Calcium current inactivation in insulin-secreting cells is mediated by calcium influx and membrane depolarization

Abstract

Inactivation of voltage-dependent calcium currents was studied in single, dissociated insulin-secreting HIT cells voltage-clamped by the whole-cell patch-clamp method at room temperature. Na and K currents were suppressed by tetrodotoxin, tetraethylammonium, ATP, 4-aminopyridine and Cs. Ca currents activated in less than 10 ms by depolarizations beyond -50 mV from a holding potential of -100 mV and were identified, as in previous studies, by their sensitivity to divalent cation blockade and permeability to Ba as a charge carrier. Sustained depolarization revealed two kinetically distinct phases of inactivation: a rapid phase inactivated approximately 50% of the current in less than 100 ms while the remaining current was inactivated over the next 10-20 s. Rapid inactivation appeared to be due to Ca2+ influx since it was slowed markedly when Ba2+ was used as the current carrier, while the degree of inactivation increased and decreased with increasing depolarization in direct parallel with the U-shaped current-voltage relationship for inward Ca current. Slow inactivation appeared to be voltage-dependent since current could be inactivated (by approximately 20%) by 10 s long depolarizations to potentials below the threshold for activating Ca current, slow time constants of inactivation were voltage-dependent and slow inactivation persisted when Ca was replaced with Ba. Ca currents with low activation thresholds (in the -50 to -30 mV range) appeared to be preferentially inactivated by the rapid Ca-dependent mechanism. Recovery of slowly inactivated Ca current was very slow and currents inactivated by larger depolarizations required longer recovery time than those elicited by smaller depolarizations. Rapid and slow inactivation mechanisms may be important in understanding the fast spiking and slow plateau depolarizations seen in pancreatic B-cells exposed to stimulatory levels of glucose.