Potassium channels and genes encoding these potassium channels

Details Potassium channels and genes encoding these potassium channels Potassium channels and genes encoding these potassium channels

2000-01-27

2004-09-21

Spector, Lorraine (Department: 1646)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S325000, C530S350000, C536S023500

Reexamination Certificate

active

06794161

ABSTRACT:

TECHNICAL FIELD
This invention relates to novel potassium channels and genes encoding these channels. More specifically the invention provides isolated polynucleotides encoding the KCNQ4 potassium channel subunit, cells transformed with these polynucleotides, transgenic animals comprising genetic mutations, and the use of the transformed cells and the transgenic animals for the in vitro and in vivo screening of chemical compounds affecting KCNQ4 subunit containing potassium channels.
BACKGROUND ART
Potassium channels participate in the regulation of electrical signalling in excitable cells, and regulates the ionic composition of biological fluids. Mutations in the three known genes of the KCNQ branch of the K
+
-channel gene family underlie inherited cardiac arrhythmia's, in some cases associated with deafness, and neonatal epilepsy.
Hearing loss is the most frequent sensory defect in humans. Hearing loss can be due to environmental and genetic factors, and the progressive hearing loss of the elderly (presbyacusis) most often seems to be due to a combination of both.
Inherited deafness can be classified as non-syndromic (isolated hearing loss) or syndromic (associated with other anomalies). Several hundred syndromes, consisting of hearing loss associated with defects in a variety of other organ systems, have been described. Non-syndromic deafness is classified according to its mode of inheritance as DFN, DFNA, and DFNB (X-linked, autosomal dominant and autosomal recessive, respectively). In general, autosomal recessive deafness has an early onset and is very severe. Autosomal dominant deafness, by contrast, more often develops slowly over several decades and may become apparent only in adulthood. It is hoped that genes identified in families with dominant deafness may also—with different types of mutations—underlie some forms of presbyacusis.
A bewildering number of loci for non-syndromic deafness were identified in the last four years. There are at least 19 loci for autosomal dominant deafness (DFNA1 to DFNA19), and 22 loci for DFNB. Sometimes, depending on the particular mutation, the same gene can be involved in dominant or recessive deafness. This large number of loci reflects the complexity of the inner ear. Identification of these genes and characterisation of their products will significantly advance our understanding of the molecular basis of the physiology of this sensory organ.
Several genes involved in syndromic and non-syndromic deafness have already been identified and are reviewed by Petit [Petit C: Genes responsible for human hereditary deafness: symphony of a thousand;
Nature Genet.
1996 14 385-391] and Kalatzis & Petit [Kalatzis V & Petit C: The fundamental and medical impacts of recent progress in research on hereditary hearing loss;
Hum. Mol. Genet.
1998 7 1589-1597]. Among others, their gene products include transcription factors, unconventional myosin isoforms, &agr;-tectorin (an extracellular matrix protein), diaphanous, a protein interacting with the cytoskeleton, connexin 26 (a gap junction protein), and two genes encoding potassium channel subunits, KCNQ1 and KCNE1.
Ion channels play important roles in signal transduction and in the regulation of the ionic composition of intra- and extracellular fluids. Mutations in ion channels were since long suspected as possibly underlying some forms of hearing loss. In the cochlea (the auditory sensory organ), the transduction current through the sensory cells is carried by potassium ions and depends on the high concentration of that ion in the endolymph. So far only two genes encoding potassium channel subunits, KCNQ1 and KCNE1, were found to be mutated in syndromic hereditary deafness. The gene products of both genes, the KCNQ1 (or KvLQT1) and the mink (or IsK) protein, respectively, form heteromeric potassium channels.
KCNQ1 is a typical member of the voltage-gated potassium channel superfamily with 6 transmembrane domains and a pore region situated between the fifth and the sixth transmembrane domain. The minK protein has a single transmembrane span and cannot form potassium channels on its own. However, as a &bgr;-subunit it enhances and modifies currents mediated by KCNQ1. These heteromeric channels participate in the repolarization of the heart action potential. Certain mutations in either KCNQ1 or KCNE1 cause a form of the autosomal dominant long QT syndrome (LQTS), a disease characterised by repolarization anomalies of cardiac action potentials resulting in arrhythmias and sudden death. Interestingly, other mutations in either gene lead to the recessive Jervell and Lange-Nielsen (JLN) syndrome that combines LQTS with congenital deafness. In order to cause deafness, KCNQ1/minK currents must be reduced below levels that are already sufficiently low to cause cardiac arrhythmia.
SUMMARY OF THE INVENTION
We have now cloned and characterised KCNQ4, a novel member of the KCNQ family of potassium channel proteins. KCNQ4 has been mapped to the DFNA2 locus for autosomal dominant hearing loss, and a dominant negative KCNQ4 mutation that causes deafness in a DFNA2 pedigree was identified.
KCNQ4 is the first potassium channel gene underlying non-syndromic deafness. KCNQ4 forms heteromeric channels with other KCNQ channel subunits, in particular KCNQ3.
The present invention has important implications for the characterisation and exploitation of this interesting branch of the potassium channel super family, as well as for the understanding of the cochlear physiology, and for human deafness and progressive hearing loss.
Accordingly, in its first aspect, the invention provides an isolated polynucleotide having a nucleic acid sequence which is capable of hybridising under high stringency conditions with the polynucleotide sequence presented as SEQ ID NO: 1, its complementary strand, or a sub-sequence thereof.
In another aspect the invention provides a recombinantly produced polypeptide encoded by the .polynucleotide of the invention.
In a third aspect the invention provides a cell genetically manipulated by the incorporation of a heterologous polynucleotide of the invention.
In a fourth aspect the invention provides a method of screening a chemical compound for inhibiting or activating or otherwise modulating the activity on a potassium channel comprising at least one KCNQ4 channel subunit, which method comprises the steps of subjecting a KCNQ4 channel subunit containing cell to the action of the chemical compound; and monitoring the membrane potential, the current, the potassium flux, or the secondary calcium influx of the KCNQ4 channel subunit containing cell.
In a fifth aspect the invention relates to the use of a polynucleotide sequence of the invention for the screening of genetic materials from humans suffering from loss of hearing (e.g. dominant, recessive, or otherwise), tinnitus, and other neurological diseases for mutations in the KCNQ4 gene.
In a sixth aspect the invention relates to the chemical compound identified by the method of the invention, in particular to the use of such compounds for diagnosis, treatment or alleviation of a disease related to tinnitus; loss of hearing, in particular progressive hearing loss, neonatal deafness, and presbyacusis (deafness of the elderly); and diseases or adverse conditions of the CNS, including affective disorders, Alzheimer's disease, anxiety, ataxia, CNS damage caused by trauma, stroke or neurodegenerative illness, cognitive deficits, compulsive behaviour, dementia, depression, Huntington's disease, mania, memory impairment, memory disorders, memory dysfunction, motion disorders, motor disorders, neurodegenerative diseases, Parkinson's disease and Parkinson-like motor disorders, phobias, Pick's disease, psychosis, schizophrenia, spinal cord damage, stroke, and tremor.
In a seventh aspect the invention provides a transgenic animal comprising a knock-out mutation of the endogenous KCNQ4 gene, a replacement by or an additional expression of a mutated KCNQ4 gene, or genetically manipulated in order to over-express the

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