The mitochondrial Na + /Ca 2+ exchanger (mito-NCX) is the protein that mediates mitochondrial Ca 2+ efflux, a process that is crucial for the regulation of intracellular Ca 2+ signaling, oxidative phosphorylation, and cell death pathways. NCLX has been proposed as the mito-NCX. However, we found that NCLX deletion did not affect mito-NCX activity, a result excluding NCLX as the mito-NCX. Recent studies have suggested that TMEM65 plays a critical role in mito-NCX activity, but the specific role of this protein remains unclear. We thus carried out studies to investigate the properties and mechanisms of TMEM65. We found that TMEM65 knockout abolished mito-NCX activity. Moreover, TMEM65 is highly expressed in the heart and brain but undetectable in the liver, correlating well with mito-NCX activity in these tissues. Biochemical analyses showed that TMEM65 forms a homodimer, and AlphaFold predictions suggested plausible ion-coordinating residues critical for function. To further define TMEM65’s function, we expressed TMEM65 in Sf9 cells, a heterologous system lacking endogenous mito-NCX, and observed robust mito-NCX activity. Furthermore, purified TMEM65 reconstituted into liposomes demonstrated that TMEM65 alone is sufficient to catalyze mito-NCX. We then searched for the binding site of CGP-37157, a commonly used mito-NCX inhibitor, on TMEM65. CGP-37157 is structurally analogous to diltiazem, an L-type Ca 2+ channel blocker. We identified a hydrophobic pocket in the AlphaFold-predicted TMEM65 structure, similar to the diltiazem-binding site in L-type Ca 2+ channels. Mutating residues within this site greatly reduced CGP-37157 potency, suggesting that this site mediates CGP-37157 binding. Lastly, we showed that TMEM65 knockout caused elevated matrix Ca 2+ levels and an increased tendency toward permeability transition, consistent with TMEM65 mediating mitochondrial Ca 2+ efflux. Collectively, these results establish TMEM65 as the bona fide mito-NCX, revealing a new therapeutic target for diseases linked to mitochondrial Ca 2+ dysregulation.
Lai et al. (Sun,) studied this question.