Mitochondria serve as central hubs for Ca2+ signaling, which is critical for metabolism, intercellular communication, and cell fate determination. Mitochondrial Ca2+ homeostasis is maintained through tightly coordinated influx and efflux processes, with NCLX long recognized as the primary Ca2+ extruder operating via Na+/Ca2+ exchange. Despite its physiological significance, the molecular basis of NCLX function has remained unclear. Here, we report cryo-EM structures of rat NCLX in cytosolic-facing occluded and open states. The central transmembrane (TM) module of NCLX comprises 10 helices organized into two structurally similar halves with inverted orientations. Two characteristic α-repeats form a central ion-binding pocket, while peripheral TMs 1 and 6 are loosely associated with the core, likely mediating alternative access to the binding site. These structural features closely resemble those of NCXs, revealing a conserved mechanism underlying ion exchange. While NCLX retains the canonical Ca2+-binding site, it lacks several key Na+-binding residues found in NCXs, suggesting it functions as a non-selective cation/Ca2+ exchanger. Consistent with this, cell-based Ca2+ uptake assays show that NCLX mediates slower Ca2+ exchange than NCX and can utilize Na+, K+, Li+, and potentially protons as counterions. Leveraging the structural symmetry of NCLX and its bidirectional exchange capability, we propose a model for the matrix-facing state and an alternating-access mechanism in which the sliding-door motions of TMs 1 and 6 enable ion access from cytosolic and matrix sides, analogous to NCX. These findings provide a structural and mechanistic framework for understanding mitochondrial NCLX function.
Zhang et al. (Sun,) studied this question.