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Na + /Ca 2+ exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj ( Methanococcus jannaschii NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na + or Ca 2+ specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity ( K m ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

In eukaryotic Na(+)/Ca(2+) exchangers (NCX) the Ca(2+) binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca(2+) sensor (Ca3-Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca(2+)-driven interdomain switch has been described, albeit how it couples Ca(2+) binding with signal propagation remains unclear. To resolve the dynamic features of Ca(2+)-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium (45)Ca(2+) binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg(2+)-bound forms of CBD12 are highly flexible, whereas Ca(2+) binding to the Ca3-Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca(2+) is observed on the globally derived Dmax (maximal interatomic distance), although under comparable conditions a normal [Ca(2+)]-dependent allosteric regulation occurs. Low affinity sites (Ca1-Ca2) of CBD1 do not contribute to Ca(2+)-induced population shift, but the occupancy of these sites by 1 mM Mg(2+) shifts the Ca(2+) affinity (Kd) at the neighboring Ca3-Ca4 sites from ∼ 50 nM to ∼ 200 nM and thus, keeps the primary Ca(2+) sensor (Ca3-Ca4 sites) within a physiological range. Thus, Ca(2+) binding to the Ca3-Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.

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.

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.

The crystal structures of the CBD1 and CBD2 domains of the Na+/Ca2+ exchanger protein (NCX1) provided a major breakthrough in Ca2+-dependent regulation of NCX1, although the dynamic aspects of the underlying molecular mechanisms are still not clear. Here we provide new experimental approaches for evaluating the kinetic and equilibrium properties of Ca2+ interaction with regulatory sites by using purified preparations of CBD1, CBD2, and CBD12 proteins. CBD12 binds approximately 6 Ca2+ ions (mol/mol), whereas the binding of only approximately 2 Ca2+ ions is observed (with a Hill coefficient of nH=approximately 2) either for CBD1 or CBD2. In the absence of Mg2+, CBD1 has a much higher affinity for Ca2+ (Kd=0.3+/-1.2 microm) than CBD2 (Kd=5.0+/-1.2 microm). The Ca2+ dissociation from CBD2 (koff=230+/-70 s(-1)) is at least 25 times faster than from CBD1 (koff=10+/-3 s(-1)), whereas the kon values indicate fast kinetics for Ca2+ binding (kon=koff/Kd=10(7)-10(8) m(-1) s(-1)) for both CBDs. At 2-5 mm Mg2+, both CBDs bind Ca2+, with a Kd of 1-2 microm (Mg2+ has very little effect on Ca2+ off rates). Mg2+ cannot occupy the primary site of CBD2, whereas the other Ca2+ sites of CBDs interact with Mg2+ as well. There is no competition between Na+ and Ca2+ for any CBD site. The kinetically diverse Ca2+ sensors may sense differentially the dynamic swings in [Ca2+] within specific subcellular compartments (dyadic cleft, submembrane space, bulk cytosol, etc.).