Gating and permeation mechanisms of ion channels will be studied. Voltage-gated ion channels open or close in a fraction of a millisecond, controlling the ion flows that generate electrical signals. These channels are present in most cells of the body, and are vital to many processes, including thought, movement, timing of the heartbeat, and control of secretion of hormones e.g. insulin. In nature ion channels are separated into two component parts. A permeation module or pore is found in isolation in bacteria and elsewhere. To this is added a gating module to form the voltage-gated channel found, for example, in nerve and muscle fibers. The mechanism of the gating module is understood on in part. It is proposed here to investigate the molecule events that link the gating module to the pore. Gating occurs in steps, beginning with movements in the four individual subunits that constitute a channel, and culminating in a concerted step that opens the pore. The last step will be examined in potassium channel mutants in which the concerted step is conveniently isolated from the earlier steps. These channels will be prepared by site-directed-mutagenesis, expressed in a cell line, and examined in patch-clamp experiments. Particular attention will be given to the effects of pore occupancy on the gating apparatus, and to the hypothesis that the concerted step that opens the pore involves a change in occupancy. Sodium channel experiments to examine occupancy effects will be performed primarily on voltage-clamped internally perfused giant axons of the squid, the best preparation for measuring the gating currents that signal the conformation changes in the gating module.
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