Bakowski D, Parekh A B
Laboratory of Molecular and Cellular Signalling, Department of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, UK.
J Physiol. 2000 Dec 1;529 Pt 2(Pt 2):295-306. doi: 10.1111/j.1469-7793.2000.00295.x.
Tight-seal whole-cell patch-clamp experiments were carried out in order to investigate the effects of different holding potentials on the rate of development and amplitude of the Ca2+ release-activated Ca2+ current ICRAC in rat basophilic leukaemia (RBL-1) cells. ICRAC was monitored at -80 mV from fast voltage ramps, spanning 200 mV in 50 ms. At hyperpolarised potentials, the macroscopic CRAC conductance was lower than that seen at depolarised potentials. The conductance increased almost 5-fold over the voltage range -60 to +40 mV and was seen when the stores were depleted either by the combination of IP3 and thapsigargin in high Ca2+ buffer, or passively with 10 mM EGTA or BAPTA. The voltage-dependent conductance of the CRAC channels could not be fully accounted for by Ca2+-dependent fast inactivation, nor by other slower inhibitory mechanisms. It also did not seem to involve intracellular Mg2+ or the polycations spermine and spermidine. Voltage step relaxation experiments revealed that the voltage-dependent conductance changes developed and reversed slowly, with a time constant of several seconds at -60 mV. In the presence of physiological levels of intracellular Ca2+ buffers, ICRAC was barely detectable when cells were clamped at -60 mV and dialysed with IP3 and thapsigargin, but at 0 mV the current in low Ca2+ buffer was as large as that seen in high Ca2+ buffer. Our results suggest that CRAC channels exhibit slow voltage-dependent conductance changes which can triple the current amplitude over the physiological range of voltages normally encountered by these cells. The role of this conductance change and possible underlying mechanisms are discussed.
为了研究不同钳制电位对大鼠嗜碱性粒细胞白血病（RBL-1）细胞中Ca2+释放激活的Ca2+电流ICRAC的发育速率和幅度的影响，进行了紧密封全细胞膜片钳实验。通过快速电压斜坡在-80 mV监测ICRAC，斜坡在50 ms内跨越200 mV。在超极化电位下，宏观CRAC电导低于去极化电位下的电导。在-60至+40 mV的电压范围内，电导增加了近5倍，并且当在高Ca2+缓冲液中通过IP3和毒胡萝卜素的组合使储存耗尽时，或者在被动使用10 mM EGTA或BAPTA时可以观察到。CRAC通道的电压依赖性电导不能完全由Ca2+依赖性快速失活或其他较慢的抑制机制来解释。它似乎也不涉及细胞内Mg2+或多阳离子精胺和亚精胺。电压阶跃松弛实验表明，电压依赖性电导变化发展和逆转缓慢，在-60 mV时时间常数为几秒。在存在生理水平的细胞内Ca2+缓冲液的情况下，当细胞钳制在-60 mV并用IP3和毒胡萝卜素透析时，几乎检测不到ICRAC，但在0 mV时，低Ca2+缓冲液中的电流与高Ca2+缓冲液中的电流一样大。我们的结果表明，CRAC通道表现出缓慢的电压依赖性电导变化，这可以使电流幅度在这些细胞通常遇到的生理电压范围内增加两倍。讨论了这种电导变化的作用和可能的潜在机制。
