The whole-cell Ca2+ release-activated Ca2+ current, ICRAC , is regulated by the mitochondrial Ca2+ uniporter channel and is independent of extracellular and cytosolic Na.

Abstract

Key points Ca2+ entry through Ca2+ release‐activated Ca2+ channels activates numerous cellular responses. Under physiological conditions of weak intracellular Ca2+ buffering, mitochondrial Ca2+ uptake regulates CRAC channel activity.Knockdown of the mitochondrial Ca2+ uniporter channel prevented the development of I CRAC 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+–Ca2+ exchanger did not prevent the development of I CRAC in strong or weak Ca2+ buffer.Whole cell CRAC current is Ca2+‐selective.Mitochondrial Ca2+ channels, and not Na+‐dependent transport, regulate CRAC channels under physiological conditions. Ca2+ entry through store‐operated Ca2+ release‐activated Ca2+ (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 Ca2+, are important regulators of CRAC channel activity under physiological conditions of weak intracellular Ca2+ buffering. The mitochondrial Ca2+ transporter(s) that regulates CRAC channels is unclear and could involve the 40 kDa mitochondrial Ca2+ uniporter (MCU) channel or the Na+–Ca2+–Li+ exchanger (NCLX). Here, we have investigated the involvement of these mitochondrial Ca2+ transporters in supporting the CRAC current (I CRAC) under a range of conditions in RBL mast cells. Knockdown of the MCU channel impaired the activation of I CRAC under physiological conditions of weak intracellular Ca2+ buffering. In strong Ca2+ buffer, knockdown of the MCU channel did not inhibit I CRAC development demonstrating that mitochondria regulate CRAC channels under physiological conditions by buffering of cytosolic Ca2+ via the MCU channel. Surprisingly, manipulations that altered extracellular Na+, cytosolic Na+ or both failed to inhibit the development of I CRAC in either strong or weak intracellular Ca2+ buffer. Knockdown of NCLX also did not affect I CRAC. Prolonged removal of external Na+ also had no significant effect on store‐operated Ca2+ entry, on cytosolic Ca2+ oscillations generated by receptor stimulation or on CRAC channel‐driven gene expression. In the RBL mast cell, Ca2+ flux through the MCU but not NCLX is indispensable for activation of I CRAC.