Structure-dynamic and functional relationships in a Li-transporting sodium‑calcium exchanger mutant.

The cell membrane (NCX) and mitochondrial (NCLX) Na/Ca exchangers control Ca homeostasis. Eleven (out of twelve) ion-coordinating residues are highly conserved among eukaryotic and prokaryotic NCXs, whereas in NCLX, nine (out of twelve) ion-coordinating residues are different. Consequently, NCXs exhibit high selectivity for Na and Ca, whereas NCLX can exchange Ca with either Na or Li. However, the underlying molecular mechanisms and physiological relevance remain unresolved. Here, we analyzed the NCX_Mj-derived mutant NCLX_Mj (with nine substituted residues) imitating the ion selectivity of NCLX. Site-directed fluorescent labeling and ion flux assays revealed the nearly symmetric accessibility of ions to the extracellular and cytosolic vestibules in NCLX_Mj (K = 0.8-1.4), whereas the extracellular vestibule is predominantly accessible to ions (K = 0.1-0.2) in NCX_Mj. HDX-MS (hydrogen-deuterium exchange mass-spectrometry) identified symmetrically rigidified core helix segments in NCLX_Mj, whereas the matching structural elements are asymmetrically rigidified in NCX_Mj. The HDX-MS analyses of ion-induced conformational changes and the mutational effects on ion fluxes revealed that the "Ca-site" (S) of NCLX_Mj binds Na, Li, or Ca, whereas one or more additional Na/Li sites of NCLX_Mj are incompatible with the Na sites (S and S) of NCX_Mj. Thus, the replacement of ion-coordinating residues in NCLX_Mj alters not only the ion selectivity of NCLX_Mj, but also the capacity and affinity for Na/Li (but not for Ca) binding, bidirectional ion-accessibility, the response of the ion-exchange to membrane potential changes, and more. These structure-controlled functional features could be relevant for differential contributions of NCX and NCLX to Ca homeostasis in distinct sub-cellular compartments.