19F-NMR Probing of Ion-Induced Conformational Changes in Detergent-Solubilized and Nanodisc-Reconstituted NCX_Mj.

Consecutive interactions of 3Na+ or 1Ca2+ with the Na+/Ca2+ exchanger (NCX) result in an alternative exposure (access) of the cytosolic and extracellular vestibules to opposite sides of the membrane, where ion-induced transitions between the outward-facing (OF) and inward-facing (IF) conformational states drive a transport cycle. Here, we investigate sub-state populations of apo and ion-bound species in the OF and IF states by analyzing detergent-solubilized and nanodisc-reconstituted preparations of NCX_Mj with 19F-NMR. The 19F probe was covalently attached to the cysteine residues at entry locations of the cytosolic and extracellular vestibules. Multiple sub-states of apo and ion-bound species were observed in nanodisc-reconstituted (but not in detergent-solubilized) NCX_Mj, meaning that the lipid-membrane environment preconditions multiple sub-state populations toward the OF/IF swapping. Most importantly, ion-induced sub-state redistributions occur within each major (OF or IF) state, where sub-state interconversions may precondition the OF/IF swapping. In contrast with large changes in population redistributions, the sum of sub-state populations within each inherent state (OF or IF) remains nearly unchanged upon ion addition. The present findings allow the further elucidation of structure-dynamic modules underlying ion-induced conformational changes that determine a functional asymmetry of ion access/translocation at opposite sides of the membrane and ion transport rates concurring physiological demands.

Dynamic distinctions in the Na+/Ca2+ exchanger adopting the inward- and outward-facing conformational states.

Na+/Ca2+ 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 Ca2+ 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 (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.

Structure-dynamic basis of splicing-dependent regulation in tissue-specific variants of the sodium-calcium exchanger.

Tissue-specific splice variants of Na(+)/Ca(2+) exchangers contain 2 Ca(2+)-binding regulatory domains (CBDs), CBD1 and CBD2. Ca(2+) interaction with CBD1 activates sodium-calcium exchangers (NCXs), and Ca(2+) binding to CBD2 alleviates Na(+)-dependent inactivation. A combination of mutually exclusive (A, B) and cassette (C-F) exons in CBD2 raises functionally diverse splice variants through unknown mechanisms. Here, the effect of exons on CBDs backbone dynamics were investigated in the 2-domain tandem (CBD12) of the brain, kidney, and cardiac splice variants by using hydrogen-deuterium exchange mass spectrometry and stopped-flow techniques. Mutually exclusive exons stabilize interdomain interactions in the apoprotein, which primarily predefines the extent of responses to Ca(2+) binding. Deuterium uptake levels were up to 20% lower in the cardiac vs. the brain CBD12, reveling that elongation of the CBD2 FG loop by cassette exons rigidifies the interdomain Ca(2+) salt bridge at the 2-domain interface, which secondarily modulates the Ca(2+)-bound states. In matching splice variants, the extent of Ca(2+)-induced rigidification correlates with decreased (up to 10-fold) Ca(2+) off rates, where the cardiac CBD12 exhibits the slowest Ca(2+) off rates. Collectively, structurally disordered/dynamic segments at mutually exclusive and cassette exons have local and distant effects on the folded structures nearby the Ca(2+) binding sites, which may serve as a structure-dynamic basis for splicing-dependent regulation of NCX.

Sodium recognition by the Na+/Ca2+ exchanger in the outward-facing conformation.

Na(+)/Ca(2+) exchangers (NCXs) are ubiquitous membrane transporters with a key role in Ca(2+) homeostasis and signaling. NCXs mediate the bidirectional translocation of either Na(+) or Ca(2+), and thus can catalyze uphill Ca(2+) transport driven by a Na(+) gradient, or vice versa. In a major breakthrough, a prokaryotic NCX homolog (NCX_Mj) was recently isolated and its crystal structure determined at atomic resolution. The structure revealed an intriguing architecture consisting of two inverted-topology repeats, each comprising five transmembrane helices. These repeats adopt asymmetric conformations, yielding an outward-facing occluded state. The crystal structure also revealed four putative ion-binding sites, but the occupancy and specificity thereof could not be conclusively established. Here, we use molecular-dynamics simulations and free-energy calculations to identify the ion configuration that best corresponds to the crystallographic data and that is also thermodynamically optimal. In this most probable configuration, three Na(+) ions occupy the so-called Sext, SCa, and Sint sites, whereas the Smid site is occupied by one water molecule and one H(+), which protonates an adjacent aspartate side chain (D240). Experimental measurements of Na(+)/Ca(2+) and Ca(2+)/Ca(2+) exchange by wild-type and mutagenized NCX_Mj confirm that transport of both Na(+) and Ca(2+) requires protonation of D240, and that this side chain does not coordinate either ion at Smid. These results imply that the ion exchange stoichiometry of NCX_Mj is 3:1 and that translocation of Na(+) across the membrane is electrogenic, whereas transport of Ca(2+) is not. Altogether, these findings provide the basis for further experimental and computational studies of the conformational mechanism of this exchanger.

Structure-Based Engineering of Lithium-Transport Capacity in an Archaeal Sodium-Calcium Exchanger.

Members of the Ca(2+)/cation exchanger superfamily (Ca(2+)/CA) share structural similarities (including highly conserved ion-coordinating residues) while exhibiting differential selectivity for Ca(2+), Na(+), H(+), K(+), and Li(+). The archaeal Na(+)/Ca(2+) exchanger (NCX_Mj) and its mammalian orthologs are highly selective for Na(+), whereas the mitochondrial ortholog (NCLX) can transport either Li(+) or Na(+) in exchange with Ca(2+). Here, structure-based replacement of ion-coordinating residues in NCX_Mj resulted in a capacity for transporting either Na(+) or Li(+), similar to the case for NCLX. This engineered protein may serve as a model for elucidating the mechanisms underlying ion selectivity and ion-coupled alternating access in NCX and similar proteins.

Structure-dynamic determinants governing a mode of regulatory response and propagation of allosteric signal in splice variants of Na+/Ca2+ exchange (NCX) proteins.

The Ca(2+)-dependent allosteric regulation of Na(+)/Ca(2+) exchanger (NCX) proteins represents Ca(2+) interaction with the cytosolic domains, CBD1 (calcium-binding domain 1) and CBD2, which is associated either with activation, inhibition or no response to regulatory Ca(2+) in a given splice variant. CBD1 contains a high affinity Ca(2+)-sensor (which is highly conserved among splice variants), whereas primary information upon Ca(2+) binding to CBD1 is modified by alternative splicing of CBD2, yielding the diverse regulatory responses to Ca(2+). To resolve the structure-dynamic determinants of splicing-dependent regulation, we tested two-domain tandem (CBD12) constructs possessing either positive, negative or no response to Ca(2+) using hydrogen-deuterium exchange MS (HDX-MS), SAXS, equilibrium 45Ca(2+) binding and stopped-flow kinetics. Taken together with previously resolved crystallographic structures of CBD12, the data revealed that Ca(2+) binding to CBD1 rigidifies the main-chain flexibility of CBD2 (but not of CBD1), whereas CBD2 stabilizes the apo-CBD1. Strikingly, the extent and strength of Ca(2+)-dependent rigidification of CBD2 is splice-variant dependent, where the main-chain rigidification spans from the Ca(2+)-binding sites of CBD1, through a helix of CBD2 (positioned at the domains' interface) up to the tip of CBD2 [>50 Å (1 Å = 0.1 nm)] or alternatively, it stops at the CBD2 helix in the splice variant exhibiting an inhibitory response to regulatory Ca(2+). These results provide a structure-dynamic basis by which alternative splicing diversifies the regulatory responses to Ca(2+) as well as controls the extent and strength of allosteric signal propagation over long distance.

Population shift underlies Ca2+-induced regulatory transitions in the sodium-calcium exchanger (NCX).

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.

Structural dynamics of Na+ and Ca2+ interactions with full-size mammalian NCX.

Cytosolic Ca2+ and Na+ allosterically regulate Na+/Ca2+ exchanger (NCX) proteins to vary the NCX-mediated Ca2+ entry/exit rates in diverse cell types. To resolve the structure-based dynamic mechanisms underlying the ion-dependent allosteric regulation in mammalian NCXs, we analyze the apo, Ca2+, and Na+-bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Ca2+ binding to the cytosolic regulatory domains (CBD1 and CBD2) rigidifies the intracellular regulatory loop (5L6) and promotes its interaction with the membrane domains. Either Na+ or Ca2+ stabilizes the intracellular portions of transmembrane helices TM3, TM4, TM9, TM10, and their connecting loops (3L4 and 9L10), thereby exposing previously unappreciated regulatory sites. Ca2+ or Na+ also rigidifies the palmitoylation domain (TMH2), and neighboring TM1/TM6 bundle, thereby uncovering a structural entity for modulating the ion transport rates. The present analysis provides new structure-dynamic clues underlying the regulatory diversity among tissue-specific NCX variants.

G503 is obligatory for coupling of regulatory domains in NCX proteins.

In multidomain proteins, interdomain linkers allow an efficient transfer of regulatory information, although it is unclear how the information encoded in the linker structure coins dynamic coupling. Allosteric regulation of NCX proteins involves Ca(2+)-driven tethering of regulatory CBD1 and CBD2 (through a salt bridge network) accompanied by alignment of CBDs and Ca(2+) occlusion at the interface of the two CBDs. Here we investigated "alanine-walk" substitutions in the CBD1-CBD2 linker (501-HAGIFT-506) and found that among all linker residues, only G503 is obligatory for Ca(2+)-induced reorientations of CBDs and slow dissociation of occluded Ca(2+). Moreover, swapping between positions A502 and G503 in the CBD1-CBD2 linker results in a complete loss of slow dissociation of occluded Ca(2+), meaning that dynamic coupling of CBDs requires an exact pose of glycine at position 503. Therefore, accumulating data revealed that position 503 occupied by glycine is absolutely required for Ca(2+)-driven tethering of CBDs, which in turn limits the linker's flexibility and, thus, restricts CBD movements. Because G503 is extremely well conserved in eukaryotic NCX proteins, the information encoded in G503 is essential for dynamic coupling of the two-domain CBD tandem and, thus, for propagation of the allosteric signal.

Proton-sensing Ca2+ binding domains regulate the cardiac Na+/Ca2+ exchanger.

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.

Characteristic attributes limiting the transport rates in NCX orthologs.

The Na+/Ca2+ exchangers (NCXs) modulate the Ca2+ signaling and homeostasis in health and disease. The transport cycle turnover rates (kcat) and the kcat/Km values of eukaryotic NCXs are ~104-times higher than those of prokaryotic NCXs. Three ion-coordinating residues (out of twelve) differ between eukaryotic NCXs and NCX_Mj. The replacement of three ion-coordinating residues in NCX_Mj does not increase kcat, probably due to the structural rigidity of NCX_Mj. Phospholipids and cholesterol increase (up to 10-fold) the transport rates in the cardiac NCX1.1, but not in NCX_Mj. A lipid environment can partially contribute to the huge kinetic variances among NCXs.

Exploring the Li+ transporting mutant of NCX_Mj for assigning ion binding sites of mitochondrial NCLX.

The plasma membrane (NCX) and mitochondrial (NCLX) Na+/Ca2+ exchangers are structurally related proteins, although they operate under strictly different ionic conditions and membrane potentials. In contrast with NCX, NCLX can transport either Li+ or Na+ in exchange for Ca2+. Whereas the crystal structure of the archaeal NCX (NCX_Mj) describes the binding sites for alternative binding of 3Na+ or 1Ca2+, these features remain elusive for NCLX due to the lack of structural information. To elucidate the ion-binding features of mitochondrial NCLX, we analyzed here the Li+-transporting NCLX_Mj mutant, produced by replacing the ion-coordinating residues in the archaeal NCX (NCX_Mj) to match the ion-coordinating residues of human NCLX. The NCLX_Mj-mediated Na+/Ca2+ or Li+/Ca2+ exchange rates are insensitive to varying voltage, consistent with an electroneutral ion exchange. Molecular dynamics (MD) simulations revealed that NCLX_Mj contains two novel Li+ binding sites with four ion-coordinating residues, derived from the three Na+ binding sites of NCX_Mj. The ion-coordination modes, observed in the MD simulations, were further supported by two-dimensional infrared (2D IR) spectroscopy and by testing the mutational effects on the ion fluxes. Collectively, our results revealed a structural basis for Li+ binding and electroneutral transport (2Na+/Li+:1Ca2+) by NCLX_Mj, meaning that the NCLX-mediated electroneutral transport may predefine mitochondrial Ca2+ and Na+ signaling to modulate cellular functions.

Essential role of the CBD1-CBD2 linker in slow dissociation of Ca2+ from the regulatory two-domain tandem of NCX1.

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.

Proton-modulated interactions of ions with transport sites of prokaryotic and eukaryotic NCX prototypes.

The cytosolic pH decline from 7.2 to 6.9 results in 90% inactivation of mammalian Na+/Ca2+ exchangers (NCXs) due to protons interactions with regulatory and transport domains ("proton block"). Remarkably, the pH titration curves of mammalian and prokaryotic NCXs significantly differ, even after excluding the allosteric effects through regulatory domains. This is fascinating since "only" three (out of twelve) ion-coordinating residues (T50S, E213D, and D240N) differ between the archaeal NCX_Mj and mammalian NCXs although they contain either three or two carboxylates, respectively. To resolve the underlying mechanisms of pH-dependent regulation, the ion-coordinating residues of NCX_Mj were mutated to imitate the ion ligation arrays of mammalian NCXs; the mutational effects were tested on the ion binding/transport by using ion-flux assays and two-dimensional infrared (2D IR) spectroscopy. Our analyses revealed that two deprotonated carboxylates ligate 3Na+ or 1Ca2+ in NCX prototypes with three or two carboxylates. The Na+/Ca2+ exchange rates of NCX_Mj reach saturation at pH 5.0, whereas the Na+/Ca2+ exchange rates of the cardiac NCX1.1 gradually increase even at alkaline pHs. The T50S replacement in NCX_Mj "recapitulates" the pH titration curves of mammalian NCX by instigating an alkaline shift. Proteolytic shaving of regulatory CBD domains activates NCX1.1, although the normalized pH-titration curves are comparable in trypsin treated and untreated NCX1.1. Thus, the T50S-dependent alkaline shift sets a dynamic range for "proton block" function at physiological pH, whereas the CBDs (and other regulatory modes) modulate incremental changes in the transport rates rather than affect the shape of pH dependent curves.

Structure-affinity insights into the Na+ and Ca2+ interactions with multiple sites of a sodium-calcium exchanger.

Selective recognition and transport of Na+ and Ca2+ ions by sodium-calcium exchanger (NCX) proteins is a primary prerequisite for Ca2+ signaling and homeostasis. Twelve ion-coordinating residues are highly conserved among NCXs, and distinct NCX orthologs contain two or three carboxylates, while sharing a common ion-exchange stoichiometry (3Na+ :1Ca2+ ). How these structural differences affect the ion-binding affinity, selectivity, and transport rates remains unclear. Here, the mutational effects of three carboxylates (E54, E213, and D240) were analyzed on the ion-exchange rates in the archaeal NCX from Methanococcus jannaschii and ion-induced structure-affinity changes were monitored by attenuated total reflection-Fourier-transform infrared spectroscopy (ATR-FTIR). The D240N mutation elevated the ion-transport rates by twofold to threefold, meaning that the deprotonation of D240 is not essential for transport catalysis. In contrast, mutating E54 or E213 to A, D, N, or Q dramatically decreased the ion-transport rates. ATR-FTIR revealed high- and low-affinity binding of Na+ or Ca2+ with E54 and E213, but not with D240. These findings reveal distinct structure-affinity states at specific ion-binding sites in the inward-facing (IF) and outward-facing orientation. Collectively, two multidentate carboxylate counterparts (E54 and E213) play a critical role in determining the ion coordination/transport in prokaryotic and eukaryotic NCXs, whereas the ortholog substitutions in prokaryotes (aspartate) and eukaryotes (asparagine) at the 240 position affect the ion-transport rates differently (kcat ), probably due to the structural differences in the transition state.

Structure-dynamic and functional relationships in a Li+-transporting sodium­calcium exchanger mutant.

The cell membrane (NCX) and mitochondrial (NCLX) Na+/Ca2+ exchangers control Ca2+ 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 Ca2+, whereas NCLX can exchange Ca2+ 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 (Kint = 0.8-1.4), whereas the extracellular vestibule is predominantly accessible to ions (Kint = 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 "Ca2+-site" (SCa) of NCLX_Mj binds Na+, Li+, or Ca2+, whereas one or more additional Na+/Li+ sites of NCLX_Mj are incompatible with the Na+ sites (Sext and Sint) 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 Ca2+) 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 Ca2+ homeostasis in distinct sub-cellular compartments.

Direct Loading of the purified endogenous inhibitor into the cytoplasm of patched cardiomyocytes blocks the ion currents and calcium transport through the NCX1 protein.

The Na(+)-Ca(2+) exchanger in mammalian heart muscle (NCX1) is the central transporter protein that regulates extrusion of Ca(2+) from the heart cell. However, the functional biochemistry and physiology of NCX1 have been severely hampered by the absence of any specific high-affinity inhibitor. Here we describe advanced procedures for purifying a candidate inhibitor, previously called endogenous inhibitor factor (NCX(IF)), and demonstrate its direct actions on NCX1 activities in the single-cell system. A combination of advanced HILIC (hydrophilic interaction liquid chromatography) procedures with analytical tests suggests that the properties of NCX(IF) resemble those of a small (disaccharide size) polar molecule lacking any aromatic rings, conjugated bonds, or a primary amino group. The effects of NCX(IF) on the NCX1-mediated ion currents (I(NCX)) and cytosolic Ca(2+) extrusion were detected by a combination of patch-clamp and confocal microscopy under conditions in which the purified NCX(IF) was directly loaded into the cytoplasm of patched cardiomyocytes. It was demonstrated that cytosolic NCX(IF) blocks the Ca(2+)-activated NCX1 inward current and the accompanying extrusion of Ca(2+) from the cell with high efficacy. A constant fraction of NCX1 inhibition was observed under conditions in which the cytosolic [Ca(2+)](i) was varied at fixed doses of NCX(IF), suggesting that the degree of inhibition is controlled by NCX(IF) dose and not by cytosolic Ca(2+) concentration. NCX(IF) blocks equally well both the Ca(2+) extrusion and Ca(2+) entry modes of NCX1, consistent with thermodynamic principles expected for the functioning of a bidirectional "carrier-type" transport system. We concluded that NCX(IF) interacts with a putative regulatory domain from the cytosolic side and, thus, may play an important regulatory role in controlling Ca(2+) signaling in the heart. This may represent a new potential tool for developing novel treatments for cardiac Ca(2+) signaling dysfunction.

Key residues controlling bidirectional ion movements in Na+/Ca2+ exchanger.

Prokaryotic and eukaryotic Na+/Ca2+ exchangers (NCX) control Ca2+ homeostasis. NCX orthologs exhibit up to 104-fold differences in their turnover rates (kcat), whereas the ratios between the cytosolic (cyt) and extracellular (ext) Km values (Kint = KmCyt/KmExt) are highly asymmetric and alike (Kint ≤ 0.1) among NCXs. The structural determinants controlling a huge divergence in kcat at comparable Kint remain unclear, although 11 (out of 12) ion-coordinating residues are highly conserved among NCXs. The crystal structure of the archaeal NCX (NCX_Mj) was explored for testing the mutational effects of pore-allied and loop residues on kcat and Kint. Among 55 tested residues, 26 mutations affect either kcat or Kint, where two major groups can be distinguished. The first group of mutations (14 residues) affect kcat rather than Kint. The majority of these residues (10 out of 14) are located within the extracellular vestibule near the pore center. The second group of mutations (12 residues) affect Kint rather than kcat, whereas the majority of residues (9 out 12) are randomly dispersed within the extracellular vestibule. In conjunction with computational modeling-simulations and hydrogen-deuterium exchange mass-spectrometry (HDX-MS), the present mutational analysis highlights structural elements that differentially govern the intrinsic asymmetry and transport rates. The key residues, located at specific segments, can affect the characteristic features of local backbone dynamics and thus, the conformational flexibility of ion-transporting helices contributing to critical conformational transitions. The underlying mechanisms might have a physiological relevance for matching the response modes of NCX variants to cell-specific Ca2+ and Na+ signaling.

Effects of purified endogenous inhibitor of the Na+/Ca2+ exchanger on ouabain-induced arrhythmias in the atria and ventricle strips of guinea pig.

Previous studies demonstrated that the purified endogenous inhibitor (NCX(IF)) of the cardiac Na(+)/Ca(2+) exchanger (NCX1) has the capacity to modulate cardiac muscle contractility. Here, we tested the effects of purified NCX(IF) on arrhythmias induced by ouabain in the atria and ventricle strips of guinea pig. For the sake of comparison NCX(IF) was compared to lidocaine and KB-R7943. In the ventricle strip, NCX(IF) ( approximately 10 U/ml) results in rapid, complete and stable inhibition of ouabain-induced arrhythmias (the inhibition of arrhythmia is not followed by revival of irregular contractions). Under similar experimental conditions the atria strips require somewhat higher doses of NCX(IF) (25-50 U/ml) for complete suppression of arrhythmia. In the atria strip, NCX(IF) (10-25 U/ml) increases the threshold dose (1 microM) of ouabain for arrhythmia onset 2.2+/-0.5-fold (n=5, p<0.05) as well as prolongs the lag-phase for arrhythmia appearance 4.0+/-0.5-fold (n=5, p<0.01). The lag period for arrhythmia onset was also lengthened (2.0+/-0.4-fold) by NCX(IF) in the ventricle strips (n=6, p<0.002). At low frequency of pacing (1 Hz), all three tested substances, lidocaine, KB-R7943, and NCX(IF) can effectively suppress the ouabain-induced arrhythmia. However, at higher frequency (2 Hz), lidocaine is ineffective in suppressing arrhythmia, whereas KB-R7943 becomes pro-arrhythmic. In contrast to reference drugs, NCX(IF) retains its anti-arrhythmic capacity at high frequencies, either in the atria (n=6, p<0.01) or ventricle (n=5, p<0.05) strips. In conclusion, NCX(IF) results in rapid, effective and stable suppression of arrhythmia both in the atria and ventricle preparations under conditions at which the reference drugs become ineffective.

Asymmetric Preorganization of Inverted Pair Residues in the Sodium-Calcium Exchanger.

In analogy with many other proteins, Na(+)/Ca(2+) exchangers (NCX) adapt an inverted twofold symmetry of repeated structural elements, while exhibiting a functional asymmetry by stabilizing an outward-facing conformation. Here, structure-based mutant analyses of the Methanococcus jannaschii Na(+)/Ca(2+) exchanger (NCX_Mj) were performed in conjunction with HDX-MS (hydrogen/deuterium exchange mass spectrometry) to identify the structure-dynamic determinants of functional asymmetry. HDX-MS identified hallmark differences in backbone dynamics at ion-coordinating residues of apo-NCX_Mj, whereas Na(+)or Ca(2+) binding to the respective sites induced relatively small, but specific, changes in backbone dynamics. Mutant analysis identified ion-coordinating residues affecting the catalytic capacity (kcat/Km), but not the stability of the outward-facing conformation. In contrast, distinct "noncatalytic" residues (adjacent to the ion-coordinating residues) control the stability of the outward-facing conformation, but not the catalytic capacity. The helix-breaking signature sequences (GTSLPE) on the α1 and α2 repeats (at the ion-binding core) differ in their folding/unfolding dynamics, while providing asymmetric contributions to transport activities. The present data strongly support the idea that asymmetric preorganization of the ligand-free ion-pocket predefines catalytic reorganization of ion-bound residues, where secondary interactions with adjacent residues couple the alternating access. These findings provide a structure-dynamic basis for ion-coupled alternating access in NCX and similar proteins.