This proposal is the initial project of a research program dedicated to the combined biochemical and biophysical analysis of ion channels which regulate the electrical activity of excitable tissues. The specific goals of the present project include in vitro experiments designed to investigate the Na-channel underlying the action potential of skeletal muscle. The primary method to be employed is the direct electrical recording of currents through individual Na-channels that have been incorporated into planar lipid bilayers. This method has been established and used by the principal investigator during a previous year of preliminary work to study guanidinium-toxin blockade of batrachotoxin-modified Na-channels from the plasma membrane of rat skeletal muscle. A primary advantage of studying the batrachotoxin-modified Na-channel is that voltage-dependent inactivation of the channel is eliminated by this toxin. This permits voltage-dependent activation and the effects of blocking agents to be conveniently studied under steady-state conditions at constant voltage. Using this technique, the structure of the channel's entryway to ions will be probed by specific toxins which act from the extracellular side and also by guanidinium compounds and other local anesthetics which may act from either side of the channel. Voltage-dependent gating will be studied by statistical analysis of steady-state current fluctuations as a function of voltage. Other experiments designed to investigate structure and mechanism include studies of variant toxin-insensitive Na-channels, mapping of the channel polypeptide(s) by specific proteolysis and chemical labeling, and the effect of neutral versus negatively charged lipids on gating, conduction and blocking behavior. This project will advance basic understanding of the mechanism of the Na-channel and its behavior in the presence of local anesthetics. In addition, methods necessary for applying the technique to other important ion channels will be developed. The possible relationships of this project to human health include neurological diseases, anesthesia and nervous system physiology.
