TMEM65 functions as the mitochondrial Na⁺/Ca²⁺ exchanger

Mitochondria export Ca²⁺ via Na⁺/Ca²⁺ exchange machinery (mito-NCX) to regulate intracellular Ca²⁺ signalling and mitochondrial Ca²⁺ homeostasis. TMEM65 has recently been implicated as essential for mito-NCX, but its mechanisms and roles remain unclear. Here we show that TMEM65 depletion severely impairs mito-NCX. TMEM65 is highly expressed in the heart and brain but absent in the liver, correlating with mito-NCX activity in these tissues. Biochemical and functional analyses reveal that TMEM65 forms a homodimer, containing plausible ion-coordinating residues critical for function. Heterologous expression of TMEM65 induces Na⁺/Ca²⁺ exchange in cells lacking native mito-NCX activity. Moreover, purified, liposome-reconstituted TMEM65 exhibits key mito-NCX features. We further identify the binding site for CGP-37157, a potent, widely used mito-NCX inhibitor. Finally, TMEM65 deletion elevates mitochondrial Ca²⁺ and primes mitochondria to permeability transition. These findings firmly establish TMEM65 as the protein mediating mito-NCX, offering a new therapeutic target for diseases associated with mitochondrial Ca²⁺ dysregulation.