Voltage sensitive ionic channels in excitable membranes are thought to be opened and closed by the movement of charged "gates." Asymmetric currents attributed to the movement of Na channel gates have been measured in various tissues. A difficulty with all these observations is the inability to separate true gating current from other possible contaminating asymmetric currents. This proposal presents a new approach to this problem. Membrane electrical properties will be measured in frog slow muscle fibers before and at varying intervals following denervation. Prior to denervation frog slow fibers have no Na channels and thus asymmetric current measured in these preparations will serve as a control measurement of non-Na channel asymmetric current. Following denervation Na channels appear in the slow fiber membrane. Asymmetric currents and Na currents will be measured in these preparations, and that portion of asymmetric current that increases in proportion to Na conductance will give a more accurate estimate of true gating current than has been possible previously. In other experiments, channel number will be determined by fluctuation analysis. By measuring channel number and gating current in the same cell, it will be possible to determine the amount of gating charge associated with an individual channel. Gating currents are the most direct measure of the molecular events that cause channel opening. Determination of the magnitude and kinetics of true gating current will lead to a better understanding of the molecular mechanisms underlying membrane excitability.