TMEM65 regulates NCLX-dependent mitochondrial calcium efflux.

The balance between mitochondrial calcium (Ca) uptake and efflux regulates ATP production, but if perturbed causes energy starvation or Ca overload and cell death. The mitochondrial sodium-calcium exchanger, NCLX, is a critical route of Ca efflux in excitable tissues, such as the heart and brain, and animal models support NCLX as a promising therapeutic target to limit pathogenic Ca overload. However, the mechanisms that regulate NCLX activity remain largely unknown. We used proximity biotinylation proteomic screening to identify the NCLX interactome and define novel regulators of NCLX function. Here, we discover the mitochondrial inner membrane protein, TMEM65, as an NCLX-proximal protein that potently enhances sodium (Na)-dependent Ca efflux. Mechanistically, acute pharmacologic NCLX inhibition or genetic deletion of NCLX ablates the TMEM65-dependent increase in Ca efflux. Further, loss-of-function studies show that TMEM65 is required for Na-dependent Ca efflux. Co-fractionation and structural modeling of TMEM65 and NCLX suggest these two proteins exist in a common macromolecular complex in which TMEM65 directly stimulates NCLX function. In line with these findings, knockdown of in mice promotes Ca overload in the heart and skeletal muscle and impairs both cardiac and neuromuscular function. We further demonstrate that deletion causes excessive mitochondrial permeability transition, whereas TMEM65 overexpression protects against necrotic cell death during cellular Ca stress. Collectively, our results show that loss of TMEM65 function in excitable tissue disrupts NCLX-dependent Ca efflux, causing pathogenic Ca overload, cell death and organ-level dysfunction, and that gain of TMEM65 function mitigates these effects. These findings demonstrate the essential role of TMEM65 in regulating NCLX-dependent Ca efflux and suggest modulation of TMEM65 as a novel strategy for the therapeutic control of Ca homeostasis.