Zweifach A, Lewis R S
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, California 94305, USA.
J Gen Physiol. 1995 Feb;105(2):209-26. doi: 10.1085/jgp.105.2.209.
Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.
利用全细胞膜片钳记录和[Ca2+]i测量技术，在Jurkat白血病T淋巴细胞中研究了Ca2+释放激活的Ca2+（CRAC）通道的快速失活。在细胞外Ca2+浓度为22 mM的情况下，当超极化脉冲至比-40 mV更负的电位时，Ca2+电流以双指数时间进程下降（时间常数分别为8 - 30 ms和50 - 150 ms）。多条证据表明，快速失活过程是Ca2+依赖性而非电压依赖性的。首先，通过开放通道增加Ca2+进入速率的条件会增强失活的速度和程度。其次，当Ba2+作为电荷载体时，失活显著降低。第三，用快速Ca2+缓冲剂BAPTA（12 mM）进行细胞内透析会减缓失活，但将较慢的螯合剂EGTA的胞质浓度从1.2 mM提高到12 mM则不会。复极化至-12 mV后200 ms内快速失活完全恢复。快速失活不受开放CRAC通道数量或整体[Ca2+]i变化的影响。这些结果表明，ICRAC的快速失活是由单个通道细胞内口附近的Ca作用引起的，并且通过一个CRAC通道进入的Ca不会影响相邻通道。一个在存在移动缓冲剂情况下Ca2+扩散的简单模型预测，多个Ca2+失活位点位于距孔细胞内口3 - 4 nm处，这与CRAC通道本身的位置一致。
