Department of Physiology, Anatomy and Genetics, University of Oxford, Sherrington Building, Parks Road, Oxford, OX1 3PT, UK.
Chemistry Research Laboratory, Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.
J Physiol. 2020 May;598(9):1753-1773. doi: 10.1113/JP276551. Epub 2019 Feb 6.
Ca entry through Ca release-activated Ca channels activates numerous cellular responses. Under physiological conditions of weak intracellular Ca buffering, mitochondrial Ca uptake regulates CRAC channel activity. Knockdown of the mitochondrial Ca uniporter channel prevented the development of I in weak buffer but not when strong buffer was used instead. Removal of either extracellular or intra-pipette Na had no effect on the selectivity, kinetics, amplitude, rectification or reversal potential of whole-cell CRAC current. Knockdown of the mitochondrial Na -Ca exchanger did not prevent the development of I in strong or weak Ca buffer. Whole cell CRAC current is Ca -selective. Mitochondrial Ca channels, and not Na -dependent transport, regulate CRAC channels under physiological conditions.
Ca entry through store-operated Ca release-activated Ca (CRAC) channels plays a central role in activation of a range of cellular responses over broad spatial and temporal bandwidths. Mitochondria, through their ability to take up cytosolic Ca , are important regulators of CRAC channel activity under physiological conditions of weak intracellular Ca buffering. The mitochondrial Ca transporter(s) that regulates CRAC channels is unclear and could involve the 40 kDa mitochondrial Ca uniporter (MCU) channel or the Na -Ca -Li exchanger (NCLX). Here, we have investigated the involvement of these mitochondrial Ca transporters in supporting the CRAC current (I ) under a range of conditions in RBL mast cells. Knockdown of the MCU channel impaired the activation of I under physiological conditions of weak intracellular Ca buffering. In strong Ca buffer, knockdown of the MCU channel did not inhibit I development demonstrating that mitochondria regulate CRAC channels under physiological conditions by buffering of cytosolic Ca via the MCU channel. Surprisingly, manipulations that altered extracellular Na , cytosolic Na or both failed to inhibit the development of I in either strong or weak intracellular Ca buffer. Knockdown of NCLX also did not affect I . Prolonged removal of external Na also had no significant effect on store-operated Ca entry, on cytosolic Ca oscillations generated by receptor stimulation or on CRAC channel-driven gene expression. In the RBL mast cell, Ca flux through the MCU but not NCLX is indispensable for activation of I .
钙通过钙释放激活的钙通道（CRAC）进入细胞会激活许多细胞反应。在细胞内钙缓冲能力较弱的生理条件下，线粒体摄取钙可调节 CRAC 通道活性。在弱缓冲液中，敲低线粒体钙单向转运蛋白通道会阻止 I 的产生，但在使用强缓冲液时则不会。去除细胞外或细胞内的 Na 对全细胞 CRAC 电流的选择性、动力学、幅度、整流或反转电位均没有影响。敲低线粒体 Na -Ca 交换器并不能阻止强或弱钙缓冲液中 I 的产生。全细胞 CRAC 电流是钙选择性的。在线粒体钙通道，而不是钠依赖性转运的作用下，在生理条件下调节 CRAC 通道。
通过储存操作的钙释放激活的钙（CRAC）通道进入的钙在广泛的时空带宽内激活一系列细胞反应中起着核心作用。在线粒体在弱细胞内钙缓冲条件下，通过摄取胞质钙，是调节 CRAC 通道活性的重要调节因子。调节 CRAC 通道的线粒体钙转运蛋白尚不清楚，可能涉及 40 kDa 线粒体钙单向转运蛋白（MCU）通道或 Na -Ca -Li 交换器（NCLX）。在这里，我们研究了这些线粒体钙转运蛋白在 RBL 肥大细胞中在一系列条件下对支持 CRAC 电流（I ）的作用。在弱细胞内钙缓冲条件下，敲低 MCU 通道会损害 I 的激活。在强钙缓冲液中，敲低 MCU 通道并不能抑制 I 的发展，这表明在线粒体通过 MCU 通道缓冲胞质钙来调节 CRAC 通道在生理条件下。令人惊讶的是，改变细胞外 Na 、胞质 Na 或两者都不能抑制在强或弱细胞内钙缓冲液中 I 的发展。敲低 NCLX 也不会影响 I 。长时间去除外部 Na 也对受刺激受体产生的钙内流、胞质 Ca 振荡或 CRAC 通道驱动的基因表达没有显著影响。在 RBL 肥大细胞中，通过 MCU 而不是 NCLX 的钙通量对于 I 的激活是必不可少的。
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