Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-09-09
2003-03-25
Eyler, Yvonne (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...
C435S006120, C435S320100, C435S325000, C536S023100, C536S023500, C530S350000, C530S387100
Reexamination Certificate
active
06537778
ABSTRACT:
CROSS-REFERENCES TO RELATED APPLICATIONS
Not applicable.
FIELD OF THE INVENTION
This invention provides isolated nucleic acid and amino acid sequences of a novel family of eukaryotic mechanosensory ion channels that are designated mechanosensory transduction channels (MSC).
BACKGROUND OF THE INVENTION
The ability to detect mechanical stimuli is an essential and prevalent characteristic of living organisms, and is found from bacteria to simple metazoans to the most complex of mammals. Indeed, the ability to detect mechanical stimuli and convert them into electrical signals forms the basis of many central aspects of animal life, such as light touch, heavy touch, proprioception, baroreception, balance, and the crown jewel, hearing. Even the ability of cells to stop growing when in contact with neighboring cells is likely dependent on mechanical stimuli. Not surprisingly, therefore, numerous human conditions result at least in part from an inability to detect mechanical stimuli, such as Meniere's Disease, sensorineural deafness, blood pressure disorders, and various types of cancers.
In general, the variety of known mechanosensory modalities are thought to be mediated by mechanically-gated cation channels present within the membrane of receptor cells. This view has come in large part from detailed studies into the physiology of mechanosensation using various cell types involved in mechanosensory detection, such as the hair cells of the vertebrate inner ear, single-celled ciliates such as Paramecium, or the sensory neurons of Drosophila (see, e.g., Keman et al.,
Neuron
12:1195-1206 (1994)). In Drosophila, the dendrite of the sensory neuron is enclosed in a cavity filled with a specialized receptor lymph, which is unusually rich in potassium ions, and is functionally equivalent to the potassium-rich endolyniph of the vertebrate cochlea. These potassium ions produce a transepithelial potential difference, with the apical side of the epithelium being positively charged. Mechanical stimulation of the bristle, which is adjacent to the sensory neuron, generates a mechanoreceptor potential within the neuron, detectable as a negative deflection of the transepithelial potential, which reflects the flow of cations from the receptor lymph into the sensory neuron.
Activation of the hair cells of vertebrates also result in the influx of cations into cells (see, e.g., Hudspeth,
Nature
, 341:397-404 (1989)). Each hair cell has a number of specialized microvillar structures, called stereocilia, whose deflection results in the activation of a putative channel present on the surface of the cell. Interestingly, electrophysiological studies have suggested that these cells contain a similar number of receptor channels as they do stereocilia, suggesting that perhaps each receptor channel is coupled to a single stereocilium. In addition, studies of the kinetics of hair-cell activation have suggested that the putative mechanosensory receptors are directly stimulated by mechanical force, resulting in the direct opening of the channel without the involvement of second messengers.
Despite the great importance of mechanosensation for animal behavior and health, and the detailed electrophysiological understanding that has been gained from the above-described studies, almost nothing is known about the molecular basis of mechanosensory detection in eukaryotes. Several mutations and distantly related molecules involved in this process have, however, been found. In Drosophila, for example, a number of mutations have been isolated that disrupt mechanoreception, resulting in a variety of phenotypes such as reduced locomotor activity, total uncoordination, and even death (Keman et al.,
Neuron
12:1195-1206 (1994)). Also, mutations have been identified in the nematode
C. elegans
that result in a loss of sensitivity to gentle touch (reviewed in Garcia-Aanoveros & Corey,
Ann. Rev. Neurosci
. 20:567-594 (1997)). In addition, a prokaryotic mechanosensory channel has been identified (Sukarev et al.,
Nature
368:265-268 (1994)). Still, despite these advances, the principle molecule of the mechanosensory transduction process in eukaryotes, the mechanically gated channel, has yet to be isolated or identified.
The identification and isolation of eukaryotic mechanosensory transduction channels would allow for the development of new methods of pharmacological and genetic modulation of mechanosensory transduction pathways. For example, availability of mechanosensory transduction channel proteins would permit screening for high-affinity agonists, antagonists, and modulators of mechanosensation in animals. Such molecules could then be used, e.g., in the pharmaceutical industry, to treat one or more of the many human conditions involving loss or hyperactivation of mechanosensation. In addition, the determination of nucleotide and amino acid sequences of mechanosensory transduction channels associated with a human condition would provide new tools for the diagnosis and/or treatment, e.g., gene-based treatment, of the condition.
SUMMARY OF THE INVENTION
The present invention provides for the first time nucleic acids encoding a eukaryotic mechanosensory transduction protein. The nucleic acids and the polypeptides they encode are referred herein as mechanosensory channel (MSC) nucleic acids and proteins. In vivo, MSC proteins form mechanosensory transduction channels that play a central role in many critical processes such as hearing, proprioception, and tactile sensation.
In one aspect, the present invention provides an isolated nucleic acid encoding a mechanosensory transduction protein, the protein having at least one of the following characteristics: (i) comprising greater than about 70% amino acid sequence identity to SEQ ID NO:2 or SEQ ID NO:4; (ii) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; or (iii) specifically binding to polyclonal antibodies generated against a polypeptide comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4; wherein the protein does not comprise the polypeptide sequence of SEQ ID NO:6.
In one embodiment, the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. In another embodiment, the nucleic acid comprises a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, but not SEQ ID NO:5.
In another embodiment, the nucleic acid selectively hybridizes under moderately stringent wash conditions to a nucleic acid comprising a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. In another embodiment, the nucleic acid selectively hybridizes under stringent wash conditions to a nucleic acid comprising a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, but not SEQ ID NO:5.
In another embodiment, the nucleic acid is amplified by primers that selectively hybridize under stringent hybridization conditions to the same sequence as degenerate primer sets encoding an amino acid sequence selected from the group consisting of: LDVLIENEQKEV (SEQ ID NO:7), HHLFGPWAIII (SEQ ID NO:8), and VLINLLIAMMSDTYQRIQ (SEQ ID NO:9).
In another embodiment, the nucleic acid is less than 120 kb. In another embodiment, the nucleic acid is less than 90 kb. In another embodiment, the nucleic acid is less than 60 kb. In another embodiment, the nucleic acid is less than 30 kb. In another embodiment, the nucleic acid is less than 10 kb. In another embodiment, the nucleic acid sequence encoding the MSC protein is isolated away from its genomic neighbors.
In another aspect, the present invention provides an expression cassette comprising a nucleic acid encoding a mechanosensory transduction protein, the protein having at least one of the following characteristics: (i) comprising greater than about 70% amino acid sequence identity to SEQ ID NO:2 or SEQ ID NO:4; (ii) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; or (iii) specifically binding to polyclonal antibodies generated against a polypeptide comprising an amino acid sequence of SEQ ID NO:2 or
Walker Richard G.
Willingham Aarron
Zuker Charles S.
Eyler Yvonne
Murphy Joseph F.
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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