Institut für Physiologie und Pathophysiologie, Universität Marburg, Deutschhausstr. 2, 35037 Marburg, Germany.
Cell Calcium. 2010 Jul;48(1):19-27. doi: 10.1016/j.ceca.2010.06.002. Epub 2010 Jul 14.
STIM1 'senses' decreases in endoplasmic reticular (ER) luminal Ca(2+) and induces store-operated Ca(2+) (SOC) entry through plasma membrane Orai channels. The Ca(2+)/calmodulin-activated K(+) channel K(Ca)3.1 (previously known as SK4) has been implicated as an 'amplifier' of the Ca(2+)-release activated Ca(2+) (CRAC) current, especially in T lymphocytes. We have previously shown that human macrophages express K(Ca)3.1, and here we used the whole-cell patch-clamp technique to investigate the activity of these channels during Ca(2+) store depletion and store-operated Ca(2+) influx. Using RT-PCR, we found that macrophages express the elementary CRAC channel components Orai1 and STIM1, as well as Orai2, Orai3 and STIM2, but not the putatively STIM1-activated channels TRPC1, TRPC3-7 or TRPV6. In whole-cell configuration, a robust Ca(2+)-induced outwardly rectifying K(+) current inhibited by clotrimazole and augmented by DC-EBIO could be detected, consistent with K(Ca)3.1 channel current (also known as intermediate-conductance IK1). Introduction of extracellular Ca(2+) following Ca(2+) store depletion via P2Y(2) receptors induced a robust charybdotoxin (CTX)- and 2-APB-sensitive outward K(+) current and hyperpolarization. We also found that SOC entry induced by thapsigargin treatment induced CTX-sensitive K(+) current in HEK293 cells transiently expressing K(Ca)3.1. Our data suggest that SOC and K(Ca)3.1 channels are tightly coupled, such that a small Ca(2+) influx current induces a much large K(Ca)3.1 channel current and hyperpolarization, providing the necessary electrochemical driving force for prolonged Ca(2+) signaling and store repletion.
STIM1“感知”内质网（ER）腔中 Ca(2+) 的减少，并通过质膜 Orai 通道诱导储存操作的 Ca(2+)（SOC）进入。Ca(2+)/钙调蛋白激活的 K(+) 通道 K(Ca)3.1（以前称为 SK4）被认为是 Ca(2+)释放激活的 Ca(2+)（CRAC）电流的“放大器”，尤其是在 T 淋巴细胞中。我们之前已经表明，人类巨噬细胞表达 K(Ca)3.1，在这里我们使用全细胞膜片钳技术来研究 Ca(2+)储存耗竭和储存操作的 Ca(2+)流入期间这些通道的活性。通过 RT-PCR，我们发现巨噬细胞表达基本的 CRAC 通道成分 Orai1 和 STIM1，以及 Orai2、Orai3 和 STIM2，但不表达假定的由 STIM1 激活的通道 TRPC1、TRPC3-7 或 TRPV6。在全细胞构型中，可以检测到由钙诱导的向外整流的钾电流，该电流被克霉唑抑制并被 DC-EBIO 增强，与 K(Ca)3.1 通道电流（也称为中间电导 IK1）一致。通过 P2Y(2)受体耗尽 Ca(2+)储存后引入细胞外 Ca(2+)，会诱导出强大的河豚毒素（CTX）和 2-APB 敏感的外向 K(+)电流和超极化。我们还发现，用 thapsigargin 处理诱导 SOC 进入会在瞬时表达 K(Ca)3.1 的 HEK293 细胞中诱导 CTX 敏感的 K(+)电流。我们的数据表明，SOC 和 K(Ca)3.1 通道紧密耦联，因此少量的 Ca(2+)内流电流会诱导出更大的 K(Ca)3.1 通道电流和超极化，为长时间的 Ca(2+)信号和储存补充提供必要的电化学驱动力。
