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

摘要

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 .