The proposal is directed toward understanding the precise mechanisms of cholinergic inhibition of cochlear hair cells. ACh hyperpolarizes hair cells by activation of ligand-gated cation channels that initially depolarize the cell allowing Ca2+ entry and subsequently opening Ca2+-dependent K+ channels that hyperpolarize the cell. It is not only the Ca2+ entering the cell through the ligand-gated channels that mediates the inhibition; there is recent evidence that Ca2+ release from the hair cell's synaptic cistern can augment this effect. A combination of biophysical, molecular genetic, and histological techniques will be used to probe the underlying mechanisms of efferent inhibition. 1. Whole-cell patch clamp recordings in combination with pharmacological agents will be used to examine how inhibiting the calcium release channels affects the ACh evoked and spontaneous currents. 2. PCR cloning will be used to identify the hair cell calcium release channel with ryanodine and IP3 receptor probes. In situ hybridization will be used to map out cochlear expression of the candidate genes. 3. Immunohistochemistry will examine further the subcellular localization of ACh receptors and calcium release channels. 4. Transient transfection of a host cell line will be used to examine candidate genes for the ACh receptor and its dependent potassium channels. An interesting and potentially informative long-term goal of these experiments is to completely reconstitute the hair cell's cholinergic mechanism in a heterologous expression system.
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