The quantal gating charge of sodium channel inactivation.

Using a very low noise voltage clamp technique it has been possible to record from the squid giant axon a slow component of gating current (Ig) during the inactivation phase of the macroscopic sodium current (INa) which was hitherto buried in the baseline noise. In order to examine whether this slow Ig contains gating charge that originates from transitions between the open (O) and the inactivated (I) states, which would indicate a true voltage dependence of inactivation, or whether other transitions contribute charge to slow Ig, a new model independent analysis termed isochronic plot analysis has been developed. From a direct correlation of Ig and the time derivative of the sodium conductance dgNa/dt the condition when only O-I transitions occur is detected. Then the ratio of the two signals is constant and a straight line appears in an isochronic plot of Ig vs. dgNa/dt. Its slope does not depend on voltage or time and corresponds to the quantal gating charge of the O-I transition (qh) divided by the single channel ionic conductance (gamma). This condition was found at voltages above -10 mV up to +40 mV and a figure of 1.21 e- was obtained for qh at temperatures of 5 and 15 degrees C. At lower voltages additional charge from other transitions, e.g. closed to open, is displaced during macroscopic inactivation. This means that conventional Eyring rate analysis of the inactivation time constant tau h is only valid above -10 mV and here the figure for qh was confirmed also from this analysis. It is further shown that most of the present controversies surrounding the voltage dependence of inactivation can be clarified. The validity of the isochronic plot analysis has been confirmed using simulated gating and ionic currents.