Zweifach A, Lewis R S
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, California 94305, USA.
J Gen Physiol. 1996 May;107(5):597-610. doi: 10.1085/jgp.107.5.597.
The depletion of intracellular Ca2+ stores triggers the opening of Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane of T lymphocytes. We have investigated the additional role of extracellular Ca2+ (Ca02+) in promoting CRAC channel activation in Jurkat leukemic T cells. Ca2+ stores were depleted with 1 microM thapsigargin in the nominal absence of Ca02+ with 12 mM EGTA or BAPTA in the recording pipette. Subsequent application of Ca02+ caused ICRAC to appear in two phases. The initial phase was complete within 1 s and reflects channels that were open in the absence of Ca02+. The second phase consisted of a severalfold exponential increase in current amplitude with a time constant of 5-10 s; we call this increase Ca(2+)-dependent potentiation, or CDP. The shape of the current-voltage relation and the inferred single-channel current amplitude are unchanged during CDP, indicating that CDP reflects an alteration in channel gating rather than permeation. The extent of CDP is modulated by voltage, increasing from approximately 50% at +50 mV to approximately 350% at -75 mV in the presence of 2 mM Ca02+. The voltage dependence of CDP also causes ICRAC to increase slowly during prolonged hyperpolarizations in the constant presence of Ca02+. CDP is not affected by exogenous intracellular Ca2+ buffers, and Ni2+, a CRAC channel blocker, can cause potentiation. Thus, the underlying Ca2+ binding site is not intracellular. Ba2+ has little or no ability to potentiate CRAC channels. These results demonstrate that the store-depletion signal by itself triggers only a small fraction of capacitative Ca2+ entry and establish Ca2+ as a potent cofactor in this process. CDP confers a previously unrecognized voltage dependence and slow time dependence on CRAC channel activation that may contribute to the dynamic behavior of ICRAC.
细胞内Ca2+储备的耗尽会触发T淋巴细胞质膜中Ca2+释放激活的Ca2+（CRAC）通道的开放。我们研究了细胞外Ca2+（Ca02+）在促进Jurkat白血病T细胞中CRAC通道激活方面的额外作用。在记录微管中使用12 mM乙二醇双四乙酸（EGTA）或1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA），在名义上不存在Ca02+的情况下，用1 microM毒胡萝卜素耗尽Ca2+储备。随后施加Ca02+导致CRAC电流（ICRAC）分两个阶段出现。初始阶段在1秒内完成，反映了在不存在Ca02+时开放的通道。第二阶段包括电流幅度呈几倍的指数增加，时间常数为5 - 10秒；我们将这种增加称为Ca(2+)依赖性增强，或CDP。在CDP期间，电流 - 电压关系的形状和推断的单通道电流幅度不变，表明CDP反映的是通道门控的改变而非通透的改变。CDP的程度受电压调节，在存在2 mM Ca02+的情况下，从 +50 mV时的约50%增加到 -75 mV时的约350%。CDP的电压依赖性还会导致在持续存在Ca02+的情况下长时间超极化期间ICRAC缓慢增加。CDP不受外源性细胞内Ca2+缓冲剂的影响，并且CRAC通道阻滞剂Ni2+可导致增强作用。因此，潜在的Ca2+结合位点不在细胞内。Ba2+几乎没有或没有增强CRAC通道的能力。这些结果表明，储备耗尽信号本身仅触发了一小部分容量性Ca2+内流，并确立了Ca +作为该过程中一种有效的辅助因子。CDP赋予了CRAC通道激活一种先前未被认识到的电压依赖性和缓慢的时间依赖性，这可能有助于ICRAC的动态行为。
