino acid sequence as depicted in SEQ ID NO:2 by culturing a host cell under conditions suitable for the expression of the polypeptide, and recovering the polypeptide from the host cell culture.

It is another object of the present invention to provide methods for detecting nucleic acids that code for KCNQ5 proteins, as well as processes for detecting molecules that bind to and/or otherwise modulate (e.g., activate, up-regulate, increase, or inhibit, block, or down-regulate) the activity of the KCNQ5 protein. As used herein, “modulate” encompasses both channel openers/activators, and the like, as well as channel closers/inactivators/blockers, and the like.

A further object of the present invention is to provide methods of modulating KCNQ5 proteins, specifically methods of opening/activating or closing/inactivating/blocking KCNQ5 potassium channels. Moreover, the present invention encompasses a method of treating a disease or disorder, preferably, a neuroaffective disorder, such as a neurological, neurophysiological, or neuropsychological disease or disorder, by modulating the activity of the KCNQ5 protein. Thus, the present invention is directed to a treatment of an individual in need of such treatment for a condition that is mediated by a potassium channel, particularly, the KCNQ5 potassium channel, or for a condition that is mediated by the biological activity of human KCNQ5, comprising administering to the individual a potassium channel modulating compound in an amount effective to modulate the activity of the potassium channel as described herein.

Another object of the present invention is to provide methods for identifying compounds (e.g., small molecules, peptides, analogs, mimetics) that modulate the biological activity of a potassium channel, preferably the hKCNQ5 potassium channel, comprising: combining a candidate compound modulator of a potassium channel biological activity with the KCNQ5 potassium channel polypeptide having the amino acid sequence set forth in SEQ ID NO:2; and measuring an effect of the candidate compound modulator on the biological activity of the potassium channel polypeptide. According to the present invention, the potassium channel polypeptide may be expressed by a recombinant host cell.

It is a further object of the present invention to provide an antisense polynucleotide molecule comprising substantially the complement of the KCNQ5 nucleic acid sequence set forth in SEQ ID NO:1, or a biologically effective portion thereof. Also, in accordance with the present invention is provided a method for modulating, e.g., inhibiting or downregulating, the expression and/or activity of the KCNQ5 potassium channel polypeptide using the antisense polynucleotide molecule, comprising administering to an individual in need of such modulating an amount of the antisense molecule effect to modulate the expression and/or activity of the KCNQ5 potassium channel polypeptide.

Yet a further object of the present invention is to provide an antibody having specificity toward an isolated and purified KCNQ5 polypeptide, preferably the hKCNQ5 polypeptide, having the amino acid sequence as shown in SEQ ID NO:2, or an immunoreactive peptide fragment thereof.

Additional objects and advantages afforded by the present invention will be apparent from the detailed description hereinbelow.

• Provisional Patent
• Utility Patent

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