The topology of the C-terminal sections of the NCX1 Na+/Ca2+ exchanger and the NCKX2 Na+/Ca2+-K+ exchanger

Mammalian Na⁺/Ca²⁺ (NCX) and Na⁺/Ca²⁺-K⁺ exchangers (NCKX) are polytopic membrane proteins that play critical roles in calcium homeostasis in many cells. Although hydropathy plots for NCX and NCKX are very similar, reported topological models for NCX1 and NCKX2 differ in the orientation of the three C-terminal transmembrane segments (TMS). NCX1 is thought to have 9 TMS and a re-entrant loop, whereas NCKX2 is thought to have 10 TMS. The current topological model of NCKX2 is very similar to the 10 membrane spanning helices seen in the recently reported crystal structure of NCX_MJ, a distantly related archaebacterial Na⁺/Ca²⁺ exchanger. Here we reinvestigate the orientation of the three C-terminal TMS of NCX1 and NCKX2 using mass-tagging experiments of substituted cysteine residues. Our results suggest that NCX1, NCKX2 and NCX_MJ all share the same 10 TMS topology.     

Identification of an endogenous inhibitor of the cardiac Na+/Ca2+ exchanger, phospholemman

Rapid and precise control of Na(+)/Ca(2+) exchanger (NCX1) activity is essential in the maintenance of beat-to-beat Ca(2+) homeostasis in cardiac myocytes. Here, we show that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, is a novel endogenous protein inhibitor of cardiac NCX1. Using a heterologous expression system that is devoid of both endogenous PLM and NCX1, we first demonstrated by confocal immunofluorescence studies that both exogenous PLM and NCX1 co-localized at the plasma membrane. Reciprocal co-immunoprecipitation studies revealed specific protein-protein interaction between PLM and NCX1. The functional consequences of direct association of PLM with NCX1 was the inhibition of NCX1 activity, as demonstrated by whole-cell patch clamp studies to measure NCX1 current density and radiotracer flux assays to assess Na(+)-dependent (45)Ca(2+) uptake. Inhibition of NCX1 by PLM was specific, because a single mutation of serine 68 to alanine in PLM resulted in a complete loss of inhibition of NCX1 current, although association of the PLM mutant with NCX1 was unaltered. In native adult cardiac myocytes, PLM co-immunoprecipitated with NCX1. We conclude that PLM, a member of the FXYD family of small ion transport regulators known to modulate Na(+)-K(+)-ATPase, also regulates Na(+)/Ca(2+) exchange in the heart.     

Shank2 associates with and regulates Na+/H+ exchanger 3

Na+/H+ exchanger 3 (NHE3) plays a pivotal role in transepithelial Na+ and HCO3(-) absorption across a wide range of epithelia in the digestive and renal-genitourinary systems. Accumulating evidence suggests that PDZ-based adaptor proteins play an important role in regulating the trafficking and activity of NHE3. A search for NHE3-binding modular proteins using yeast two-hybrid assays led us to the PDZ-based adaptor Shank2. The interaction between Shank2 and NHE3 was further confirmed by immunoprecipitation and surface plasmon resonance studies. When expressed in PS120/NHE3 cells, Shank2 increased the membrane expression and basal activity of NHE3 and attenuated the cAMP-dependent inhibition of NHE3 activity. Furthermore, knock-down of native Shank2 expression in Caco-2 epithelial cells by RNA interference decreased NHE3 protein expression as well as activity but amplified the inhibitory effect of cAMP on NHE3. These results indicate that Shank2 is a novel NHE3 interacting protein that is involved in the fine regulation of transepithelial salt and water transport through affecting NHE3 expression and activity.     

Colocalization of voltage-gated Na+ channels with the Na+/Ca2+ exchanger in rabbit cardiomyocytes during development

Reverse-mode activity of the Na(+)/Ca(2+) exchanger (NCX) has been previously shown to play a prominent role in excitation-contraction coupling in the neonatal rabbit heart, where we have proposed that a restricted subsarcolemmal domain allows a Na(+) current to cause an elevation in the Na(+) concentration sufficiently large to bring Ca(2+) into the myocyte through reverse-mode NCX. In the present study, we tested the hypothesis that there is an overlapping expression and distribution of voltage-gated Na(+) (Na(v)) channel isoforms and the NCX in the neonatal heart. For this purpose, Western blot analysis, immunocytochemistry, confocal microscopy, and image analyses were used. Here, we report the robust expression of skeletal Na(v)1.4 and cardiac Na(v)1.5 in neonatal myocytes. Both isoforms colocalized with the NCX, and Na(v)1.5-NCX colocalization was not statistically different from Na(v)1.4-NCX colocalization in the neonatal group. Western blot analysis also showed that Na(v)1.4 expression decreased by sixfold in the adult (P < 0.01) and Na(v)1.1 expression decreased by ninefold (P < 0.01), whereas Na(v)1.5 expression did not change. Although Na(v)1.4 underwent large changes in expression levels, the Na(v)1.4-NCX colocalization relationship did not change with age. In contrast, Na(v)1.5-NCX colocalization decreased ～50% with development. Distance analysis indicated that the decrease in Na(v)1.5-NCX colocalization occurs due to a statistically significant increase in separation distances between Na(v)1.5 and NCX objects. Taken together, the robust expression of both Na(v)1.4 and Na(v)1.5 isoforms and their colocalization with the NCX in the neonatal heart provides structural support for Na(+) current-induced Ca(2+) entry through reverse-mode NCX. In contrast, this mechanism is likely less efficient in the adult heart because the expression of Na(v)1.4 and NCX is lower and the separation distance between Na(v)1.5 and NCX is larger.     

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.     

The Na+/H+ exchanger isoform 1

The Na+/H+ exchanger (NHE) isoform 1 is a ubiquitously expressed integral membrane protein which regulates intracellular pH in mammalian cells. Nine isoforms of the Na+/H+ exchanger have been identified. The isoform first discovered has two domains: an N-terminal membrane domain containing approximately 500 amino acids and a C-terminal regulatory domain containing approximately 315 amino acids. The exchanger, which resides in the plasma membrane, exchanges an intracellular proton for an extracellular sodium, thereby regulating intracellular pH. It is involved in cell growth and differentiation, cell migration, and regulation of sodium fluxes. The Na+/H+ exchanger plays an important role in myocardial damage during ischemia and reperfusion and has recently been implicated as a mediator of cardiac hypertrophy. Inhibitors of the Na+/H+ exchanger, which may prove useful in the clinical treatment of these conditions, are currently being developed and clinical trials are underway.     

Identification of the protein and cDNA of the cardiac Na+/H+ exchanger.

We examined the myocardial form of the Na+/H+ exchanger. A partial length cDNA clone was isolated from a rabbit cardiac library and it encoded for a Na+/H+ exchange protein. In comparison with the human Na+/H+ exchanger, the sequence of the 5' end of the cDNA was highly conserved, much more than the 3' region, while the deduced amino acid sequence was also highly conserved. To further characterize the myocardial Na+/H+ exchange protein, we examined Western blots of isolated sarcolemma with antibody produced against a fusion protein of the Na+/H+ exchanger. The antibodies reacted with a sarcolemma protein of 50 kDa and with a protein of 70 kDa. The results show that the rabbit myocardium does possess a Na+/H+ exchanger protein homologous to the known human Na+/H+ exchanger.     

Stimulation of Na+-Ca2+ exchange in heart sarcolemma by insulin

Insulin was found to stimulate Na+-dependent Ca2+ uptake in dog heart sarcolemma in a concentration dependent manner (0.001 to 1 milliunits/ml). Maximal stimulation (160 to 170%) was seen at 0.1 to 1 milliunits/ml of insulin. Unlike Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ uptake was unaltered by 1 microunit/ml of insulin. However, high concentrations of insulin (0.01 to 1 milliunits/ml) significantly increased the ATP-dependent Ca2+ uptake activity of heart sarcolemma; maximal increase (60%) was observed at 1 milliunit/ml of insulin. The Na+ K+-ATPase activity did not change upon incubating sarcolemma with insulin. The membrane preparation exhibited specific insulin binding characteristics. The Scatchard plot analysis of the data indicated two binding sites for insulin; the association constants for the high and low affinity sites were 2 X 10(9) M-1 and 4.4 X 10(8) M-1, respectively. These results support the view regarding the presence of insulin receptors in the heart cell membrane and indicate a dramatic effect of insulin on the sarcolemmal Ca2+ transport systems.     

Cleavage of the plasma membrane Na+/Ca2+ exchanger in excitotoxicity

In brain ischemia, gating of postsynaptic glutamate receptors and other membrane channels triggers intracellular Ca2+ overload and cell death. In excitotoxic settings, the initial Ca2+ influx through glutamate receptors is followed by a second uncontrolled Ca2+ increase that leads to neuronal demise. Here we report that the major plasma membrane Ca2+ extruding system, the Na+/Ca2+ exchanger (NCX), is cleaved during brain ischemia and in neurons undergoing excitotoxicity. Inhibition of Ca2+-activated proteases (calpains) by overexpressing their endogenous inhibitor protein, calpastatin or the expression of an NCX isoform not cleaved by calpains, prevented Ca2+ overload and rescued neurons from excitotoxic death. Conversely, down-regulation of NCX by siRNA compromised neuronal Ca2+ handling, transforming the Ca2+ transient elicited by non-excitotoxic glutamate concentrations into a lethal Ca2+overload. Thus, proteolytic inactivation of NCX-driven neuronal Ca2+ extrusion is responsible for the delayed excitotoxic Ca2+ deregulation and neuronal death.     

Amiloride and its analogs as tools to inhibit Na+ transport via the Na+ channel, the Na+/H+ antiport and the Na+/Ca2+ exchanger

Amiloride analogs inhibit a number of transmembrane Na+ transport systems: 1) the epithelium Na+ channel, 2) the Na+/H+ exchange system and 3) the Na+/Ca2+ exchange system. Structure--activity relationships using amiloride derivatives with selected modification of each of the functional groups of the molecule indicate that the 3 Na+ transporting systems have distinct pharmacological profiles. 5-N Disubstituted derivatives of amiloride, such as ethylisopropylamiloride are the most potent inhibitors of the Na+/H+ exchange system. Conversely, amiloride derivatives that are substituted on the guanidino moiety, such as phenamil, are potent inhibitors of the epithelium Na+ channel. It is thus possible, by using selected amiloride derivatives to inhibit selectively one or another of the Na+ transport systems.     

Modification of sarcolemmal Na+-K+-ATPase and Na+/Ca2+ exchanger expression in heart failure by blockade of renin-angiotensin system

The activities of both sarcolemmal (SL) Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchanger, which maintain the intracellular cation homeostasis, have been shown to be depressed in heart failure due to myocardial infarction (MI). Because the renin-angiotensin system (RAS) is activated in heart failure, this study tested the hypothesis that attenuation of cardiac SL changes in congestive heart failure (CHF) by angiotensin-converting enzyme (ACE) inhibitors is associated with prevention of alterations in gene expression for SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchanger. CHF in rats due to MI was induced by occluding the coronary artery, and 3 wk later the animals were treated with an ACE inhibitor, imidapril (1 mg.kg(-1).day(-1)), for 4 wk. Heart dysfunction and cardiac hypertrophy in the infarcted animals were associated with depressed SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities. Protein content and mRNA levels for Na(+)/Ca(2+) exchanger as well as Na(+)-K(+)-ATPase alpha(1)-, alpha(2)- and beta(1)-isoforms were depressed, whereas those for alpha(3)-isoform were increased in the failing heart. These changes in SL activities, protein content, and gene expression were attenuated by treating the infarcted animals with imidapril. The beneficial effects of imidapril treatment on heart function and cardiac hypertrophy as well as SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities in the infarcted animals were simulated by enalapril, an ACE inhibitor, and losartan, an angiotensin receptor antagonist. These results suggest that blockade of RAS in CHF improves SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities in the failing heart by preventing changes in gene expression for SL proteins.     

Effect of 6-hydroxydopamine on the gene expression of Na+/Ca2+ exchanger in the rat heart

The Na+/Ca2+ exchanger (NCX) is an important component of the process of excitation-contraction coupling in the heart muscle. The level of gene expression as well as transport activities of this membrane structure is changed under pathological conditions like ischemic injury, myocardial infarction or diabetes. In this work we focused on the question whether the adrenergic modulation affects gene expression of the NCX in rat hearts. NCX mRNA levels were studied in the left cardiac atrium (divided into ganglionic and nonganglionic part) and also in the left ventricle of rats treated with 6-hydroxydopamine (6-OHDA) in control and stressed conditions. We have shown that administration of 6-OHDA decreases mRNA levels of NCX in both ganglionic and nonganglionic part of the left atrium and also in the left ventricle. This effect was not altered under combined administration of 6-OHDA and single immobilization stress. These data suggest that an activation of the adrenergic system can potentiate gene expression of the cardiac NCX.     

Contribution of the Na+/Ca2+ exchanger to rapid Ca2+ release in cardiomyocytes

Trigger Ca(2+) is considered to be the Ca(2+) current through the L-type Ca(2+) channel (LTCC) that causes release of Ca(2+) from the sarcoplasmic reticulum. However, cell contraction also occurs in the absence of the LTCC current (I(Ca)). In this article, we investigate the contribution of the Na(+)/Ca(2+) exchanger (NCX) to the trigger Ca(2+). Experimental data from rat cardiomyocytes using confocal microscopy indicating that inhibition of reverse mode Na(+)/Ca(2+) exchange delays the Ca(2+) transient by 3-4 ms served as a basis for the mathematical model. A detailed computational model of the dyadic cleft (fuzzy space) is presented where the diffusion of both Na(+) and Ca(2+) is taken into account. Ionic channels are included at discrete locations, making it possible to study the effect of channel position and colocalization. The simulations indicate that if a Na(+) channel is present in the fuzzy space, the NCX is able to bring enough Ca(2+) into the cell to affect the timing of release. However, this critically depends on channel placement and local diffusion properties. With fuzzy space diffusion in the order of four orders of magnitude lower than in water, triggering through LTCC alone was up to 5 ms slower than with the presence of a Na(+) channel and NCX.     

Physiological role and regulation of the Na+/H+ exchanger

In mammalian eukaryotic cells, the Na+/H+ exchanger is a family of membrane proteins that regulates ions fluxes across membranes. Plasma membrane isoforms of this protein extrude 1 intracellular proton in exchange for 1 extracellular sodium. The family of Na+/H+ exchangers (NHEs) consists of 9 known isoforms, NHE1-NHE9. The NHE1 isoform was the first discovered, is the best characterized, and exists on the plasma membrane of all mammalian cells. It contains an N-terminal 500 amino acid membrane domain that transports ions, plus a 315 amino acid C-terminal, the intracellular regulatory domain. The Na+/H+ exchanger is regulated by both post-translational modifications including protein kinase-mediated phosphorylation, plus by a number of regulatory-binding proteins including phosphatidylinositol-4,5-bisphosphate, calcineurin homologous protein, ezrin, radixin and moesin, calmodulin, carbonic anhydrase II, and tescalcin. The Na+/H+ exchanger is involved in a variety of complex physiological and pathological events that include regulation of intracellular pH, cell movement, heart disease, and cancer. This review summarizes recent advances in the understanding of the physiological role and regulation of this protein.     

Regulation and characterization of the Na+/H+ exchanger.

The Na+/H+ exchanger is a ubiquitous protein present in all mammalian cell types that functions to remove one intracellular H+ for one extracellular Na+. Several isoforms of the protein exist, which are referred to as NHE1 to NHE6 (for Na+/H+ exchanger one through six). The NHE1 protein was the first isoform cloned and studied in a variety of systems. This review summarizes recent papers on this protein, particularly those that have examined regulation of the protein and its expression and activity.