State-dependent inactivation of the alpha 1G T-type calcium channel

We have examined the kinetics of whole-cell T-current in HEK 293 cells stab ly expressing the alpha 1G channel, with symmetrical Na-i(+) and Na-o(+) an d 2 mM Ca-o(2+). After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential -100 mV), currents relaxed exponentiall y at all voltages. The time constant of the relaxation was exponentially vo ltage dependent from -120 to -70 mV (e-fold for 31 mV; tau = 2.5 ms at -100 mV), but tau = 12-17 ms from -40 to +60 mV. This suggests a mixture of vol tage-dependent deactivation (dominating at very negative voltages) and near ly voltage-independent inactivation. Inactivation measured by test pulses f ollowing that protocol was consistent with open-state inactivation. During depolarizations lasting 100-300 ms, inactivation was strong but incomplete (similar to 98%). Inactivation was also produced by long, weak depolarizati ons (tau = 220 ms at -80 mV; V-1/2 = -82 mV), which could not be explained by voltage-independent inactivation exclusively from the open state. Recove ry from inactivation was exponential and fast (tau = 85 ms at -100 mV), but weakly voltage dependent. Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and clo sed-state inactivation. There was little current at -100 mV during recovery from inactivation, consistent with less than or equal to 8% of the channel s recovering through the open state. The results are well described by a ki netic model where inactivation is allosterically coupled to the movement of the first three voltage sensors to activate. One consequence of state-depe ndent inactivation is that alpha 1G channels continue to inactivate after r epolarization, primarily from the open state, which leads to cumulative ina ctivation during repetitive pulses.