Cox D H, Dunlap K
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
J Gen Physiol. 1994 Aug;104(2):311-36. doi: 10.1085/jgp.104.2.311.
We have studied the inactivation of high-voltage-activated (HVA), omega-conotoxin-sensitive, N-type Ca2+ current in embryonic chick dorsal root ganglion (DRG) neurons. Voltage steps from -80 to 0 mV produced inward Ca2+ currents that inactivated in a biphasic manner and were fit well with the sum of two exponentials (with time constants of approximately 100 ms and > 1 s). As reported previously, upon depolarization of the holding potential to -40 mV, N current amplitude was significantly reduced and the rapid phase of inactivation all but eliminated (Nowycky, 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 General Physiology. 92:197-218; Plummer, M. R., D. E. Logothetis, and P. Hess. 1989. Neuron. 2:1453-1463; Regan, L. J., D. W. Sah, and B. P. Bean. 1991. Neuron. 6:269-280; Cox, D. H., and K. Dunlap. 1992. Journal of Neuroscience. 12:906-914). Such kinetic properties might be explained by a model in which N channels inactivate by both fast and slow voltage-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 distinguish between these possibilities we have performed several experiments to test for the presence of Ca-dependent inactivation. Three lines of evidence suggest that N channels inactivate in a Ca-dependent manner. (a) The total extent of inactivation increased 50%, and the ratio of rapid to slow inactivation increased approximately twofold when the concentration of the Ca2+ buffer, EGTA, in the patch pipette was reduced from 10 to 0.1 mM. (b) With low intracellular EGTA concentrations (0.1 mM), the ratio of rapid to slow inactivation was additionally increased when the extracellular Ca2+ concentration was raised from 0.5 to 5 mM. (c) Substituting Na+ for Ca2+ as the permeant ion eliminated the rapid phase of inactivation. Other results do not support the notion of current-dependent inactivation, however. Although high intracellular EGTA (10 mM) or BAPTA (5 mM) concentrations suppressed the rapid phase inactivation, they did not eliminate it. Increasing the extracellular Ca2+ from 0.5 to 5 mM had little effect on this residual fast inactivation, indicating that it is not appreciably sensitive to Ca2+ influx under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
我们研究了胚胎期鸡背根神经节（DRG）神经元中高压激活（HVA）、ω-芋螺毒素敏感的N型Ca2+电流的失活情况。从-80 mV到0 mV的电压阶跃产生内向Ca2+电流，该电流以双相方式失活，并且与两个指数之和拟合良好（时间常数分别约为100 ms和>1 s）。如先前报道，当钳制电位去极化至-40 mV时，N电流幅度显著降低，且失活的快速相几乎完全消除（Nowycky，M. C.，A. P. Fox和R. W. Tsien. 1985.《自然》. 316:440 - 443；Fox，A. P.，M. C. Nowycky和R. W. Tsien. 1987a.《生理学杂志》. 394:149 - 172；Swandulla，D.和C. M. Armstrong. 1988.《普通生理学杂志》. 92:197 - 218；Plummer，M. R.，D. E. Logothetis和P. Hess. 1989.《神经元》. 2:1453 - 1463；Regan，L. J.，D. W. Sah和B. P. Bean. 1991.《神经元》. 6:269 - 280；Cox，D. H.和K. Dunlap. 1992.《神经科学杂志》. 12:906 - 914）。这种动力学特性可能由一个模型来解释，即N通道通过快速和缓慢的电压依赖性过程失活。或者，钙依赖性失活的动力学模型表明，N电流失活的双相动力学和钳制电位依赖性可能是由于钙依赖性和缓慢电压依赖性失活机制的组合。为了区分这些可能性，我们进行了几个实验来检测钙依赖性失活的存在。三条证据表明N通道以钙依赖性方式失活。（a）当膜片钳微电极内Ca²⁺缓冲剂EGTA的浓度从10 mM降至0.1 mM时，失活的总程度增加了50%，快速失活与缓慢失活的比例增加了约两倍。（b）在细胞内EGTA浓度较低（0.1 mM）时，当细胞外Ca²⁺浓度从0.5 mM升高到5 mM时，快速失活与缓慢失活的比例进一步增加。（c）用Na⁺替代Ca²⁺作为通透离子消除了失活的快速相。然而，其他结果并不支持电流依赖性失活的观点。尽管细胞内高浓度的EGTA（10 mM）或BAPTA（5 mM）抑制了快速相失活，但并未消除它。将细胞外Ca²⁺浓度从0.5 mM增加到5 mM对这种残余的快速失活影响很小，表明在这些条件下它对Ca²⁺内流不太敏感。（摘要截断于400字）
