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

The cell membrane (NCX) and mitochondrial (NCLX) Na ⁺ /Ca ²⁺ exchangers control Ca ²⁺ 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 ²⁺ , whereas NCLX can exchange Ca ²⁺ 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 _int = 0.8–1.4), whereas the extracellular vestibule is predominantly accessible to ions (K _int = 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 ²⁺ -site” (S _Ca ) of NCLX_Mj binds Na ⁺ , Li ⁺ , or Ca ²⁺ , whereas one or more additional Na ⁺ /Li ⁺ sites of NCLX_Mj are incompatible with the Na ⁺ sites (S _ext and S _int ) 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 ²⁺ ) 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 ²⁺ homeostasis in distinct sub-cellular compartments.