Transmembrane channels control the ionic permeability of many cells and play a significant role in major cell functions, from nerve excitability to fertilization and development. This project focuses on one of the most ubiquitous of such channels: the voltage-dependent Na+ channel, the molecule responsible for the Na+ transport in nerves underlying the generation of the nerve impulse. Electrophysiological, biochemical and pharmacological studies will be combined to elucidate the mechanisms controlling Na+ channel function, obtain information on its molecular structure, and further clarify the mechanisms of voltage control. These are prerequisites to the understanding and treatment of related pathological states. One aspect of this project will be concerned with improving methodology used to study voltage-dependent channels in reconstituted systems. The approach will be to: (1) Purify Na+ channels from rat brain in cholate, enabling the channel's reconstitution into artificial membranes, where electrical studies are possible, and (2) Develop model membranes containing Na+ channels obtained from native membranes as well as from purified protein, where both the unmodified gating modes of the Na+ channel as well as the channel kinetics modified by alkaloid activators can be studied. In the second part of the project, the Na+ channel will be incorporated into black-lipid bilayer membranes, a technique we have already used successfully. Objectives are to: (1) Characterize effects of lipid composition on channel behavior. (2) Clarify some of the effects of Zn++, Ca++ and ionic strength on Na+ channel gating, and the temperature dependence and the effects of membrane viscosity on gating. (3) Utilize the alkaloid-activated channel to obtain information on the conducting pathway of the Na+ channel, the similarities and differences of the activation between various alkaloids, and on the mechanism of Na+ channel inactivation. (4) To classify Na channel inactivation modifiers by their actions on molecular stages of the inactivation process and specifically test the hypothesis of Na+ channel inactivation as channel block by a polypeptide blocking particle.
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