Mitochondrial Ca ²⁺ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca ²⁺ content and cytosolic Ca ²⁺ homeostasis strictly depend on Ca ²⁺ transporters. In recent decades, the major players responsible for mitochondrial Ca ²⁺ uptake and release have been identified, except the mitochondrial Ca ²⁺ /H ⁺ exchanger (CHE). Originally identified as the mitochondrial K ⁺ /H ⁺ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell‐based and cell‐free biochemical assays demonstrate the absence or greatly reduced Na ⁺ ‐independent mitochondrial Ca ²⁺ release in TMBIM5 knockout or pH‐sensing site mutants, respectively, and pH‐dependent Ca ²⁺ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long‐sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca ²⁺ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca ²⁺ exchange.

A direct link between Ca²⁺ and lipid homeostasis has not been definitively demonstrated. In this study, we show that manipulation of ER Ca²⁺ causes the re-distribution of a portion of the intracellular unesterified cholesterol to a pool that is not available to the SCAP-SREBP complex. The SREBP processing pathway in ER Ca²⁺ depleted cells remained fully functional and responsive to changes in cellular cholesterol status but differed unexpectedly in basal activity. These findings establish the role of Ca²⁺ in determining the reference set-point for controlling cellular lipid homeostasis. We propose that ER Ca²⁺ status is an important determinant of the basal sensitivity of the sterol sensing mechanism inherent to the SREBP processing pathway.

Efficient immune responses require Ca²⁺ fluxes across ORAI1 channels during engagement of T cell receptors (TCR) at the immune synapse (IS) between T cells and antigen presenting cells. Here, we show that ZDHHC20-mediated S-acylation of the ORAI1 channel at residue Cys143 promotes TCR recruitment and signaling at the IS. Cys143 mutations reduced ORAI1 currents and store-operated Ca²⁺ entry in HEK-293 cells and nearly abrogated long-lasting Ca²⁺ elevations, NFATC1 translocation, and IL-2 secretion evoked by TCR engagement in Jurkat T cells. The acylation-deficient channel remained in cholesterol-poor domains upon enforced ZDHHC20 expression and was recruited less efficiently to the IS along with actin and TCR. Our results establish S-acylation as a critical regulator of ORAI1 channel trafficking and function at the IS and reveal that ORAI1 S-acylation enhances TCR recruitment to the synapse.

The coronavirus SARS-CoV-2, the agent of the deadly COVID-19 pandemic, is an enveloped virus propagating within the endocytic and secretory organelles of host mammalian cells. Enveloped viruses modify the ionic homeostasis of organelles to render their intra-luminal milieu permissive for viral entry, replication and egress. Here, we show that infection of Vero E6 cells with the delta variant of the SARS-CoV-2 alkalinizes the endoplasmic reticulum (ER)–Golgi intermediate compartment (ERGIC) as well as lysosomes, mimicking the effect of inhibitors of vacuolar proton ATPases. We further show the envelope protein of SARS-CoV-2 accumulates in the ERGIC when expressed in mammalian cells and selectively dissipates the ERGIC pH. This viroporin action is prevented by mutations of Val25 but not Asn15 within the channel pore of the envelope (E) protein. We conclude that the envelope protein acts as a proton channel in the ERGIC to mitigate the acidity of this intermediate compartment. The altered pH homeostasis of the ERGIC likely contributes to the virus fitness and pathogenicity, making the E channel an attractive drug target for the treatment of COVID-19.