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 (I_g) during the inactivation phase of the macroscopic sodium current (I_Na) which was hitherto buried in the baseline noise. In order to examine whether this slowI_g 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 slowI_g, a new model independent analysis termed isochronic plot analysis has been developed. From a direct correlation ofI_g and the time derivative of the sodium conductance dg_Na/d 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 ofI_g vs. dg_Na/d. Its slope does not depend on voltage or time and corresponds to the quantal gating charge of the O-I transition (q_h) divided by the single channel ionic conductance (). This condition was found at voltages above – 10 mV up to + 40 mV and a figure of 1.21e^– was obtained forq_h at temperatures of 5 and 15°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 _h is only valid above – 10 mV and here the figure forq_h 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.Abbreviations I_g gating current - I_Na sodium ionic current - g_Na macroscopic sodium conductance - single channel conductance - C, O, I closed, open, inactivated state occupancy of channels - g_h quantal charge displaced in a single O-I transition of Na channel - e^– equivalent electron charge - h index referring to inactivation process - S_l limiting slope in isochronic plot see Eq.(3) - fractional distance, see Fig. 4 and (4, 5) - TMA tetramethylammonium - TTX tetrodotoxin - Tris tris(hydroxymethyl)aminomethane - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid