The whole‐cell Ca(2+) release‐activated Ca(2+) current, I (CRAC), is regulated by the mitochondrial Ca(2+) uniporter channel and is independent of extracellular and cytosolic Na(+)

KEY POINTS: Ca(2+) entry through Ca(2+) release‐activated Ca(2+) channels activates numerous cellular responses. Under physiological conditions of weak intracellular Ca(2+) buffering, mitochondrial Ca(2+) uptake regulates CRAC channel activity. Knockdown of the mitochondrial Ca(2+) 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(+)–Ca(2+) exchanger did not prevent the development of I (CRAC) in strong or weak Ca(2+) buffer. Whole cell CRAC current is Ca(2+)‐selective. Mitochondrial Ca(2+) channels, and not Na(+)‐dependent transport, regulate CRAC channels under physiological conditions. ABSTRACT: Ca(2+) entry through store‐operated Ca(2+) release‐activated Ca(2+) (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(2+), are important regulators of CRAC channel activity under physiological conditions of weak intracellular Ca(2+) buffering. The mitochondrial Ca(2+) transporter(s) that regulates CRAC channels is unclear and could involve the 40 kDa mitochondrial Ca(2+) uniporter (MCU) channel or the Na(+)–Ca(2+)–Li(+) exchanger (NCLX). Here, we have investigated the involvement of these mitochondrial Ca(2+) 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 Ca(2+) buffering. In strong Ca(2+) 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 Ca(2+) 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 Ca(2+) buffer. Knockdown of NCLX also did not affect I (CRAC). Prolonged removal of external Na(+) also had no significant effect on store‐operated Ca(2+) entry, on cytosolic Ca(2+) oscillations generated by receptor stimulation or on CRAC channel‐driven gene expression. In the RBL mast cell, Ca(2+) flux through the MCU but not NCLX is indispensable for activation of I (CRAC).