Nongenomic Actions of Thyroid Hormone and Intracellular Calcium Metabolism

Nongenomic actions of thyroid hormone include several that involve or require calcium. Actions of thyroid hormone at the plasma or intracellular membranes include stimulation of membrane glucose transport and of the Na⁺/H⁺ antiporter (exchanger) by mechanisms that require liberation of intracellular calcium and stimulation of the cell membrane and sarcoplasmic reticulum calcium pumps (Ca²⁺-ATPases). These pumps not only transport Ca²⁺, but also are regulated by the intracellular calmodulin-Ca²⁺ complex (plasma membrane/sarcolemma) or calmodulin-dependent protein kinase II phosphorylation of phospholamban (sarcoplasmic reticulum). Intracellular calcium ion concentration may also be subject to regulation by other nongenomic effects of iodothyronines, such as those on the Na⁺/H⁺ antiporter or sodium current, that secondarily affect the Na⁺/Ca²⁺ exchanger. Certain of these nongenomic actions of thyroid hormone, e.g., Na⁺/H⁺ exchanger, Ca²⁺-ATPase, are now recognized to begin at a recently described hormone receptor on a heterodimeric structural membrane protein, integrin αvβ3. The thyroid hormone signal at this receptor is further transduced by the mitogen-activated protein kinase (MAPK; extracellular regulated kinase1/2, ERK1/2) pathway.     

Crystal structure of Ca2+/H+ antiporter protein YfkE reveals the mechanisms of Ca2+ efflux and its pH regulation

Ca²⁺ efflux by Ca²⁺ cation antiporter (CaCA) proteins is important for maintenance of Ca²⁺ homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na⁺/Ca²⁺ exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca²⁺ efflux. To demonstrate conformational changes essential for the CaCA mechanism, we present the crystal structure of the Ca²⁺/H⁺ antiporter protein YfkE from Bacillus subtilis at 3.1-Å resolution. YfkE forms a homotrimer, confirmed by disulfide crosslinking. The protonated state of YfkE exhibits an inward-facing conformation with a large hydrophilic cavity opening to the cytoplasm in each protomer and ending in the middle of the membrane at the Ca²⁺-binding site. A hydrophobic “seal” closes its periplasmic exit. Four conserved α-repeat helices assemble in an X-like conformation to form a Ca²⁺/H⁺ exchange pathway. In the Ca²⁺-binding site, two essential glutamate residues exhibit different conformations compared with their counterparts in NCX_Mj, whereas several amino acid substitutions occlude the Na⁺-binding sites. The structural differences between the inward-facing YfkE and the outward-facing NCX_Mj suggest that the conformational transition is triggered by the rotation of the kink angles of transmembrane helices 2 and 7 and is mediated by large conformational changes in their adjacent transmembrane helices 1 and 6. Our structural and mutational analyses not only establish structural bases for mechanisms of Ca²⁺/H⁺ exchange and its pH regulation but also shed light on the evolutionary adaptation to different energy modes in the CaCA protein family.     

Na+/H+ antiporter properties in peripheral blood lymphocytes from normotensive obese and type 2 diabetic patients do not differ significantly from controls

It has been hypothesized that some genetic factors link different conditions characterized by the presence of insulin resistance: among them, obesity, type 2 (non-insulin-dependent) diabetes mellitus and arterial hypertension. A good candidate could be the Na⁺/H⁺ exchanger, the increased activity of which is considered a genetic marker of essential hypertension. In this study we looked at whether the Na⁺ dependence of the Na⁺/H⁺ antiporter is modified in obese and type 2 diabetic patients, in the absence of arterial hypertension. The activity of this ion exchanger was measured in peripheral blood lymphocytes by acidifying them in Na⁺-free buffer and then monitoring the recovery of intracellular pH after Na⁺ addition. Quiescent lymphocytes were used because they do not have insulin receptors, thus ruling out the effects of the elevated insulin concentrations on the Na⁺/H⁺ exchanger activity. Antiport activity, measured as the ability to extrude H⁺ in the presence of external Na⁺, showed no differences in normotensive obese and type 2 diabetic patients when compared with healthy subjects. Our data therefore suggest that an altered Na⁺/H⁺ exchange activity cannot be considered a common feature of insulin-resistant states.     

Activation of Na+,H+ exchanger produces vasoconstriction of renal resistance vessels

To evaluate the influence of the sodium/proton exchanger (Na⁺,H⁺ exchanger) on the constriction of rat resistance vessels and on the iliac artery, the isometric vasoconstrictions of renal resistance vessels and strips from iliac artery derived from Wistar-Kyoto rats were measured using a vessel myograph. The Na⁺,H⁺ exchanger was activated by intracellular acidification using propionic acid. Cytosolic pH (pH_i) and cytosolic free sodium concentration ([Na⁺]_i) in vascular smooth muscle cells were measured using the fluorescent dye technique. The activation of the Na⁺,H⁺ exchanger increased the [Na⁺]_i by 12.4 ± 1.3 mmol/L (n = 8). The activation of the Na⁺,H⁺ exchanger caused a contractile response of the renal resistance vessels (increase of tension, 1.5 ± 0.1 × 10⁻³ N; n = 13) and of the rat iliac artery (increase of tension, 7.5 ± 0.8 × 10⁻³ N; n = 5). The contractile response after activation of the Na⁺,H⁺ exchanger was significantly inhibited in the absence of external sodium or in the presence of amiloride, confirming the involvement of the Na⁺,H⁺ exchanger. The contractile response after activation of the Na⁺,H⁺ exchanger was significantly reduced in the absence of external calcium, after inhibition of calcium channels by nifedipine, and in the presence of an intracellular calcium antagonist 8-(diethylamino-)-octyl-3,4,5-trimethoxybenzoate (TMB-8), indicating that the activation of the Na⁺,H⁺ exchanger consecutively caused transplasma membrane calcium influx. On the other hand, the inhibition of the Na⁺,Ca²⁺ exchanger by NiCl₂ significantly increased the vasoconstriction of renal resistance vessels after activation of the Na⁺,H⁺ exchanger. The activation of the Na⁺,H⁺ exchanger produces vasoconstriction by an increased cytosolic sodium concentration, inhibition of the Na⁺,Ca²⁺ exchanger, and activation of transplasma membrane calcium influx through potential dependent calcium channels.     

The Autocrine/Paracrine Loop After Myocardial Stretch: Mineralocorticoid Receptor Activation

The stretch of cardiac muscle increases developed force in two phases. The first phase, which occurs rapidly, constitutes the well-known Frank-Starling mechanism and it is generally attributed to enhanced myofilament responsiveness to Ca²⁺. The second phase or slow force response (SFR) occurs gradually and is due to an increase in the calcium transient amplitude as a result of a stretch-triggered autocrine/paracrine mechanism. We previously showed that Ca²⁺ entry through reverse Na⁺/Ca²⁺ exchange underlies the SFR, as the final step of an autocrine/paracrine cascade involving release of angiotensin II/endothelin, and a Na⁺/H⁺ exchanger (NHE-1) activation-mediated rise in Na⁺. In the present review we mainly focus on our three latest contributions to the understanding of this signalling pathway triggered by myocardial stretch: 1) The finding that an increased production of reactive oxygen species (ROS) from mitochondrial origin is critical in the activation of the NHE-1 and therefore in the genesis of the SFR; 2) the demonstration of a key role played by the transactivation of the epidermal growth factor receptor; and 3) the involvement of mineralocorticoid receptors (MR) activation in the stretch-triggered cascade leading to the SFR. Among these novel contributions, the critical role played by the MR is perhaps the most important one. This finding may conceivably provide a mechanistic explanation to the recently discovered strikingly beneficial effects of MR antagonism in humans with cardiac hypertrophy and failure.     

Myocardial recovery during post-ischemic reperfusion: Optimal concentrations of Na+ and Ca2+ in the reperfusate and protective effects of amiloride added to cardioplegic solution

The effects of Na⁺ and Ca²⁺ concentrations in the reperfusate on post-ischemic myocardial recovery were examined. Also, the myocardial protective effects of amiloride, an inhibitor of the Na⁺/Ca²⁺ and Na⁺/H⁺ exchange systems, added to cardioplegic solutions were assessed, using an isolated working rat heart perfusion system. Global myocardial ischemia was induced by 30-min normothermic cardioplegic arrest, using St. Thomas’ solution. The concentration of Na⁺ in the reperfusate varied, stepwise, from 75 to 145 mM/l, and that of Ca²⁺, from 0.1 to 2.5 mM/l. In this study post-ischemic functional recovery was best at 110 mM/l Na⁺ and 1.2–1.8 mM/l Ca²⁺ in the reperfusate. A significantly greater postischemic functional recovery and a lower creatine kinase release were observed when amiloride was added to the cardioplegic solution. Ca²⁺ overload via Na⁺/Ca²⁺ and Na⁺/H⁺ exchange systems would, thus, appear to be due, at least in part, to post-ischemic reperfusion injury.     

Protein Kinase C Induced Changes in Erythrocyte Na/H Exchange and Cytosolic Free Calcium in Humans

To investigate the interrelationship between erythrocyte Na⁺/H⁺ exchange rate and free cytosolic Ca²⁺ concentration, the effect of the (non)selective protein kinase C inhibitors staurosporine, Ro 31-8220 and CGP 41251 (1 μmol/L) and of the protein kinase C activator phorbol-12-myristate-13-acetate (PMA, 1 μmol/L) was studied in vitro on these variables. PKC depleted erythrocytes were obtained after 24 h PMA down-regulation of the cells and intracellular Ca²⁺ clamping was obtained using quin-2 AM and fluo-3 AM.PMA increased (P < .05) the erythrocyte Na⁺/H⁺ exchange activity and this rise was accompanied by an increase in the free cytosolic Ca²⁺ concentration. When staurosporine and Ro 31-8220 were added to erythrocytes in suspension, a decrease in free cytosolic Ca²⁺ concentration was also found, whereas no significant change was observed after CGP 41251 administration. The Na⁺/H⁺ exchange rate was decreased in the 24 h PMA down-regulated erythrocytes as well as in Ca²⁺-clamped cells.Addition of Ca²⁺ in a concentration range of 0 to 1 mmol/L in the presence of calcimycin resulted (P < .001) in a stimulation of Na⁺/H⁺ exchange by 74%. Calcium increased the V_max for cellular pH_i or external Na⁺ activation of Na⁺/H⁺ exchange, whereas it did not affect the K_m for H_i⁺ or external Na⁺ activation. However, in PKC down-regulated cells, calcium did not activate the Na⁺/H⁺ exchange in erythrocytes and the calpain inhibitor E-64d did not prevent this inactivation.Our data show a concomitant increase in free cytosolic Ca²⁺ concentration and Na⁺/H⁺-exchange rate upon protein kinase C activation and a corresponding decrease in both variables upon PKC inhibition, indicating a Ca²⁺ requirement for protein kinase C activation of Na⁺/H⁺ exchange.     

Intracellular Levels of Na+ and TTX-sensitive Na+ Channel Current in Diabetic Rat Ventricular Cardiomyocytes

Intracellular Na⁺ ([Na⁺] _i ) is an important modulator of excitation–contraction coupling via regulating Ca²⁺ efflux/influx, and no investigation has been so far performed in diabetic rat heart. Here, we examined whether any change of [Na⁺] _i in paced cardiomyocytes could contribute to functional alterations during diabetes. Slowing down in depolarization phase of the action potential, small but significant decrease in its amplitude with a slight depolarized resting membrane potential was traced in live cardiomyocytes from diabetic rat, being parallel with a decreased TTX-sensitive Na⁺ channel current (I _Na) density. We recorded either [Na⁺] _i or [Ca²⁺] _i by using a fluorescent Na⁺ indicator (SBFI-AM or Na-Green) or a Ca²⁺ indicator (Fura 2-AM) in freshly isolated cardiomyocytes. We examined both [Na⁺] _i and [Ca²⁺] _i at rest, and also [Na⁺] _i during pacing with electrical field stimulation in a range of 0.2–2.0 Hz stimulation frequency. In order to test the possible contribution of Na⁺/H⁺ exchanger (NHE) to [Na⁺] _i , we examined the free cytoplasmic [H⁺] _i changes from time course of [H⁺] _i recovery in cardiomyocytes loaded with SNARF1-AM by using ammonium prepulse method. Our data showed that [Na⁺] _i in resting cells from either diabetic or control group was not significantly different, whereas the increase in [Na⁺] _i was significantly smaller in paced diabetic cardiomyocytes compared to that of the controls. However, resting [Ca²⁺] _i in diabetic cardiomyocytes was significantly higher than that of the controls. Here, a lower basal pH _i in diabetics compared with the controls correlates also with a slightly higher but not significantly different NHE activity and consequently a similar Na⁺ loading rate at resting state with a leftward shift in pH sensitivity of NHE-dependent H⁺-flux. NHE protein level remained unchanged, while protein levels of Na⁺/K⁺ ATPase and Na⁺/Ca²⁺ exchanger were decreased in the diabetic cardiomyocytes. Taken together, the present data indicate that depressed I _Na plays an important role in altered electrical activity with less Na⁺ influx during contraction, and an increased [Ca²⁺] _i load in these cells seems to be independent of [Na⁺] _i . The data with insulin treatment suggest further a recent balance between Na⁺ influx and efflux proteins associated with the [Na⁺] _i , particularly during diabetes.     

The role of Na+/H+ exchange in ischemia-reperfusion

In ischemia the cytosol of cardiomyocytes acidifies; this is reversed upon reperfusion. One of the major pH_i-regulating transport systems involved is the Na⁺/H⁺ exchanger. Inhibitors of the Na⁺/H⁺ exchanger have been found to more effectively protect ischemic-reperfused myocardium when administered before and during ischemia than during reperfusion alone. It has been hypothesized that the protection provided by pre-ischemic administration is due to a reduction in Na⁺ and secondary Ca²⁺ influx. Under reperfusion conditions Na⁺/H⁺ exchange inhibition also seems protective since it prolongs intracellular acidosis which can prevent hypercontracture. In detail, however, the mechanisms by which Na⁺/H⁺ exchange inhibition provides protection in ischemic-reperfused myocardium are still not fully identified.     

NCLX is an essential component of mitochondrial Na+/Ca2+ exchange

Mitochondrial Ca²⁺ efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an Na⁺-dependent mechanism mediates mitochondrial Ca²⁺ efflux, the molecular identity of this transporter has remained elusive. Here we show that the Na⁺/Ca²⁺ exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing Ca²⁺ and Na⁺ fluorescent imaging, we demonstrate that mitochondrial Na⁺-dependent Ca²⁺ efflux is enhanced upon overexpression of NCLX, is reduced by silencing of NCLX expression by siRNA, and is fully rescued by the concomitant expression of heterologous NCLX. NCLX-mediated mitochondrial Ca²⁺ transport was inhibited, moreover, by CGP-37157 and exhibited Li⁺ dependence, both hallmarks of mitochondrial Na⁺-dependent Ca²⁺ efflux. Finally, NCLX-mediated mitochondrial Ca²⁺ exchange is blocked in cells expressing a catalytically inactive NCLX mutant. Taken together, our results converge to the conclusion that NCLX is the long-sought mitochondrial Na⁺/Ca²⁺ exchanger.     

Mechanisms of Ca2+ overload induced by extracellular H2O2 in quiescent isolated rat cardiomyocytes

Rat cardiomyocytes were exposed to H₂O₂ (1–100 μmol/L) for 10 min with washout for 10 min. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using fluo-3. [Ca²⁺]_i increased with 100 μmol/L H₂O₂ and further increased during washout, causing irreversible contracture in one-half of the cells. The increase in [Ca²⁺]_i with 10 μmol/L H₂O₂ was modest with few cells showing irreversible contracture and attenuated by caffeine, and [Ca²⁺]_i gradually decreased during washout and this decrease was accelerated by a calcium-free solution, while 1 μmol/L H₂O₂ did not have any effects on [Ca²⁺]_i or cell viability. Ca²⁺ overload caused during exposure to 100 μmol/L H₂O₂ was attenuated by caffeine with improved cellular viability but not by chelerythrine, KB-R7943 or nifedipine. With 100 μmol/L H₂O₂ calcium-free solution attenuated the increase during exposure and washout while KB-R7943 or chelerythrine partly attenuated further increase during washout but not improved cell viability, but chelerythrine did not have additional effect on calcium-free treatment. Catalase abolished the effects of H₂O₂. We concluded that the increased [Ca²⁺]_i during exposure to 100 μmol/L H₂O₂ was caused both by release of Ca²⁺ from the intracellular store sites including the sarcoplasmic reticulum and by influx through route(s) other than the voltage-dependent Ca²⁺ channels or Na⁺/Ca²⁺ exchanger, although the Na⁺/Ca²⁺ exchanger or protein kinase C-mediated mechanism was partly responsible for a further increase during washout. Received: 1 February 2001, Returned for revision: 19 February 2001, Revision received: 11 April 2001, Accepted: 11 April 2001     

Regulation of myocardial Na+/H+ exchanger activity

The Na⁺/H⁺ exchanger is a plasma membrane protein, present in the myocardium, which removes intracellular protons and exchanges them with extracellular Na⁺. The protein comprises an N-terminal, hydrophobic, integral membrane domain that transports the ions and a C-terminal, hydrophilic region that regulates the N-terminal domain. The C-terminal domain has several sub-domains, including one region that binds calmodulin and another that is phosphorylated by protein kinases. The Na⁺/H⁺ exchanger is activated by angiotensin, endothelin and α1-adrenergic stimulation. These effectors increase phosphorylation of the C-terminal domain by protein kinases, and G proteins have been implicated in this, but their role remains to be defined. It has recently been shown that ischemia and other stimuli lead to an increased expression of the Na⁺/H⁺ exchanger in the myocardium. The role of this increased expression in the pathology of ischemia and reperfusion-mediated myocardial damage has yet to be determined. Recent evidence suggests that the Na⁺/H⁺ exchanger may play a key role in hypertrophy of the myocardium, and that its activation through G protein-coupled receptors may be important in mediating its effects. Received: 23 April 2001 / Accepted: 14 May 2001     

Regulation and Role of the Presynaptic and Myocardial Na+/H+ Exchanger NHE1: Effects on the Sympathetic Nervous System in Heart Failure

In acute myocardial ischemia and in chronic heart failure, sympathetic activation with excessive norepinephrine (NE) release from and reduced NE reuptake into sympathetic nerve endings is a prominent cause of arrhythmias and cardiac dysfunction. The Na⁺/H⁺ exchanger NHE1 is the predominant isoform in the heart. It contributes to cellular acid–base balance, and electrolyte, and volume homeostasis, and is activated in response to intracellular acidosis and/or activation of guanine nucleotide binding (G) protein‐coupled receptors. NHE1 mediates its signaling via protein kinases A (PKA) or C (PKC). In cardiomyocytes, NHE1 is restricted to specialized membrane domains, where it regulates the activity of pH‐sensitive proteins and modulates the driving force of the Na⁺/Ca²⁺ exchanger. During acute ischemia/reperfusion and in heart failure the activity/amount of NHE1 is increased, leading to intracellular Ca²⁺ overload and promoting structural (apoptosis, hypertrophy) and functional (arrhythmias, hypercontraction) myocardial damage. In sympathetic nerve endings, increased NHE1 activity results in the accumulation of axoplasmic Na⁺ that diminishes the inward and/or favors the outward transport of NE via the neuronal norepinephrine transporter (NET). The increased NE levels within the nerve–muscle junction facilitate the sustained stimulation of myocardial α‐ and β‐adrenoceptors (ARs), which in turn aggravate the increases in myocardial NHE1 activity and the associated deleterious effects. Furthermore, the responsiveness of the β‐AR declines overtime, which results in further release of NE, initiating a vicious cycle. Accordingly, NHE1 is a potential candidate for targeted intervention to suppress this feedback loop.     

Mechanism of Ca2+ resistance in adult heart cells isolated with trypsin plus Ca2+

Ca²⁺-resistant heart cells prepared with trypsin and Ca²⁺ leak Na⁺ and K⁺ more slowly than Ca²⁺-susceptible cells prepared without trypsin and Ca²⁺. The two preparations show similar leak rates for amino acids and nucleotides. Cells prepared with Ca²⁺ alone show low ion leak rates, but the yield of rod-shaped cells is less than half that when trypsin is present. Cells prepared with trypsin alone show high ion leak rates. The Na⁺-K⁺ ATPase activity of Ca²⁺-susceptible cells appears to be approximately three-fold greater than that of Ca²⁺-resistant cells. Imposing a Na⁺-K⁺ leak by the addition of gramicidin D causes no stimulation of Na⁺-K⁺ ATPase in Ca²⁺-susceptible cells, but stimulates the activity of Ca²⁺-resistant cells up to that of the Ca²⁺-susceptible cells. Ca²⁺-resistant cells appear to contain more K⁺ and less Na⁺ than Ca²⁺-susceptible cells. Treatment of Ca²⁺-resistant cells with ouabain (1 mm) for 5 min changes the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ balance to approximately that of the Ca²⁺-susceptible cells, and induces a similar degree of Ca²⁺ susceptibility. We therefore conclude that treatment with trypsin plus Ca²⁺ confers Ca²⁺ resistance by keeping the permeability of the sarcolemma to Na⁺ and K⁺ sufficiently low to allow the Na⁺-K⁺ ATPase and $\text{Na}{}^{\mathrm{+}}\text{Ca}{}^{\mathrm{+}}$ exchanger to maintain normal gradients of Na⁺, K⁺ and Ca²⁺. The agent responsible for maintaining low ion permeability appears to be Ca²⁺ itself, while trypsin increases the yield and purity of the Ca²⁺-resistant cells.     

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

Na⁺/Ca²⁺ exchangers (NCXs) are ubiquitous membrane transporters with a key role in Ca²⁺ homeostasis and signaling. NCXs mediate the bidirectional translocation of either Na⁺ or Ca²⁺, and thus can catalyze uphill Ca²⁺ 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 S_ext, S_Ca, and S_int sites, whereas the S_mid site is occupied by one water molecule and one H⁺, which protonates an adjacent aspartate side chain (D240). Experimental measurements of Na⁺/Ca²⁺ and Ca²⁺/Ca²⁺ exchange by wild-type and mutagenized NCX_Mj confirm that transport of both Na⁺ and Ca²⁺ requires protonation of D240, and that this side chain does not coordinate either ion at S_mid. 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 Ca²⁺ is not. Altogether, these findings provide the basis for further experimental and computational studies of the conformational mechanism of this exchanger.Ca²⁺ signals control a variety of cellular processes essential for the basic function of multiple organs. In cardiac cells, for example, Ca²⁺ release from the sarcoplasmic reticulum is a necessary step for heart contraction, whereas Ca²⁺ extrusion from the cell is required for cardiac relaxation. These fluctuations in the cytosolic Ca²⁺ concentration underlie the initiation of the heartbeat (1, 2). Na⁺/Ca²⁺ exchangers (NCXs) play a central role in the homeostasis of cellular Ca²⁺ (3–5). These integral membrane proteins are ubiquitous in many types of tissues including the heart, brain, and kidney (4), and consequently their dysfunction is associated with numerous human pathologies such as cardiac arrhythmia, hypertension, skeletal muscle dystrophy, and postischemic brain damage (5). NCXs facilitate the translocation of either Ca²⁺ or Na⁺ across the membrane; thus, they can harness a transmembrane sodium motive force to energize Ca²⁺ transport against a concentration gradient. For example, the cardiac exchanger NCX1 mediates the extrusion of intracellular Ca²⁺ driven by a Na⁺ transmembrane gradient maintained by the Na⁺/K⁺ ATPase (3, 6).Numerous electrophysiological studies over the past three decades have analyzed the functional and regulatory properties of these important exchangers. It is well established that NCXs are reversible and electrogenic, but it has been debated whether the Na⁺/Ca²⁺ exchange stoichiometry is 3:1 or 4:1 (3, 5, 7–12). In any case, NCXs can also facilitate Na⁺/Na⁺ and Ca²⁺/Ca²⁺ exchange, implying that the translocation of Na⁺ and Ca²⁺ are two distinct reactions (3, 5, 6, 13). NCXs are regulated by several factors, such as cytosolic Na⁺ and Ca²⁺ concentrations, pH, ATP, and PIP₂ (5, 6). Ca²⁺ regulation in particular involves accessory cytoplasmic domains not directly implicated in the ion-exchange function of the transmembrane domain (5, 14, 15). It is in highly conserved regions within the latter domain, known as α1 and α2 (in transmembrane helices TM2/TM3 and TM7/TM8, respectively), where specific polar and carboxylic amino acids have been identified to be crucial for ion binding and transport (6, 16–18). Similar sequence motifs are found in related antiporters that exchange Na⁺ for K⁺ and Ca²⁺ (NCKX), and Ca²⁺ for H⁺ (CAX) (19–21).In a recent breakthrough, the NCX exchanger from the archaeobacterium Methanocaldococcus jannaschii was isolated and functionally reconstituted, and its crystal structure determined at 1.9-Å resolution (22). The structure of NCX_Mj revealed an intriguing architecture consisting of two inverted topological repeats of five transmembrane helices each (Fig. 1A), which adopt similar but not identical conformations (Fig. S1). This structural asymmetry is likely to underlie the ability of the exchanger to adopt outward- and inward-open conformations alternately, as is mandatory in secondary-transport processes (23, 24). Importantly, the electron density map for NCX_Mj also revealed clear peaks for four putative ion-binding sites in the α1 and α2 regions, involving several of the residues previously identified as essential (Fig. 1B). One of these sites, referred to as S_Ca, also produced an anomalous scattering signal, somewhat weak but indicative of the presence of a bound Ca²⁺. The sites referred to as S_ext and S_int appeared to be Na⁺ sites, based on chemical and geometric considerations. The fourth site, S_mid, was tentatively interpreted also as a Na⁺ site, although in this case the ion-coordination number and coordination geometry would be atypical (22).Open in a separate windowFig. 1.Structure of the Na⁺/Ca²⁺ exchanger from M. jannaschii in an outward-facing conformation. (A) The architecture of NCX_Mj (PDB entry 3V5U) consists of two repeats of five transmembrane helices each (orange, marine cartoons), which adopt opposite orientations in the membrane (the cytoplasmic side is below). Four putative ion-binding sites (indicated by gray spheres) are found halfway across the transmembrane region, flanked by helices TM2–TM3 and TM7–TM8. The structures of the five-helix repeats are highly similar but not identical (Fig. S1). The major differences are the orientation of TM1/TM6 relative to the rest of the repeat, and a bend in the periplasmic half of TM7, not observed in the equivalent region of TM2. These structural asymmetries likely underlie the ability of the exchanger to alternatively adopt outward- and inward-facing conformations (24). (B) Close-up of the four putative ion-binding sites. The side-chain and backbone groups lining these sites are highlighted; the hypothetical ion coordination contacts are indicated in each case. (C) All-atom simulation model of NCX_Mj embedded in a phospholipid membrane. The membrane consists of 208 POPC molecules, and protein and membrane are hydrated by ∼14,900 water molecules. Chloride ions (omitted for clarity) were also included to counter the net charge of the ion–protein complex. The overall system size is ∼77,300 atoms. (D) Ion configuration that is most likely to correspond to the crystal structure of NCX_Mj, according to the structural and thermodynamic analyses described in Figs. 2 and ​and3.3. A water molecule (W) occupies the S_mid site, while Na⁺ ions occupy S_ext, S_Ca, and S_int. Note that D240 is protonated, and that the side-chain carboxamide group of N81 is inverted relative to the original assignment. The figure shows the conformation that is most frequently observed in a 200-ns MD simulation, according to an RMSD-based clustering analysis comprising the residues highlighted in B.If one assumes that the exchange of Na⁺ and Ca²⁺ mediated by NCX_Mj proceeds according to a “ping-pong” mechanism, in analogy with other NCXs (13), binding of Na⁺ and Ca²⁺ ought to be mutually exclusive (22), even if the exchange stoichiometry is 3:1 rather than 4:1. Thus, the detection of concurrent electron density signals for three Na⁺ and one Ca²⁺ must reflect the coexistence of protein molecules with either of these two ion configurations in the crystal lattice. On the other hand, configurations in which Na⁺ and Ca²⁺ are simultaneously bound cannot be entirely ruled out; indeed, a minor transport mode has been reportedly observed for cardiac NCX1 whereby one Na⁺ and one Ca²⁺ are cotranslocated (9). Either way, it is puzzling that the local structure of the binding sites revealed by the electron density is uniquely defined, as it seems unlikely that this structure is identical irrespective of which ion type is bound. This lack of disorder could be explained if only one of the ion configurations that hypothetically coexist in the protein crystal was significantly populated, but it is not immediately apparent which of these configurations is represented by the data. Thus, notwithstanding the groundbreaking insights provided by the NCX_Mj structure, the interpretation of the electron density in regard to the ion configuration is, in our view, not straightforward: could the proposed Na⁺ occupancy of the suboptimal S_mid site result from the concurrent binding of Ca²⁺, explaining the minor transport mode observed for NCX1? If so, would S_mid remain occupied by Na⁺ in the absence of Ca²⁺? Or is the S_Ca site alternatively occupied by Na⁺ and Ca²⁺? If so, does the electron density signal detected in S_mid reflect a fourth Na⁺ ion? Or could it be a water molecule, or another cation, such as K⁺? It is worth noting that the amino acid composition of the binding sites in NCX_Mj is more akin to that of NCKX exchangers, which cotransport K⁺ along with Ca²⁺, than to NCX homologs (Fig. S2), including those of Escherichia coli and Methanosarcina acetivorans (25). The functional reconstitution of NCX_Mj showed that Na⁺/Ca²⁺ exchange in NCX_Mj is not influenced by a K⁺ gradient (22), but it is conceivable that K⁺ is constitutively bound throughout the transport cycle.Here, we use all-atom molecular-dynamics (MD) simulations and free-energy calculations of NCX_Mj to systematically assess a series of possible ion configurations, both in terms of their consistency with the protein conformation observed in the experimental crystal structure, and from a thermodynamic standpoint. This computational analysis yields a clear-cut testable hypothesis, which we examine experimentally via Na⁺/Ca²⁺ and Ca²⁺/Ca²⁺ exchange assays for wild-type and mutagenized NCX_Mj.     

Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism

Regulation of ion-transport in the Na⁺/Ca²⁺ exchanger (NCX) occurs via its cytoplasmic Ca²⁺-binding domains, CBD1 and CBD2. Here, we present a mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). We initially determined the structure of the Ca²⁺-free form of CBD2-AD and the structure of CBD2-BD that represent the two major splice variant classes in NCX1. Although the apo-form of CBD2-AD displays partially disordered Ca²⁺-binding sites, those of CBD2-BD are entirely unstructured even in an excess of Ca²⁺. Striking differences in the electrostatic potential between the Ca²⁺-bound and -free forms strongly suggest that Ca²⁺-binding sites in CBD1 and CBD2 form electrostatic switches analogous to C₂-domains. SAXS analysis of a construct containing CBD1 and CBD2 reveals a conformational change mediated by Ca²⁺-binding to CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca²⁺ is influenced by the closely located cassette exons. We further propose that Ca²⁺-binding to CBD2 induces a second electrostatic switch, required to alleviate Na⁺-dependent inactivation of Na⁺/Ca²⁺ exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform- and splice variant-specific and allows for tailored exchange activities.