The object of this project is to describe and characterize the molecular basis of voltage dependent processes. In particular, the aim is an understanding of the physical events responsible for the voltage dependence of sodium and potassium channels and the sodium/calcium exchange. The experiments use electrophysiological techniques to describe the currents through a large number of channels (macroscopic currents), through a few channels (noise measurements), through single channels and the currents produced by the rearrangement of the charged groups of the channel macromolecule. Channels will be modified (BTX for sodium and ATP for potassium) in an attempt to gain more information about their physical states. Results will be interpreted using kinetic models with energy barriers between the physical states represented by the positions of the charged particles in the macromolecule. Parameters will be fitted to these models using all the information from the electrical measurements and they will be modified according to the results of the fitting. In addition, labelled ATP will be used to mark the K channel and attempt its extraction and characterization. The Na/Ca exchange will be characterized by measuring isotopic fluxes under membrane potential control and the currents produced by the exchange. We will attempt the determination of its stoichiometry and voltage dependence. Experiments will be performed using the giant axon of the squid in perfused, dialyzed or cut-open configurations. This latter technique allows patch clamping from the internal side of the membrane. The experiments requiring larger amounts of membrane material will be performed in membranes extracted from the retinal nerve of the squid. This research is expected to provide new information on the detailed mechanisms of voltage gated channels and on the operation of the sodium/calcium exchange which will help in the understanding of the molecular mechanisms of voltage dependent processes. These studies are expected to have relevance in the physiology of nerve, muscle, excitable tissues and cells in general.
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