Reuveny E, Narahashi T
Department of Pharmacology, Northwestern University Medical School, Chicago, IL 60611.
Brain Res. 1993 Feb 12;603(1):64-73. doi: 10.1016/0006-8993(93)91300-h.
Voltage-activated calcium channel currents were recorded from differentiated human neuroblastoma cells. SK-N-SH-SY5Y (SH-SY5Y) line, using patch-clamp techniques. Experimental solutions were designed to suppress sodium and potassium channel currents, and barium ions were used as the charge carrier. Two distinct types of calcium channel currents (N- and L-like) were identified based on their time-dependent inactivation, pharmacology and single-channel conductances. N- and L-like calcium channel currents were evoked by step depolarizing pulses to potentials more positive than -40 mV from a holding potential of -100 mV. The N-like component showed time-dependent inactivation during maintained depolarization with a time constant of tau f approximately 100 ms, whereas the L-like currents showed very slow inactivation with a time constant of tau s approximately 1,000 ms. Steady-state inactivation of currents evoked from a holding potential of -100 mV had two distinct components. One component involved the reduction of the transient current and had a half-maximal current at approximately -66 mV, whereas the other component involved the reduction of the steady-state current in the range of -35 to 0 mV with a half-maximal current at approximately -17 mV. Bay K 8644 (5 microM), had two distinct actions, one was the increase (50%) of the current associated with a depolarizing pulse to +10 mV. The second action was the increase in the peak amplitude of the tail current and the slowing of the deactivation kinetics. Omega-conotoxin at 1 microM irreversibly reduced the N-like current, sparing a component that was still sensitive to 5 microM Bay K 8644. The single-channel currents recorded with the cell-attached configuration of the patch clamp revealed two distinct conductances: a large approximately 28 pS and a small approximately 16 pS, corresponding to the L- and N-like channels, respectively. Bay K 8644 at 5 microM increased the mean open time of L-like single channel currents without changing single-channel conductance.
采用膜片钳技术，从分化的人神经母细胞瘤细胞SK-N-SH-SY5Y（SH-SY5Y）系中记录电压门控钙通道电流。实验溶液旨在抑制钠通道和钾通道电流，钡离子用作电荷载体。根据其时间依赖性失活、药理学特性和单通道电导，鉴定出两种不同类型的钙通道电流（N型和L型）。从-100 mV的 holding 电位通过逐步去极化脉冲将电位激发到比-40 mV更正的电位，从而诱发N型和L型钙通道电流。N型成分在持续去极化过程中表现出时间依赖性失活，时间常数tau f约为100 ms，而L型电流表现出非常缓慢的失活，时间常数tau s约为1000 ms。从-100 mV的 holding 电位诱发的电流的稳态失活有两个不同的成分。一个成分涉及瞬态电流的减少，在约-66 mV处有半数最大电流，而另一个成分涉及-35至0 mV范围内稳态电流的减少，在约-17 mV处有半数最大电流。5 microM的Bay K 8644有两种不同的作用，一种是与去极化脉冲至+10 mV相关的电流增加（50%）。第二种作用是尾电流峰值幅度的增加和失活动力学的减慢。1 microM的ω-芋螺毒素不可逆地降低N型电流，保留了对5 microM Bay K 8644仍敏感的一个成分。膜片钳细胞贴附配置记录的单通道电流显示出两种不同的电导：一个大的约28 pS和一个小的约16 pS，分别对应L型和N型通道。5 microM的Bay K 8644增加了L型单通道电流的平均开放时间，而不改变单通道电导。
