Abstract
AbstractThe balance between mitochondrial calcium (mCa2+) uptake and efflux regulates ATP production, but if perturbed causes energy starvation ormCa2+overload and cell death. The mitochondrial sodium-calcium exchanger, NCLX, is a critical route ofmCa2+efflux in excitable tissues, such as the heart and brain, and animal models support NCLX as a promising therapeutic target to limit pathogenicmCa2+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+)-dependentmCa2+efflux. Mechanistically, acute pharmacologic NCLX inhibition or genetic deletion of NCLX ablates the TMEM65-dependent increase inmCa2+efflux. Further, loss-of-function studies show that TMEM65 is required for Na+-dependentmCa2+efflux. Co-fractionation andin silicostructural 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 ofTmem65in mice promotesmCa2+overload in the heart and skeletal muscle and impairs both cardiac and neuromuscular function. We further demonstrate thatTMEM65deletion causes excessive mitochondrial permeability transition, whereas TMEM65 overexpression protects against necrotic cell death during cellular Ca2+stress. Collectively, our results show that loss of TMEM65 function in excitable tissue disrupts NCLX-dependentmCa2+efflux, causing pathogenicmCa2+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-dependentmCa2+efflux and suggest modulation of TMEM65 as a novel strategy for the therapeutic control ofmCa2+homeostasis.
Publisher
Cold Spring Harbor Laboratory