Adenylyl cyclase (AC)-mediated signal transduction in hair cells and their neural contacts, both efferent and afferent, is predicted to impact mechanosensory transduction and rcceptoneural transmission. AC enzymatic activity is attributable to nine AC isoforms, each with specific pharmacology, G-protein coupling, and ultrastructural localization. (1) A first goal is to obtain full-length cDNA sequence for AC isoforms in hair cells, determine properties for individual expressed i so form variants, and localise the i so forms at the ultrastructural level. Hair cells in the cochlea appear poised to respond to Ca2+. AC isoforms 1 and 8, activated by Ca2+/calmodulin, and AC9, inhibited by Ca'Vcalcineurin, are expressed potentially regulating phosphorylation/dephosphorylation cascades. Hair cells in vestibular end organs, as represented by a trout saccular hair cell preparation, utilize AC isoforms 9, 7 and 6, with AC9 and 6 inhibited by Ca2+. Molecular analysis of unusual splice variants will provide key information on mechanisms of control. (2) A second goal is lo determine the nature of G-protein coupling to AC isoforms in hair cells and their efferent and afferent contacts. The isoforms expressed in saccular hair cells are activated via Galphas/olf, net previously detected in hair cells. A preliminary finding of Gas/olf cDNA in saccular hair cells will be followed by full-length sequence determination for analysis of molecular domains underlying function. G protein coupling to AC enzymatic activity in vestibular and auditory end organs will be histocytochemically examined, allowing ultrastructural localization of specific pharmacology. AC regulation via G-protein-coupling to efferent neurotransinitters, serotonin and dopamine, in saccular hair cells, will additionally be examined in vitro, complementing ultrastructural studies. (3) A third goal is to characterize protein tirgets of the AC signal transduction pathway in hair cells, focusing on two ion channels directly gated by cAMP. Subunits comprising cyclic nucleotide-gated (CNG) channels in hair cells, potentially involved in mechanosensory transduction, will be analyzed for the saccular hair cell preparation, which contains sufficient numbers of hair cells for full-length sequencing. Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels underlying IH, predicted to influence hair cell spontaneous release of transmitter, will be similarly studied. Molecular sequence of respective CNG and HCN transcripts will predict molecular function and sensitivity to cAMP. The elucidation of these AC-mediated signal transduction pathways in inner ear sensory epithelia may allow eventual pharmacological amelioration of tinnitus, sensorinetiral deafness and vertigo.
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