Target Proteins for Linkages in Membranes of Hair Cells

University Of Louisiana At Lafayette, Lafayette LA

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Abstract

DESCRIPTION (provided by applicant): Hair bundle mechanoreceptors detect sound, motion, acceleration and gravity. They are missing, damaged or nonfunctioning in millions of Americans suffering from hearing or balance disorders. Hair bundles are composed of numerous stereocilia interconnected by extracellular linkages. Whereas most of the linkages may tie the hair bundle together so that it behaves as a single, mechanical unit, certain linkages may function as "gating springs" that open transduction channels. Hair bundle mechanoreceptors on tentacles of sea anemones are similar to those of the acousticolateralis system except that anemone hair bundles self-repair following extensive trauma. Repair is accomplished by "repair proteins" that apparently include replacement linkages for those lost during trauma. Exogenously supplied repair proteins rapidly restore structural integrity and function to traumatized hair bundles. According to the primary hypothesis of this proposal, extracellular linkages are not themselves integral proteins, but instead are attached to integral proteins called, "membrane target proteins." In this proposal, a specific chromatographic fraction of the repair protein mixture will be used as an affinity trap to isolate candidate membrane target proteins. These proteins will be blotted to PVDF membrane and partially sequenced. The resulting information will be used to synthesize peptides that will be used as immunogens. Peptide-specific Antibodies are expected to label specific domains of hair bundles. A cDNA library to tentacle tissue will be constructed and then used to transfect bacteria. Recombinant clones will be screened using the peptide-specific antibodies. Single stranded DNA will be isolated from immunopositive clones and then sequenced. This research may lead to the isolation of the transduction channel among other important membrane proteins in hair cells. This information will likely lead to a better understanding of the molecular basis of certain forms of deafness and balance disorders.