Molecular mechanisms of mitochondrial Ca2+ exchanger NCLX

Abstract

Mitochondrial Ca2+ homeostasis is maintained through coordinated influx and efflux processes, with NCLX long recognized as the primary Ca2+ extruder operating via Na+/Ca2+ exchange. Here, we report cryo-EM structures of rat NCLX in cytosolic-facing occluded and open states. The central transmembrane (TM) domain of NCLX comprises ten helices arranged in two inverted, structurally similar halves, with two -repeats forming a central ion-binding pocket. Peripheral TMs 1 and 6 are loosely associated with the core and likely mediate alternative access to this site. These structural features closely resemble those of NCXs, indicating a conserved ion exchange mechanism. While NCLX retains the canonical Ca2+-binding site, it lacks several key Na+-binding residues found in NCXs, suggesting broader ion selectivity. Consistently, cell-based Ca2+ uptake assays show that NCLX mediates Ca2+ exchange using Na+, K+, Li+, and potentially protons as counterions. Based on the structural symmetry of NCLX and its bidirectional exchange capability, we propose a matrix-facing model and an alternating-access mechanism in which TMs 1 and 6 undergo sliding motions to enable ion exchange between cytosolic and matrix sides, analogous to NCX. These findings provide a structural and mechanistic framework for understanding NCLX-mediated Ca2+ transport in mitochondria.