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

Abstract 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.