Significance: Na ⁺ /Ca ²⁺ exchangers (NCXs) have a key role in the homeostasis of cellular Ca ²⁺ and consequently are implicated in diverse human-health disorders, including neurodegenerative and cardiovascular diseases. A detailed understanding of the molecular mechanisms of these membrane proteins is therefore of interest from fundamental and biomedical standpoints. Here, we establish the structural mechanism of Na ⁺ recognition in a prokaryotic NCX homolog, using atomistic molecular-dynamics simulations based on recently reported crystallographic data, as well as experimental transport assays of wild-type and mutagenized exchangers. The results have general implications pertaining to the ion exchange stoichiometry and electrogenicity of the Na ⁺ /Ca ²⁺ transport cycle across the NCX family, and provide the basis for future investigations of the conformational mechanism of these important transporters.

The cardiac Na(+)/Ca(2+) exchanger (NCX) regulates cellular [Ca(2+)](i) and plays a central role in health and disease, but its molecular regulation is poorly understood. Here we report on how protons affect this electrogenic transporter by modulating two critically important NCX C(2) regulatory domains, Ca(2+) binding domain-1 (CBD1) and CBD2. The NCX transport rate in intact cardiac ventricular myocytes was measured as a membrane current, I(NCX), whereas [H(+)](i) was varied using an ammonium chloride "rebound" method at constant extracellular pH 7.4. At pH(i) = 7.2 and [Ca(2+)](i) < 120 nM, I(NCX) was less than 4% that of its maximally Ca(2+)-activated value. I(NCX) increases steeply at [Ca(2+)](i) between 130-150 nM with a Hill coefficient (n(H)) of 8.0 ± 0.7 and K(0.5) = 310 ± 5 nM. At pH(i) = 6.87, the threshold of Ca(2+)-dependent activation of I(NCX) was shifted to much higher [Ca(2+)](i) (600-700 nM), and the relationship was similarly steep (n(H) = 8.0±0.8) with K(0.5) = 1042 ± 15 nM. The V(max) of Ca(2+)-dependent activation of I(NCX) was not significantly altered by low pH(i). The Ca(2+) affinities for CBD1 (0.39 ± 0.06 μM) and CBD2 (K(d) = 18.4 ± 6 μM) were exquisitely sensitive to [H(+)], decreasing 1.3-2.3-fold as pH(i) decreased from 7.2 to 6.9. This work reveals for the first time that NCX can be switched off by physiologically relevant intracellular acidification and that this depends on the competitive binding of protons to its C(2) regulatory domains CBD1 and CBD2.

Na+/Ca2+ exchanger (NCX) proteins extrude Ca2+ from the cell to maintain cellular homeostasis. Since NCX proteins contribute to numerous physiological and pathophysiological events, their pharmacological targeting has been desired for a long time. This intervention remains challenging owing to our poor understanding of the underlying structure-dynamic mechanisms. Recent structural studies have shed light on the structure-function relationships underlying the ion-transport and allosteric regulation of NCX. The crystal structure of an archaeal NCX (NCX_Mj) along with molecular dynamic simulations and ion flux analyses, have assigned the ion binding sites for 3Na+ and 1Ca2+, which are being transported in separate steps. In contrast with NCX_Mj, eukaryotic NCXs contain the regulatory Ca2+-binding domains, CBD1 and CBD2, which affect the membrane embedded ion-transport domains over a distance of ~80 Å. The Ca2+-dependent regulation is ortholog, isoform and splice-variant dependent to meet physiological requirements, exhibiting either a positive, negative or no response to regulatory Ca2+. The crystal structures of the two-domain (CBD12) tandem have revealed a common mechanism involving a Ca2+-driven tethering of CBDs in diverse NCX variants. However, dissociation kinetics of occluded Ca2+ (entrapped at the two-domain interface) depends on the alternative-splicing segment (at CBD2), thereby representing splicing-dependent dynamic coupling of CBDs. The HDX-MS, SAXS, NMR, FRET, equilibrium 45Ca2+ binding and stopped-flow techniques provided insights into the dynamic mechanisms of CBDs. Ca2+ binding to CBD1 results in a population shift, where more constraint conformational states become highly populated without global conformational changes in the alignment of CBDs. This mechanism is common among NCXs. Recent HDX-MS studies have demonstrated that the apo CBD1 and CBD2 are stabilized by interacting with each other, while Ca2+ binding to CBD1 rigidifies local backbone segments of CBD2, but not of CBD1. The extent and strength of Ca2+-dependent rigidification at CBD2 is splice-variant dependent, showing clear correlations with phenotypes of matching NCX variants. Therefore, diverse NCX variants share a common mechanism for the initial decoding of the regulatory signal upon Ca2+ binding at the interface of CBDs, whereas the allosteric message is shaped by CBD2, the dynamic features of which are dictated by the splicing segment.

The membrane-bound sodium–calcium exchanger (NCX) proteins shape Ca²⁺ homeostasis in many cell types, thus participating in a wide range of physiological and pathological processes. Determination of the crystal structure of an archaeal NCX (NCX_Mj) paved the way for a thorough and systematic investigation of ion transport mechanisms in NCX proteins. Here, we review the data gathered from the X-ray crystallography, molecular dynamics simulations, hydrogen–deuterium exchange mass-spectrometry (HDX-MS), and ion-flux analyses of mutants. Strikingly, the apo NCX_Mj protein exhibits characteristic patterns in the local backbone dynamics at particular helix segments, thereby possessing characteristic HDX profiles, suggesting structure-dynamic preorganization (geometric arrangements of catalytic residues before the transition state) of conserved α₁ and α₂ repeats at ion-coordinating residues involved in transport activities. Moreover, dynamic preorganization of local structural entities in the apo protein predefines the status of ion-occlusion and transition states, even though Na⁺ or Ca²⁺ binding modifies the preceding backbone dynamics nearby functionally important residues. Future challenges include resolving the structural-dynamic determinants governing the ion selectivity, functional asymmetry and ion-induced alternating access. Taking into account the structural similarities of NCX_Mj with the other proteins belonging to the Ca²⁺/cation exchanger superfamily, the recent findings can significantly improve our understanding of ion transport mechanisms in NCX and similar proteins.

In NCX proteins CBD1 and CBD2 domains are connected through a short linker (3 or 4 amino acids) forming a regulatory tandem (CBD12). Only three of the six CBD12 Ca(2+)-binding sites contribute to NCX regulation. Two of them are located on CBD1 (K(d) = approximately 0.2 microM), and one is on CBD2 (K(d) = approximately 5 microM). Here we analyze how the intrinsic properties of individual regulatory sites are affected by linker-dependent interactions in CBD12 (AD splice variant). The three sites of CBD12 and CBD1 + CBD2 have comparable K(d) values but differ dramatically in their Ca(2+) dissociation kinetics. CBD12 exhibits multiphasic kinetics for the dissociation of three Ca(2+) ions (k(r) = 280 s(-1), k(f) = 7 s(-1), and k(s) = 0.4 s(-1)), whereas the dissociation of two Ca(2+) ions from CBD1 (k(f) = 16 s(-1)) and one Ca(2+) ion from CBD2 (k(r) = 125 s(-1)) is monophasic. Insertion of seven alanines into the linker (CBD12-7Ala) abolishes slow dissociation of Ca(2+), whereas the kinetic and equilibrium properties of three Ca(2+) sites of CBD12-7Ala and CBD1 + CBD2 are similar. Therefore, the linker-dependent interactions in CBD12 decelerate the Ca(2+) on/off kinetics at a specific CBD1 site by 50-80-fold, thereby representing Ca(2+) "occlusion" at CBD12. Notably, the kinetic and equilibrium properties of the remaining two sites of CBD12 are "linker-independent," so their intrinsic properties are preserved in CBD12. In conclusion, the dynamic properties of three sites are specifically modified, conserved, diversified, and integrated by the linker in CBD12, thereby generating a wide range dynamic sensor.

Na⁺/Ca²⁺ exchanger (NCX) proteins mediate Ca²⁺-fluxes across the cell membrane to maintain Ca²⁺ homeostasis in many cell types. Eukaryotic NCX contains Ca²⁺-binding regulatory domains, CBD1 and CBD2. Ca²⁺ binding to a primary sensor (Ca3-Ca4 sites) on CBD1 activates mammalian NCXs, whereas CALX, a Drosophila NCX ortholog, displays an inhibitory response to regulatory Ca²⁺. To further elucidate the underlying regulatory mechanisms, we determined the 2.7 Å crystal structure of mammalian CBD12-E454K, a two-domain construct that retains wild-type properties. In conjunction with stopped-flow kinetics and SAXS (small-angle X-ray scattering) analyses of CBD12 mutants, we show that Ca²⁺ binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca²⁺-driven interdomain switch controls slow dissociation of “occluded” Ca²⁺ from the primary sensor and thus dictates Ca²⁺ sensing dynamics. In the Ca²⁺-bound conformation, the interdomain angle of CBD12 is very similar in NCX and CALX, meaning that the interdomain distances cannot account for regulatory diversity in NCX and CALX. Since the two-domain interface is nearly identical among eukaryotic NCXs, including CALX, we suggest that the Ca²⁺-driven interdomain switch described here represents a general mechanism for initial conduction of regulatory signals in NCX variants.