INACTIVATION OF HIT CELL CA2-POTENTIALS( CURRENT BY A SIMULATED BURSTOF CA2+ ACTION)

A novel voltage-clamp protocol was developed to test whether slow inac tivation of Ca2+ current occurs during bursting in insulin-secreting c ells. Single insulin-secreting HIT cells were patch-clamped and their Ca2+ currents were isolated pharmacologically. A computed p-cell burst was used as a voltage-clamp command and the net Ca2+ current elicited was determined as a cadmium difference current. Ca2+ current rapidly activated during the computed plateau and spike depolarizations and th en slowly decayed. Integration of this Ca2+ current yielded an estimat e of total Ca influx. To further analyze Ca2+ current inactivation dur ing a burst, repetitive test pulses to +10 mV were added to the voltag e command. Current elicited by these pulses was constant during the in terburst, but then slowly and reversibly decreased during the depolari zing plateau. This inactivation was reduced by replacing external Ca2 with Ba2+ as a charge carrier, and in some cells inactivation was slo wer in Ba2+. Experimental results were compared with the predictions o f the Keizer-Smolen mathematical model of bursting, after subjecting m odel equations to identical voltage commands. In this model, bursting is driven by the slow, voltage-dependent inactivation of Ca current du ring the plateau active phase. The K-S model could account for the slo pe of the slow decay of spike-elicited Ca current, the waveform of ind ividual Ca current spikes, and the suppression of test pulse-elicited Ca current during a burst command. However, the extent and rate of fas t inactivation were underestimated by the model. Although there are si gnificant differences between the data obtained and the predictions of the K-S model, the overall results show that as predicted by the mode l, Ca current slowly inactivates during a burst of imposed spikes, and inactivation is dependent on both Ca2+ influx and membrane depolariza tion. We thus show that clamping cells to their physiological voltage waveform can be readily accomplished and is a powerful approach for un derstanding the contribution of individual ion currents to bursting.