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.
Samanta K., Bakowski D., Amin N., Parekh AB.
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 ICRAC 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 ICRAC 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 ICRAC under a range of conditions in RBL mast cells. Knockdown of the MCU impaired the activation of ICRAC under physiological conditions of weak intracellular Ca2+ buffering. In strong Ca2+ buffer, knockdown of the MCU channel did not inhibit ICRAC 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 ICRAC in either strong or weak intracellular Ca2+ buffer. Knockdown of NCLX also did not affect ICRAC . 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 ICRAC . This article is protected by copyright. All rights reserved.