Patch clamp analysis of Na channel gating in mammalian myocardium: reconstruction of double pulse inactivation and voltage dependence of Na currents.

Isolated ventricular cells of the mouse heart were prepared by an enzyme digestion procedure. Unitary Na currents were recorded with the patch clamp technique from cell attached patched. Macroscopic Na currents were obtained as mean of 38 consecutive sweeps of cell attached patches with up to 100 channels each. Double pulse inactivation of macroscopic currents showed an increase of the test current amplitude at test pulse potential Vt = -30 mV after short prepulses at prepulse potential Vp = -50 mV. The open time distribution of single channels could be fitted monoexponentially yielding a mean open time tau 0. Between -70 mV and -20 mV tau 0 showed a bell shaped voltage dependence. The probability to record an empty sweep PA (0) had a minimum value at -50 mV and increased towards less negative potentials. The current-voltage relation of the unitary current was linear between -60 mV and -20 mV yielding a slope conductance of 18.5 pS at room temperature. There was no indication of the existence of more than a single unitary current level. For the apparent peak open probability of a Na channel a sigmoidal voltage dependence was found between -70 mV and -20 mV. Single channel recording reveals that the time course of double pulse inactivation coincides with the increase in the number of empty sweeps leaving tau 0 unchanged. The Markov models with two closed states, one open and one inactivated, were used for quantitative analysis. Useful were only models allowing inactivation both from at least one of the closed states and from the open state. For both model types a set of rate constants was calculated from single channel data and from the time course of the opening probability at -50 mV and -30 mV, respectively. The model with the allowed inactivation from the second closed state (M1) was superior to that with the allowed inactivation from the first closed state (M3) by the prediction of the time course of the early double pulse inactivation at Vp = 50 mV and Vt = 30 mV and, based on these data, by the prediction of the amplitude of Na currents at -40 mV and -60 mV.