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+

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⁺ channel and not Na⁺‐dependent transport regulates 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⁺ uptake channel MCU 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 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.

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