In this study, sodium channel molecular kinetic transitions are resolved by gating current harmonics. Gating (asymmetry) currents were obtained from voltage-clamped squid giant axons in sinusoidally driven dynamic steady-states with frequency and mean membrane potential as independent variables. Harmonic content of the records as a function of the independent variables shows three kinetic sub-processes (primary and secondary activation, and inactivation), and the number of states and values of rate constants in each sub-process. Protease-treated axons have a secondary activation gating process with two states corresponding to closed and open activation gates. The strongly voltage-dependent primary process has at least five kinetic substates which determine probability for transitions in the secondary kinetics. Flickering between open and closed states is a natural kinetic consequence. The harmonic content of records from axons untreated with protease show that inactivation gating can block three of five primary activation states, thereby substantially reducing gating current. Inactivation and primary activation appear to be coupled by reciprocal steric hindrance. Inactivation gating has no direct voltage dependence. The sodium channel primary amino acid residue sequence suggests that the processes occur at four molecular locations in parallel. At present, experiments using chemical agents which specifically screen the charges on lysines and argenines are being performed to test the inferences drawn from model predictions. As expected, both argenine and lysine blockers profoundly alter channel gating kinetics in predictable ways.
