The Ca 2+ pumps and the Na + / Ca 2+ exchangers

The Ca 2+ ATPases or Ca 2+ pumps transport Ca 2+ ions out of the cytosol, by using the energy stored in ATP. The Na + / Ca 2+ exchanger uses the chemical energy of the Na + gradient (the Na + concentration is much higher outside than inside the cell) to remove Ca 2+ from the cytosol. Ca 2+ pumps are found in the plasma membrane and in the endoplasmic reticulum of the cells. The pumps are probably present in the membrane of other organelles, but little experimental information is available on this matter. The Na + / Ca 2+ exchangers are located on the plasma membrane. A Na + / Ca 2+ exchanger was found in the mitochondria, but very little is known on its structure and sequence. These transporters control the Ca 2+ concentration in the cytosol and are vital to prevent Ca 2+ overload of the cells. Their activity is controlled by different mechanisms, that are still under investigation. A number of the possible isoforms for both types of proteins has been detected.© Kluwer Academic Publishers     

Na+/Ca2+-K+ exchangers (NCKX): functional properties and physiological roles

The most numerous Ca2+ extrusion protein family, in terms of distinct genes, is the SLC24 gene family of Na+/Ca2+-K+ exchangers (NCKX). Five distinct gene products have been identified, mostly from specific animal excitable tissues such as neurons and smooth muscle, but also in places like skin pigment epithelium, signifying that NCKX proteins may play very specific roles, related to Ca2+ homeostasis, in these tissues. However, progress in elucidating the specific physiological roles of NCKX proteins has been slow in coming, largely because of challenges relating to isolating the activity of these proteins in their native tissues. Herein, we provide an overview of NCKX protein functional characteristics, highlighting properties that are unique and useful as distinguishing features over other Ca2+ handling mechanisms. We also present the first comprehensive review of the literature concerning physiological roles of NCKX proteins.     

Regulation of the cardiac Na(+)-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca(2+)-binding domain

The sarcolemmal Na(+)-Ca2+ exchanger is regulated by intracellular Ca2+ at a high affinity Ca2+ binding site separate from the Ca2+ transport site. Previous data have suggested that the Ca2+ regulatory site is located on the large intracellular loop of the Na(+)-Ca2+ exchange protein, and we have identified a high-affinity 45Ca2+ binding domain on this loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Journal of Biological Chemistry. 269:22847-22852). We now use electrophysiological and mutational analyses to further define the Ca2+ regulatory site. Wild-type and mutant exchangers were expressed in Xenopus oocytes, and the exchange current was measured using the inside- out giant membrane patch technique. Ca2+ regulation was measured as the stimulation of reverse Na(+)-Ca2+ exchange (intracellular Na+ exchanging for extracellular Ca2+) by intracellular Ca2+. Single-site mutations within two acidic clusters of the Ca2+ binding domain lowered the apparent Ca2+ affinity at the regulatory site from 0.4 to 1.1-1.8 microM. Mutations had parallel effects on the affinity of the exchanger loop for 45Ca2+ binding (Levitsky et al., 1994) and for functional Ca2+ regulation. We conclude that we have identified the functionally important Ca2+ binding domain. All mutant exchangers with decreased apparent affinities at the regulatory Ca2+ binding site also have a complex pattern of altered kinetic properties. The outward current of the wild-type Na(+)-Ca2+ exchanger declines with a half time (th) of 10.8 +/- 3.2 s upon Ca2+ removal, whereas the exchange currents of several mutants decline with th values of 0.7-4.3 s. Likewise, Ca2+ regulation mutants respond more rapidly to Ca2+ application. Study of Ca2+ regulation has previously been possible only with the exchanger operating in the reverse mode as the regulatory Ca2+ and the transported Ca2+ are then on opposite sides of the membrane. The use of exchange mutants with low affinity for Ca2+ at regulatory sites also allows demonstration of secondary Ca2+ regulation with the exchanger in the forward or Ca2+ efflux mode. In addition, we find that the affinity of wild-type and mutant Na(+)-Ca2+ exchangers for intracellular Na+ decreases at low regulatory Ca2+. This suggests that Ca2+ regulation modifies transport properties and does not only control the fraction of exchangers in an active state.     

Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport

Mammalian Na+/Ca2+ exchangers are members of three branches of a much larger family of transport proteins [the CaCA (Ca2+/cation antiporter) superfamily] whose main role is to provide control of Ca2+ flux across the plasma membranes or intracellular compartments. Since cytosolic levels of Ca2+ are much lower than those found extracellularly or in sequestered stores, the major function of Na+/Ca2+ exchangers is to extrude Ca2+ from the cytoplasm. The exchangers are, however, fully reversible and thus, under special conditions of subcellular localization and compartmentalized ion gradients, Na+/Ca2+ exchangers may allow Ca2+ entry and may play more specialized roles in Ca2+ movement between compartments. The NCX (Na+/Ca2+ exchanger) [SLC (solute carrier) 8] branch of Na+/Ca2+ exchangers comprises three members: NCX1 has been most extensively studied, and is broadly expressed with particular abundance in heart, brain and kidney, NCX2 is expressed in brain, and NCX3 is expressed in brain and skeletal muscle. The NCX proteins subserve a variety of roles, depending upon the site of expression. These include cardiac excitation-contraction coupling, neuronal signalling and Ca2+ reabsorption in the kidney. The NCKX (Na2+/Ca2+-K+ exchanger) (SLC24) branch of Na+/Ca2+ exchangers transport K+ and Ca2+ in exchange for Na+, and comprises five members: NCKX1 is expressed in retinal rod photoreceptors, NCKX2 is expressed in cone photoreceptors and in neurons throughout the brain, NCKX3 and NCKX4 are abundant in brain, but have a broader tissue distribution, and NCKX5 is expressed in skin, retinal epithelium and brain. The NCKX proteins probably play a particularly prominent role in regulating Ca2+ flux in environments which experience wide and frequent fluctuations in Na+ concentration. Until recently, the range of functions that NCKX proteins play was generally underappreciated. This situation is now changing rapidly as evidence emerges for roles including photoreceptor adaptation, synaptic plasticity and skin pigmentation. The CCX (Ca2+/cation exchanger) branch has only one mammalian member, NCKX6 or NCLX (Na+/Ca2+-Li+ exchanger), whose physiological function remains unclear, despite a broad pattern of expression.     

Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae.

The PMC1 gene in Saccharomyces cerevisiae encodes a vacuolar Ca2+ ATPase required for growth in high-Ca2+ conditions. Previous work showed that Ca2+ tolerance can be restored to pmc1 mutants by inactivation of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase sensitive to the immunosuppressive drug FK506. We now report that calcineurin decreases Ca2+ tolerance of pmc1 mutants by inhibiting the function of VCX1, which encodes a vacuolar H+/Ca2+ exchanger related to vertebrate Na+/Ca2+ exchangers. The contribution of VCX1 in Ca2+ tolerance is low in strains with a functional calcineurin and is high in strains which lack calcineurin activity. In contrast, the contribution of PMC1 to Ca2+ tolerance is augmented by calcineurin activation. Consistent with these positive and negative roles of calcineurin, expression of a vcx1::lacZ reporter was slightly diminished and a pmc1::lacZ reporter was induced up to 500-fold by processes dependent on calcineurin, calmodulin, and Ca2+. It is likely that calcineurin inhibits VCX1 function mainly by posttranslational mechanisms. Activities of VCX1 and PMC1 help to control cytosolic free Ca2+ concentrations because their function can decrease pmc1::lacZ induction by calcineurin. Additional studies with reporter genes and mutants indicate that PMR1 and PMR2A, encoding P-type ion pumps required for Mn2+ and Na+ tolerance, may also be induced physiologically in response to high-Mn2+ and -Na+ conditions through calcineurin-dependent mechanisms. In these situations, inhibition of VCX1 function may be important for the production of Ca2+ signals. We propose that elevated cytosolic free Ca2+ concentrations, calmodulin, and calcineurin regulate at least four ion transporters in S. cerevisiae in response to several environmental conditions.     

Anomalous regulation of the Drosophila Na(+)-Ca2+ exchanger by Ca2+

The Na(+)-Ca2+ exchanger from Drosophila was expressed in Xenopus and characterized electrophysiologically using the giant excised patch technique. This protein, termed Calx, shares 49% amino acid identity to the canine cardiac Na(+)-Ca2+ exchanger, NCX1. Calx exhibits properties similar to previously characterized Na(+)-Ca2+ exchangers including intracellular Na+ affinities, current-voltage relationships, and sensitivity to the peptide inhibitor, XIP. However, the Drosophila Na(+)-Ca2+ exchanger shows a completely opposite response to cytoplasmic Ca2+. Previously cloned Na(+)-Ca2+ exchangers (NCX1 and NCX2) are stimulated by cytoplasmic Ca2+ in the micromolar range (0.1- 10 microM). This stimulation of exchange current is mediated by occupancy of a regulatory Ca2+ binding site separate from the Ca2+ transport site. In contrast, Calx is inhibited by cytoplasmic Ca2+ over this same concentration range. The inhibition of exchange current is evident for both forward and reverse modes of transport. The characteristics of the inhibition are consistent with the binding of Ca2+ at a regulatory site distinct from the transport site. These data provide a rational basis for subsequent structure-function studies targeting the intracellular Ca2+ regulatory mechanism.     

Structural basis of the Ca2+ inhibitory mechanism of Drosophila Na+/Ca2+ exchanger CALX and its modification by alternative splicing

The Na(+)/Ca(2+) exchanger CALX promotes Ca(2+) efflux in Drosophila sensory neuronal cells to facilitate light-mediated Ca(2+) homeostasis. CALX activity is negatively regulated by specific Ca(2+) interaction within its two intracellular Ca(2+) regulatory domains CBD1 and CBD2, yet how the Ca(2+) binding is converted to molecular motion to operate the exchanger is unknown. Here, we report crystal structures of the entire Ca(2+) regulatory domain CBD12 from two alternative splicing isoforms, CALX 1.1 and 1.2, exhibiting distinct regulatory Ca(2+) dependency. The structures show an open V-shaped conformation with four Ca(2+) ions bound on the CBD domain interface, confirmed by LRET analysis. The structures together with Ca(2+)-binding analysis support that the Ca(2+) inhibition of CALX is achieved by interdomain conformational changes induced by Ca(2+) binding at CBD1. The conformational difference between the two isoforms also indicates that alternative splicing adjusts the interdomain orientation angle to modify the Ca(2+) regulatory property of the exchangers.     

Acidocalcisome: A novel Ca2+ storage compartment in trypanosomatids and apicomplexan parasites.

Acidocalcisomes are novel acidic Ca2+ storage organelles found in trypanosomatids and apicomplexan parasites, abundant in the intracellular stages of these parasites, and characterized by their high electron density, and high content of phosphorus, Ca2+, Mg2+, Na+ and Zn2+. A number of energy-utilizing pumps and exchangers have been found in these organelles, which underlines their importance in the homeostasis of different elements, as discussed here by Roberto Docampo and Silvia Moreno.     

Reversed Na+/Ca2+ exchange contributes to Ca2+ influx and respiratory burst in microglia

Phagocytosis and the ensuing NADPH-mediated respiratory burst are important aspects of microglial activation that require calcium ion (Ca(2+)) influx. However, the specific Ca(2+) entry pathway(s) that regulates this mechanism remains unclear, with the best candidates being surface membrane Ca(2+)-permeable ion channels or Na(+)/Ca(2+) exchangers. In order to address this issue, we used quantitative real-time RT-PCR to assess mRNA expression of the Na(+)/Ca(2+) exchangers, Slc8a1-3/NCX1-3, before and after phagocytosis by rat microglia. All three Na(+)/Ca(2+) exchangers were expressed, with mRNA levels of NCX1 > NCX3 > NCX2, and were unaltered during the one hour phagocytosis period. We then carried out a biophysical characterization of Na(+)/Ca(2+) exchanger activity in these cells. To investigate conditions under which Na(+)/Ca(2+) exchange was functional, we used a combination of perforated patch-clamp analysis, fluorescence imaging of a Ca(2+) indicator (Fura-2) and a Na(+) indicator (SBFI), and manipulations of membrane potential and intracellular and extracellular ions. Then, we used a pharmacological toolbox to compare the contribution of Na(+)/Ca(2+) exchange with candidate Ca(2+)-permeable channels, to the NADPH-mediated respiratory burst that was triggered by phagocytosis. We find that inhibiting the reversed mode of the Na(+)/Ca(2+) exchanger with KB-R7943, dose dependently reduced the phagocytosis-stimulated respiratory burst; whereas, blockers of store-operated Ca(2+) channels or L-type voltage-gated Ca(2+) channels had no effect. These results provide evidence that Na(+)/Ca(2+) exchangers are potential therapeutic targets for reducing the bystander damage that often results from microglia activation in the damaged CNS.     

Co-ordinated expression of 5-HT2C receptors with the NCX1 Na+/Ca2+ exchanger in histaminergic neurones

The different roles of Na+/Ca2+ (NCX) exchangers and Na+/Ca2+/K+ (NCKX) exchangers in regulation of the ionic homeostasis in neurones are poorly understood. We have previously shown that serotonin excites histaminergic tuberomamillary (TM) neurones by activation of 5-HT2C-receptors and Na+/Ca2+ exchange. With the help of single-cell RT-PCR (sc-RT-PCR) we have now determined the coexpression pattern of different subtypes of NCX and NCKX with serotonin receptors. The majority of TM neurones express NCX1, NCX2 and NCKX3. Serotonin 2C receptor-mRNA was detected in 70% while 5-HT2A mRNA was found in only 10% of TM neurones. In all neurones expressing the 5-HT2C receptor NCX1-mRNA was present. Double immunostaining revealed the presence of the NCX1 protein in histidine decarboxylase-positive neurones. In the majority of TM neurones one or two out of five isoforms, NCX1.4, NCX1.5, NCX1.7, NCX1.14, NCX1.15, were detected by cDNA sequencing and/or by restriction analysis. The alternative splicing region is important for the Ca2+ sensitivity and presumably for the modulation of NCX1 function by second messengers. We conclude that several exchanger-subtypes can be coexpressed in single neurones and that TM cells are heterogeneous with respect to their calcium homeostasis regulation.     

Regulation of Na+-K+-ATPase: effect of Mg and Ca ions

The participation of Mg2+ and Ca2+ in complicated mechanisms of Na+, K(+)-ATPase regulation is discussed in the survey. The regulatory actions of Mg2+ on Na+, K(+)-ATPase such as its participation in phosphorylation and dephosphorylation of the enzyme, ADP/ATP-exchange inhibition, cardiac glycosides and vanadate binding with the enzyme, conformational changes induction during ATPase cycle are reviewed in detail. Some current views of mechanisms of above mentioned Mg2+ regulatory effects are discussed. The experimental evidence of Ca2+ immediate influence on the functional activity of Na+, K(+)-ATPase (catalytic, transport and glycoside-binding) are given. It's noted that these effects are based on the conformational changes in the enzyme and also on the phase transition in membrane induced by Ca2+. Unimmediate action of Ca2+ on Na+, K(+)-ATPase is also discussed, especially due to its effect on other membrane systems functionally linked with Na(+)-pump (for instance, due to Na+/Ca(+)-exchanger activation). It's concluded that Mg2+ and Ca2+ as "universal regulators" of the cell effectively influence the functional activity and conformational states of Na+, K(+)-ATPase.     

Ca2+-pumps and Na2+-Ca2+-exchangers in coronary artery endothelium versus smooth muscle

Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation. This can be achieved by Ca2+ sequestration by the sarco-/endoplasmic reticulum Ca2+ pumps (SERCA) and Ca2+ extrusion by plasma membrane Ca2+ pumps (PMCA) and Na+-Ca2+-exchangers (NCX). Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots. The activity of NCX is higher in EC than in SMC but those of PMCA and SERCA is lower. Consistently, the protein abundance for NCX protein is higher in EC than in SMC and those of PMCA and SERCA is lower. Based on RT-PCR experiments, the types of RNA present are as follows: EC for PMCA1 while SMC for PMCA4 and PMCA1; EC for SERCA2 and SERCA3 and SMC for SERCA2. Both EC and SMC express NCX1 (mainly NCX1.3). PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation. Based on these observations and the literature, we conclude that the tightly regulated Ca2+ removal systems in SMC are consistent with the cyclical control of contractility of the filaments and those in EC are consistent with Ca2+ regulation of the endothelial nitric oxide synthase near the cell surface. The differences between EC and SMC should be considered in therapeutic interventions of cardiovascular diseases.     

The molecular determinants of ionic regulatory differences between brain and kidney Na+/Ca2+ exchanger (NCX1) isoforms

The Na(+)/Ca(2+) exchanger gene NCX1 undergoes alternative splicing leading to several isoforms that differ in a small portion of the large cytoplasmic loop. This loop is involved in many regulatory processes of NCX1, including ionic regulation by the transported substrates Na(+) and Ca(2+). High intracellular Ca(2+) can alleviate intracellular Na(+)-dependent inactivation in exon A (NCX1.4)-containing isoforms but not in those containing the mutually exclusive exon B (NCX1.3). Giant excised patches from Xenopus oocytes expressing various NCX1 constructs were used to examine the specific amino acids responsible for these observed regulatory differences. Using a chimeric approach, the region responsible was narrowed down to the small central part of exon A (IDDEEYEKNKTF). Replacing the second aspartic acid of this sequence with arginine (the corresponding amino acid in exon B) in an exon A background completely prevented the effect of Ca(2+) on intracellular Na(+)-dependent inactivation. Mutating the second lysine to cysteine (exon B) had a similar, but only partial, effect. The converse double mutant, but neither single mutation alone, introduced into an exon B background (arginine to aspartic acid and cysteine to lysine) was able to restore the NCX1.4 regulatory phenotype. These data demonstrate that aspartic acid 610 and lysine 617 (using the rat NCX1.4 numbering scheme) are critical molecular determinants of the unique Ca(2+) regulatory properties of NCX1.4.     

Novel role for K+-dependent Na+/Ca2+ exchangers in regulation of cytoplasmic free Ca2+ and contractility in arterial smooth muscle

Cytoplasmic free Ca2+ ([Ca2+]cyt) is essential for the contraction and relaxation of blood vessels. The role of plasma membrane Na+/Ca2+ exchange (NCX) activity in the regulation of vascular Ca2+ homeostasis was previously ascribed to the NCX1 protein. However, recent studies suggest that a relatively newly discovered K+-dependent Na+/Ca2+ exchanger, NCKX (gene family SLC24), is also present in vascular smooth muscle. The purpose of the present study was to identify the expression and function of NCKX in arteries. mRNA encoding NCKX3 and NCKX4 was demonstrated by RT-PCR and Northern blot in both rat mesenteric and aortic smooth muscle. NCXK3 and NCKX4 proteins were also demonstrated by immunoblot and immunofluorescence. After voltage-gated Ca2+ channels, store-operated Ca2+ channels, and Na+ pump were pharmacologically blocked, when the extracellular Na+ was replaced with Li+ (0 Na+) to induce reverse mode (Ca2+ entry) activity of Na+/Ca2+ exchangers, a large increase in [Ca2+]cyt signal was observed in primary cultured aortic smooth muscle cells. About one-half of this [Ca2+]cyt signal depended on the extracellular K+. In addition, after the activity of NCX was inhibited by KB-R7943, Na+ replacement-induced Ca2+ entry was absolutely dependent on extracellular K+. In arterial rings denuded of endothelium, a significant fraction of the phenylephrine-induced and nifedipine-resistant aortic or mesenteric contraction could be prevented by removal of extracellular K+. Taken together, these data provide strong evidence for the expression of NCKX proteins in the vascular smooth muscle and their novel role in mediating agonist-stimulated [Ca2+]cyt and thereby vascular tone.     

Gene knockout studies of Ca2+-transporting ATPases.

The biochemical functions of intracellular and plasma membrane Ca2+-transporting ATPases in the control of cytosolic and organellar Ca2+ levels are well established, but the physiological roles of specific isoforms are less well understood. There appear to be three different types of Ca2+ pumps in mammalian tissues: the sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs), which sequester Ca2+ within the endoplasmic or sarcoplasmic reticulum, the plasma membrane Ca2+-ATPases (PMCAs), which extrude Ca2+ from the cell, and the putative secretory pathway Ca2+-ATPase (SPCA), the function of which is poorly understood. This review describes the results of recent analyses of mouse models with null mutations in the genes encoding SERCA and PMCA isoforms and genetic studies of SERCA and SPCA dysfunction in both humans and model organisms. These studies are yielding important insights regarding the physiological functions of individual Ca2+-transporting ATPases in vivo.     

Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors

The plasma membrane Na+/Ca2+ exchanger (NCX) is almost certainly the major Ca2+ extrusion mechanism in cardiac myocytes. Binding of Na+ and Ca2+ ions to its large cytosolic loop regulates ion transport of the exchanger. We determined the solution structures of two Ca2+ binding domains (CBD1 and CBD2) that, together with an alpha-catenin-like domain (CLD), form the regulatory exchanger loop. CBD1 and CBD2 are very similar in the Ca2+ bound state and describe the Calx-beta motif. Strikingly, in the absence of Ca2+, the upper half of CBD1 unfolds while CBD2 maintains its structural integrity. Together with a 7-fold higher affinity for Ca2+, this suggests that CBD1 is the primary Ca2+ sensor. Specific point mutations in either domain largely allow the interchange of their functionality and uncover the mechanism underlying Ca2+ sensing in NCX.