SINGLE CHANNEL ANALYSIS OF DRUG BLOCKING ACTION

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The long term objectives are to understand the nature of channel gating and ion permeation and the molecular mechanisms by which drugs and blocking ions interact with sodium and potassiun channels. To achieve this, we will use recently developed gigaseal patch clamp technique which permits direct measurement of currents through individual channels and the suction pipette technique which allows macroscopic current measurements. Single channel recordings and macroscopic ionic currents will be compared in neuroblastoma cells in which both kinds of measurements are possible. Studies will be carried out with the following specific aims: 1) to measure single channel properties (conductance and channel open time) with and without Na inactivation, 2) to measure single channel properties in the presence of various types of Na channel blockers with and without Na inactivation, 3) to characterize the internal and external TEA receptors by examining its effect on single channel properties, and 4) to elucidate the nature of aminopyridine block and unblock of K channels by studying its effect on single K channel properties, 5) Using blockers which have affinity for both Na and K channels as probes and voltage, current and temperature changes as pertubators, we will investigate the differences between Na and K channels with respect to the location of binding sites, the size of compounds they will accommodate, and their functional relation to the gating and conductance pathways, and 6) to develop a barrier model to account for drug blocking action in Na and K channels. These studies are expected to add our knowledge of excitable membranes with respect to the mechanisms of ion permeation, gating of ionic channels and to the molecular basis for drug action.