INACTIVATION OF N-TYPE CALCIUM CURRENT IN CHICK SENSORY NEURONS - CALCIUM AND VOLTAGE-DEPENDENCE

We have studied the inactivation of high-voltage-activated (HVA), omeg a-conotoxin-sensitive, N-type Ca2+ current in embryonic chick dorsal r oot ganglion (DRG) neurons. Voltage steps from -80 to 0 mV produced in ward Ca2+ currents that inactivated in a biphasic manner and were fit well with the sum of two exponentials (with time constants of similar to 100 ms and > 1 s). As reported previously, upon depolarization of t he holding potential to - 40 mV, N current amplitude was significantly reduced and the rapid phase of inactivation all but eliminated (Nowyc ky, M. C., A. P. Fox, and R. W. Tsien. 1985. Nature. 316:440-443; Fox, A. P., M. C. Nowycky, and R. W. Tsien. 1987a. Journal of Physiology. 394:149-172; Swandulla, D., and C. M. Armstrong. 1988. Journal of Gene ral Physiology. 92:197-218; Plummer, M. R., D. E. Logothetis, and P. H ess. 1989. Neuron. 2:1453-1463; Regan, L. J., D. W. Sah, and B. P. Bea n. 1991. Neuron. 6:269-280; Cox, D. H., and K. Dunlap. 1992. Journal o f Neuroscience. 12:906-914). Such kinetic properties might be explaine d by a model in which N channels inactivate by both fast and slow volt age-dependent processes. Alternatively, kinetic models of Ca-dependent inactivation suggest that the biphasic kinetics and holding-potential -dependence of N current inactivation could be due to a combination of Ca-dependent and slow voltage-dependent inactivation mechanisms. To d istinguish between these possibilities we have performed several exper iments to test for the presence of Ca-dependent inactivation. Three li nes of evidence suggest that N channels inactivate in a Ca-dependent m anner. (a) The total extent of inactivation increased 50%, and the rat io of rapid to slow inactivation increased similar to twofold when the concentration of the Ca2+ buffer, EGTA, in the patch pipette was redu ced from 10 to 0.1 mM. (b) With low intracellular EGTA concentrations (0.1 mM), the ratio of rapid to slow inactivation was additionally inc reased when the extracellular Ca2+ concentration was raised from 0.5 t o 5 mM. (c) Substituting Naf for Ca2+ as the permeant ion eliminated t he rapid phase of inactivation. Other results do not support the notio n of current-dependent inactivation, however. Although high intracellu lar EGTA (10 mM) or BAPTA (5 mM) concentrations suppressed the rapid p hase inactivation, they did not eliminate it. Increasing the extracell ular Ca2+ from 0.5 to 5 mM had little effect on this residual fast ina ctivation, indicating that it is not appreciably sensitiv e to Ca2+ in flux under these conditions. Thus, it appeals that both voltage-and cu rrent-dependent mechanisms underlie N current inactivation in chick se nsory neurons, the latter only becoming significant under low Ca2+-buf fering conditions.