The whole‐cell Ca²⁺ release‐activated Ca²⁺ current, <i>I</i>_CRAC, is regulated by the mitochondrial Ca²⁺ uniporter channel and is independent of extracellular and cytosolic Na⁺

Abstract

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 .