Acetylcholine receptor (AChR) channels are found in the post-synaptic membranes of nerve and muscle cells. These channels translate the chemical signal of acetylcholine (ACh) release from the pre-synaptic cell into an electrical depolarization of the post-synaptic cell. AChR channels have been studied using a variety of electrophysiological biochemical and morphological techniques and the molecular mechanisms of its action are beginning to be understood. In this porposal, the patch clamp method of detecting single channels will be used to study the gating mechanisms of the AChR channel. ACh has two actions on the receptor: ACh opens the channel and prolonged exposures to ACh desensitize the receptor. Preliminary experiments have shown that it is possible to distinguish these actions in a patch clamp experiment by making rapid, reversible applications of ACh to an excised patch of membrane from cultured muscle cells. Three types of experiments will be performed in the proposed investigation. 1) The activation of large numbers of channels by repeated applications of ACh. Nonstationary fluctuation analysis will be used to calculate open channel probabilities. 2) The activation of channels by long applications of ACh produce current relaxations. The kinetics of desensitization will be deduced from these relaxations. 3) The activation of single AChR chanels under conditions of no desensitization. Activation rate constants will be obtained from open-and closed-time histograms constructed from the single channel records. All of this information will be used to construct a kinetic model of channel activation and desensitization. The effects of anesthetic agents such as alcohols on the function of the AChR will also be studied. The results of these experiments will be compared with the predictions of models for the interaction of anesthetic molecules with the receptor protein. The implications of such interactions for the mechanisms of general anesthesia will be considered.
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