The specific aim of the proposed project is to elucidate the mechanism whereby the kinetics of the gating mechanism of nerve membrane sodium channels are modified by specific chemical agents which are used as probes. This will be a step toward accomplishing our long-term goal which calls for characterization and identification of sodium channels. Kinetics of opening and closing of sodium channels as affected by these chemical agents will be analyzed using internally perfused, voltage-clamped squid and crayfish giant axons. Furthermore, the activity of single channels will be studied by patch voltage clamp techniques with cultured neuroblastoma cells. The specific chemical agents to be studied are classified into two large groups, both modifying the channel kinetics drastically. One group may be called sodium channel modulators including grayanotoxins, batrachotoxin, veratridine and aconitine, all of which modify a population of sodium channels to give rise to slow opening and closing presumably through binding to open and/or closed sodium channels. The other group is represented by sodium inactivation inhibitors, including the sea anemone toxin anthopleurin-A, N-bromoacetamide, and high and low internal pH. The specific projects are aimed at the process of channel modification, the properties of the modified channels including cation selectivity, cation binding, voltage dependence and single channel properties, the site of action of the specific agents within the sodium channel, and the gating current in the modified channel. This study is expected to determine normal physiological functioning and topography of verve membrane sodium channels which are the bases for excitation.