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Antigen receptor stimulation triggers cytosolic Ca 2+ signals, which activate transcriptional and metabolic programs critical for immune function. B-cell receptor (BCR) engagement causes rapid cytosolic Ca 2+ rise through the ubiquitous store-operated calcium entry (SOCE) pathway. Slc8b1, which encodes the mitochondrial Na + /Ca 2+ exchanger (NCLX), extrudes Ca 2+ out of the mitochondria and maintains optimal SOCE activity. Inhibition of NCLX in DT40 and A20 B lymphocyte lines was recently shown to impair cytosolic Ca 2+ transients in response to antigen-receptor stimulation, however the downstream functional consequences of this impairment remain unclear. Here, we generated Slc8b1 knockout A20 B-cell lines using CRISPR/Cas9 technology and B-cell specific Slc8b1 knockout mice. Surprisingly, while loss of Slc8b1 in B lymphocytes led to reduction in SOCE, it had a marginal effect on mitochondrial Ca 2+ extrusion, suggesting that NCLX is not the major mitochondrial Ca 2+ extrusion mechanism in B cells. Furthermore, endoplasmic reticulum (ER) Ca 2+ content and rates of ER depletion and refilling remained unaltered in Slc8b1 knockout B cells. Slc8b1 deficiency increased mitochondrial production of oxidants, reduced mitochondrial bioenergetics and altered mitochondrial ultrastructure. B-cell specific Slc8b1 knockout mice showed reduced germinal center B cell responses following foreign antigen and pathogen driven immune responses. Our studies provide novel insights into the function of Slc8b1 in germinal center B cells and its contribution to B-cell signaling and effector function., Competing Interests: Declaration of Competing Interest Mohamed Trebak is a paid consultant of Seeker Biologicals Inc. The other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022. Published by Elsevier Ltd.)

Mitochondrial Ca 2+ uptake tailors the strength of stimulation of plasma membrane phospholipase C-coupled receptors to that of cellular bioenergetics. However, how Ca 2+ uptake by the mitochondrial Ca 2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca 2+ signaling is unknown. Here, we used CRISPR/Cas9 gene knockout, subcellular Ca 2+ imaging, and mathematical modeling to show that MCU is a universal regulator of intracellular Ca 2+ signaling across mammalian cell types. MCU activity sustains cytosolic Ca 2+ signaling by preventing Ca 2+ -dependent inactivation of store-operated Ca 2+ release-activated Ca 2+ channels and by inhibiting Ca 2+ extrusion. Paradoxically, MCU knockout (MCU-KO) enhanced cytosolic Ca 2+ responses to store depletion. Physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca 2+ oscillations, endoplasmic reticulum Ca 2+ refilling, nuclear translocation of nuclear factor for activated T cells transcription factors, and cell proliferation, without altering inositol-1,4,5-trisphosphate receptor activity. Our data show that MCU has dual counterbalancing functions at the cytosol-mitochondria interface, whereby the cell-specific MCU-dependent cytosolic Ca 2+ clearance and buffering capacity of mitochondria reciprocally regulate interorganellar Ca 2+ transfer and nuclear factor for activated T cells nuclear translocation during receptor-evoked signaling. These findings highlight the critical dual function of the MCU not only in the acute Ca 2+ buffering by mitochondria but also in shaping endoplasmic reticulum and cytosolic Ca 2+ signals that regulate cellular transcription and function., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Store-operated Ca 2+ entry (SOCE) is a ubiquitous pathway for Ca 2+ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca 2+ -sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca 2+ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca 2+ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca 2+ signatures, particularly under native conditions, remain unclear. To investigate the structural properties distinguishing STIM1 and STIM2 activation of ORAI1 channels under native conditions, here we used CRISPR/Cas9 to generate STIM1 -/- , STIM2 -/- , and STIM1/2 -/- knockouts in HEK293 and colorectal HCT116 cells. We show that depending on cell type, STIM2 can significantly sustain SOCE in response to maximal store depletion. Utilizing the SOCE modifier 2-aminoethoxydiphenyl borate (2-APB), we demonstrate that 2-APB-activated store-independent Ca 2+ entry is mediated exclusively by endogenous STIM2. Using variants that either stabilize or disrupt intramolecular interactions of STIM C termini, we show that the increased flexibility of the STIM2 C terminus contributes to its selective store-independent activation by 2-APB. However, STIM1 variants with enhanced flexibility in the C terminus failed to support its store-independent activation. STIM1/STIM2 chimeric constructs indicated that coordination between N-terminal sensitivity and C-terminal flexibility is required for specific store-independent STIM2 activation. Our results clarify the structural determinants underlying activation of specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.