Wang G K, Wang S Y
Department of Anesthesia Research Laboratories, Harvard Medical School, Boston, Massachusetts.
J Gen Physiol. 1992 Jan;99(1):1-20. doi: 10.1085/jgp.99.1.1.
Batrachotoxin (BTX)-modified Na+ currents were characterized in GH3 cells with a reversed Na+ gradient under whole-cell voltage clamp conditions. BTX shifts the threshold of Na+ channel activation by approximately 40 mV in the hyperpolarizing direction and nearly eliminates the declining phase of Na+ currents at all voltages, suggesting that Na+ channel inactivation is removed. Paradoxically, the steady-state inactivation (h infinity) of BTX-modified Na+ channels as determined by a two-pulse protocol shows that inactivation is still present and occurs maximally near -70 mV. About 45% of BTX-modified Na+ channels are inactivated at this voltage. The development of inactivation follows a sum of two exponential functions with tau d(fast) = 10 ms and tau d(slow) = 125 ms at -70 mV. Recovery from inactivation can be achieved after hyperpolarizing the membrane to voltages more negative than -120 mV. The time course of recovery is best described by a sum of two exponentials with tau r(fast) = 6.0 ms and tau r(slow) = 240 ms at -170 mV. After reaching a minimum at -70 mV, the h infinity curve of BTX-modified Na+ channels turns upward to reach a constant plateau value of approximately 0.9 at voltages above 0 mV. Evidently, the inactivated, BTX-modified Na+ channels can be forced open at more positive potentials. The reopening kinetics of the inactivated channels follows a single exponential with a time constant of 160 ms at +50 mV. Both chloramine-T (at 0.5 mM) and alpha-scorpion toxin (at 200 nM) diminish the inactivation of BTX-modified Na+ channels. In contrast, benzocaine at 1 mM drastically enhances the inactivation of BTX-modified Na+ channels. The h infinity curve reaches minimum of less than 0.1 at -70 mV, indicating that benzocaine binds preferentially with inactivated, BTX-modified Na+ channels. Together, these results imply that BTX-modified Na+ channels are governed by an inactivation process.
在全细胞膜片钳条件下，对GH3细胞中具有反向钠离子梯度的经蛙毒素（BTX）修饰的钠离子电流进行了特性分析。BTX使钠离子通道激活阈值向超极化方向移动约40 mV，并几乎消除了所有电压下钠离子电流的下降相，这表明钠离子通道失活被消除。矛盾的是，通过双脉冲方案测定的经BTX修饰的钠离子通道的稳态失活（h无穷大）表明失活仍然存在，且在接近-70 mV时最大程度地发生。在该电压下，约45%的经BTX修饰的钠离子通道失活。失活的发展遵循两个指数函数之和，在-70 mV时，快速时间常数（tau d(fast)）= 10 ms，慢速时间常数（tau d(slow)）= 125 ms。将膜超极化到比-120 mV更负的电压后，可实现从失活状态的恢复。恢复的时间进程最好用两个指数之和来描述，在-170 mV时，快速恢复时间常数（tau r(fast)）= 6.0 ms，慢速恢复时间常数（tau r(slow)）= 240 ms。经BTX修饰的钠离子通道的h无穷大曲线在-70 mV达到最小值后，在高于0 mV的电压下向上转向，达到约0.9的恒定平台值。显然，失活的、经BTX修饰的钠离子通道在更正的电位下可被强制打开。失活通道的重新开放动力学在+50 mV时遵循单一指数，时间常数为160 ms。氯胺-T（0.5 mM）和α-蝎毒素（200 nM）均减少了经BTX修饰的钠离子通道的失活。相反，1 mM的苯佐卡因显著增强了经BTX修饰的钠离子通道的失活。h无穷大曲线在-70 mV时达到小于0.1的最小值，表明苯佐卡因优先与失活的、经BTX修饰的钠离子通道结合。总之，这些结果表明经BTX修饰的钠离子通道受失活过程的调控。
