Analysis of cSlo interacting proteins in the inner ear

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DESCRIPTION (from applicant's abstract): Hearing loss affects approximately 35 million Americans. It also affects approximately 35 percent of those 65 and older. The primary cause of hearing loss in this group is a result of a loss of hair cells. Our interest is in understanding the molecular events that determine hair cell function, and how these events are controlled. Hair cells are the primary transducers of sound serving to convert the mechanical energy of sound to a coded a neuronal one. This proposal seeks to understand further how hair cells discriminate between frequencies of sound. Specifically, it aims to extend on the recent experimental evidence relating the primary structure of the calcium gated potassium channel, Slo, to electrical resonance; a phenomenon that allows individual hair cells to discriminate between different frequencies of sound. We will look for proteins that bind to and alter the function of this channel thus altering its ability to respond to particular frequencies of sound. A number of strategies will be used to identify these proteins including degenerate RT PCR based on homology to proteins that re known to bind to Slo, and yeast two hybrid assays utilizing the carboxy  terminus of Slo as "bait". Once these proteins have been cloned and identified, immunoprecipitation of tagged fusion proteins will be used to confirm that the interactions are in fact real. Antibodies will be raised against these proteins after expressing them in bacteria. These proteins will be co-expressed with the Slo channel in an attempt to determine how they alter its function(s). The electophysiological properties of different isoforms of the Slo channel will be studied with and without these Slo binding proteins. In addition confocal immuno-fluorescence microscopy will be used to determine how these proteins alter the subcellular distribution of Slo in hair cells. Attempts will be made to determine if these proteins are distributed differentially within the basilar papilla. This will employ both RT PCR of individual fragments of the papilla and immunohistochemistry. Constant surveillance will be maintained to determine if the genomic locus of these proteins (revealed by the genome project) maps to any of the syndromic forms of hearing loss (in view of other potassium channel defects causing syndromic hearing loss).