Newly developed biophysical techniques allow study of the membrane processes responsible for excitability and transduction in olfactory receptor neurons (ORNs). Odorants are known to interact with the olfactory cilia and increase the activity of the enzyme adenylate cyclase. This increase is mediated by one or more GTP-binding proteins (G-proteins). The specific G-protein involved may be G-olf, a major stimulatory G-protein in olfactory cilia. However, there is no evidence that G-olf is required for olfactory transduction. The proposed experiments will determine whether G-olf mediates activation of the adenylate cyclase. G-olf in single cilia will be inactivated with specific antibodies, and the effects on the odorant response measured. cAMP produced by the cyclase is known to activate ciliary membrane channels, which allow a depolarizing receptor current to flow into the ORN. Preliminary studies suggest that divalent cations at the external surface of the ciliary membrane strongly inhibit this receptor current. The extent of inhibition by calcium and magnesium on the cAMP-activated current will be measured in single cilia. The cAMP-mediated depolarization is transient, and it is unknown how the odorant response is terminated. Preliminary studies indicate that calcium entering the cilia through the cAMP-gated channels activates a secondary chloride conductance. This calcium-activated chloride conductance may help to repolarize the neuron after the odorant response. Activation of the secondary chloride current will be demonstrated in single cilia. The hypothesis that this current repolarizes the neuron after an odorant response will bc tested in intact ORNs. Gigaseal patch-clamp recordings from isolated cilia will bc used to measure the ciliary membrane properties. Perforated-patch whole-cell recordings from the soma will measure the chloride current in intact ORNs without perturbing the normal intracellular chloride concentration. Pharmacological and immunochemical means will bc used to selectively control specific ionic currents implicated in ORN function. The olfactory sense has a major role in regulating body hormonal state, emotional disposition, hunger, and social behavior. The work proposed here will advance understanding of the molecular and cellular processes which subserve sensory function.
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