Plasma membrane Ca2+ pumps and Na+/Ca2+ exchangers

Membrane-intrinsic transport systems play an essential role in intracellular Ca2+ homeostasis. ATP-driven Ca2+ pumps and carrier-mediated Na+/Ca2+ exchangers are the two specific Ca2+ transporting systems mainly responsible for Ca2+ extrusion across the plasma membrane. Ca2+ pumps operate in all eukaryotic cell types and are characterized by their high Ca2+ affinity and their specific regulation by direct interaction with Ca2+/calmodulin. Na+/Ca2+ exchangers are particularly abundant in excitable tissues and are responsible for the bulk Ca2+ efflux in these tissues. Recent success in the molecular characterization of the pumps has led to the determination of complete amino acid sequences for several isoforms and has allowed the identification and topological assignment of important functional and regulatory domains. Genetic evidence indicates that mammalian Ca2+ pump diversity is generated from a multigene family and via alternative RNA splicing. Different isoforms may vary in their regulatory properties, presumably reflecting different physiological requirements of the tissues of their expression. Although the molecular characterization of Na+/Ca2+ exchangers is not as far advanced as that of the pumps, recent studies have established detailed kinetic, stoichiometric and regulatory properties of these systems. Together with advances in expression cloning methods these studies promise to result in a rapid improvement of our knowledge of the functional properties of these ion transporters on a molecular level.     

The Na+/Ca2+ exchangers in demyelinating diseases

Intracellular [Na⁺]_i and [Ca²⁺]_i imbalance significantly contribute to neuro-axonal dysfunctions and maladaptive myelin repair or remyelination failure in chronic inflammatory demyelinating diseases such as multiple sclerosis. Progress in recent years has led to significant advances in understanding how [Ca²⁺]_i signaling network drive degeneration or remyelination of demyelinated axons.The Na⁺/Ca²⁺ exchangers (NCXs), a transmembrane protein family including three members encoded by ncx1, ncx2, and ncx3 genes, are emerging important regulators of [Na⁺]_i and [Ca²⁺]_i both in neurons and glial cells. Here we review recent advance highlighting the role of NCX exchangers in axons and myelin-forming cells, i.e. oligodendrocytes, which represent the major targets of the aberrant inflammatory attack in multiple sclerosis. The contribution of NCX subtypes to axonal pathology and myelin synthesis will be discussed. Although a definitive understanding of mechanisms regulating axonal pathology and remyelination failure in chronic demyelinating diseases is still lacking and requires further investigation, current knowledge suggest that NCX activity plays a crucial role in these processes. Defining the relative contributions of each NCX transporter in axon pathology and myelinating glia will constitute not only a major advance in understanding in detail the intricate mechanism of neurodegeneration and remyelination failure in demyelinating diseases but also will help to identify neuroprotective or remyelinating strategies targeting selective NCX exchangers as a means of treating MS.     

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.     

The role of the Na+/Ca2+ exchangers in Ca2+ dynamics in ventricular myocytes

The role of the Na⁺/Ca²⁺ exchanger (NCX) as the main pathway for Ca²⁺ extrusion from ventricular myocytes is well established. However, both the role of the Ca²⁺ entry mode of NCX in regulating local Ca²⁺ dynamics and the role of the Ca²⁺ exit mode during the majority of the physiological action potential (AP) are subjects of controversy. The functional significance of NCXs location in T-tubules and potential co-localization with ryanodine receptors was examined using a local Ca²⁺ control model of low computational cost. Our simulations demonstrate that under physiological conditions local Ca²⁺ and Na⁺ gradients are critical in calculating the driving force for NCX and hence in predicting the effect of NCX on AP. Under physiological conditions when 60% of NCXs are located on T-tubules, NCX may be transiently inward within the first 100 ms of an AP and then transiently outward during the AP plateau phase. Thus, during an AP NCX current (I_NCX) has three reversal points rather than just one. This provides a resolution to experimental observations where Ca²⁺ entry via NCX during an AP is inconsistent with the time at which I_NCX is thought to become inward. A more complex than previously believed dynamic regulation of I_NCX during AP under physiological conditions allows us to interpret apparently contradictory experimental data in a consistent conceptual framework. Our modelling results support the claim that NCX regulates the local control of Ca²⁺ and provide a powerful tool for future investigations of the control of sarcoplasmic reticulum (SR) Ca²⁺ release under pathological conditions.     

Structure-function relationships of K+-dependent Na+/Ca2+ exchangers (NCKX)

K⁺-dependent Na⁺/Ca²⁺ exchanger proteins (NCKX1-5) of the SLC24 gene family play important roles in a wide range of biological processes including but not limited to rod and cone photoreceptor vision, olfaction, enamel formation and skin pigmentation. NCKX proteins are also widely expressed throughout the brain and NCKX2 and NCKX4 knockouts in mice have specific phenotypes. Here we review our work on structure-function relationships of NCKX proteins. We discuss membrane topology, domains critical to transport function, and residues critical to cation binding and transport function, all in the context of crystal structures that were obtained for the archaeal Na⁺/Ca²⁺ exchanger NCX_Mj.     

The co-presence of Na+/Ca2+-K+ exchanger and Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

We have previously shown that there is high Na(+)/Ca(2+) exchange (NCX) activity in bovine adrenal chromaffin cells. In this study, by monitoring the [Ca(2+)](i) change in single cells and in a population of chromaffin cells, when the reverse mode of exchanger activity has been initiated, we have shown that the NCX activity is enhanced by K(+). The K(+)-enhanced activity accounted for a significant proportion of the Na(+)-dependent Ca(2+) uptake activity in the chromaffin cells. The results support the hypothesis that both NCX and Na(+)/Ca(2+)-K(+) exchanger (NCKX) are co-present in chromaffin cells. The expression of NCKX in chromaffin cells was further confirmed using PCR and northern blotting. In addition to the plasma membrane, the exchanger activity, measured by Na(+)-dependent (45)Ca(2+) uptake, was also present in membrane isolated from the chromaffin granules enriched fraction and the mitochondria enriched fraction. The results support that both NCX and NCKX are present in bovine chromaffin cells and that the regulation of [Ca(2+)](i) is probably more efficient with the participation of NCKX.     

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.     

Na+/Ca2+ Exchange and Cellular Ca2+ Homeostasis

The Na⁺/Ca²⁺ exchange system is the primary Ca²⁺ efflux mechanism in cardiac myocytes, and plays an important role in controlling the force of cardiac contraction. The exchanger protein contains 11 transmembrane segments plus a large hydrophilic domain between the 5th and 6th transmembrane segments; the transmembrane regions are reponsible for mediating ion translocation while the hydrophilic domain is responsible for regulation of activity. Exchange activity is regulated in vitro by interconversions between an active state and either of two inactive states. High concentrations of cytosolic Na⁺ or the absence of cytosolic Ca²⁺ promote the formation of the inactive states; phosphatidylinositol-(4,5)bisphosphate (or other negatively charged phospholipids) and cytosolic Ca²⁺ counteract the inactivation process. The importance of these mechanisms in regulating exchange activity under normal physiological conditions is uncertain. Exchanger function is also dependent upon cytoskeletal interactions, and the exchanger's location with respect to intracellular Ca²⁺-sequestering organelles. An understanding of the exchanger's function in normal cell physiology will require more detailed information on the proximity of the exchanger and other Ca²⁺-transporting proteins, their interactions with the cytoskeleton, and local concentrations of anionic phospholipids and transported ions.     

Regulation of Na+/Ca2+ exchange activity by cytosolic Ca2+ in transfected Chinese hamster ovary cells

Transfected Chinese hamster ovary cells stably expressing thebovine cardiacNa⁺/Ca²⁺exchanger (CK1.4 cells) were used to determine the range of cytosolic Ca²⁺ concentrations([Ca²⁺]_i)that activateNa⁺/Ca²⁺exchange activity. Ba²⁺ influx wasmeasured in fura 2-loaded, ionomycin-treated cells under conditions inwhich the intracellular Na⁺concentration was clamped with gramicidin at ~20 mM.[Ca²⁺]_iwas varied by preincubating ionomycin-treated cells with either theacetoxymethyl ester of EGTA or medium containing 0-1 mM added CaCl₂. The rate ofBa²⁺ influx increased in asaturable manner with[Ca²⁺]_i,with the half-maximal activation value of 44 nM and a Hill coefficientof 1.6. When identical experiments were carried out with cellsexpressing a Ca²⁺-insensitivemutant of the exchanger, Ba²⁺influx did not vary with[Ca²⁺]_i.The concentration for activation of exchange activity was similar tothat reported for whole cardiac myocytes but approximately an order ofmagnitude lower than that reported for excised, giant patches. Thereason for the difference in Ca²⁺regulation between whole cells and membrane patches is unknown.

     

Multipurpose Na+ ions mediate excitation and cellular homeostasis: Evolution of the concept of Na+ pumps and Na+/Ca2+ exchangers

Ionic signalling is the most ancient form of regulation of cellular functions in response to environmental challenges. Signals, mediated by Na⁺ fluxes and spatio-temporal fluctuations of Na⁺ concentration in cellular organelles and cellular compartments contribute to the most fundamental cellular processes such as membrane excitability and energy production. At the very core of ionic signalling lies the Na⁺-K⁺ ATP-driven pump (or NKA) which creates trans-plasmalemmal ion gradients that sustain ionic fluxes through ion channels and numerous Na⁺-dependent transporters that maintain cellular and tissue homeostasis. Here we present a brief account of the history of research into NKA, Na⁺ -dependent transporters and Na⁺ signalling.     

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.     

Ni2+ transport by the human Na+/Ca2+ exchanger expressed in Sf 9cells

The mechanism ofNi²⁺ block of theNa⁺/Ca²⁺exchanger was examined in Sf 9 cells expressing the human heartNa⁺/Ca²⁺exchanger (NCX1-NACA1). As predicted from the reported actions ofNi²⁺, its application reducedextracellular Na⁺-dependentchanges in intracellular Ca²⁺concentration (measured by fluo 3 fluorescence changes). However, contrary to expectation, the reduced fluorescence was accompanied bymeasured⁶³Ni²⁺entry. The⁶³Ni²⁺entry was observed in Sf 9 cells expressing theNa⁺/Ca²⁺exchanger but not in control cells. The established sequential transport mechanism of theNa⁺/Ca²⁺exchanger could be compatible with these results if one of the two iontranslocation steps is blocked byNi²⁺ and the other permitsNi²⁺ translocation. We concludethat, because Ni²⁺ entry wasinhibited by extracellular Ca²⁺and enhanced by extracellular Na⁺,the Ca²⁺ translocation step movedNi²⁺, whereas theNa⁺ translocation step wasinhibited by Ni²⁺. A model ispresented to discuss these findings.     

Regulation of the Na+/H+ exchanger in fibroblasts overexpressing the Na+/Ca2+ exchanger

To assess the role of Ca²⁺in regulation of theNa⁺/H⁺exchanger (NHE1), we used CCL-39 fibroblasts overexpressing theNa⁺/Ca²⁺exchanger (NCX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca²⁺ rise andlong-lasting cytoplasmic alkalinization (60-80% inhibition) induced by -thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant withthe calmodulin (CaM)-binding domain deleted (amino acids 637-656),suggesting that the effect of NCX1 transfection involves Ca²⁺-CaM binding. Expression ofNCX1 only slightly inhibited platelet-derived growth factor BB-inducedalkalinization and did not affect hyperosmolarity- or phorbol12-myristate 13-acetate-induced alkalinization. Downregulation ofprotein kinase C (PKC) inhibited thrombin-induced alkalinization partially in control cells and abolished it completely inNCX1-transfected cells, suggesting that the thrombin effect is mediatedexclusively via Ca²⁺ and PKC. Onthe other hand, deletion mutant study revealed that PKC-dependentregulation occurs through a small cytoplasmic segment (amino aids566-595). These data suggest that a mechanism involving directCa²⁺-CaM binding lasts for arelatively long period after agonist stimulation, despite apparentshort-lived Ca²⁺ mobilization, andfurther support our previous conclusion that Ca²⁺- and PKC-dependent mechanismsare mediated through distinct segments of the NHE1 cytoplasmic domain.

     

Modulation of cytosolic and nuclear Ca2+ and Na+ transport by taurine in heart cells

Brain membranes contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. We have previously postulated: (a) that this kind of tubulin is a peripheral membrane protein that arises from microtubules that in vivo interact with membranes and (b) that the hydrophobic behaviour is due to the interaction of tubulin with a membrane component. Here we report the in vitro conversion of hydrophilic into hydrophobic tubulin by incubating microtubule associated proteins (MAPs) free taxol-stabilized microtubules with Triton X-100 solubilized membranes. After incubation, the microtubules were sedimented, depolymerized and subjected to partition into Triton X-114. Part of the tubulin was isolated in the detergent phase and contained, as observed in native membranes, a high proportion of the acetylated isotype. Because of the high proportion of acetylated tubulin the in vitro conversion resembles the in vivo interaction. Electrophoretic analysis of the detergent phase shows, besides tubulin, two major protein bands of 29 and 100 kDa molecular mass. The ability of the solubilized membranes to convert hydrophilic into hydrophobic tubulin is greatly diminished if the solubilized membrane preparation is preincubated in the presence of trypsin or heated at 90°C for 5 min, indicating that the membrane component that confers the hydrophobic behaviour to tubulin is of proteinaceous nature.     

Na+/Ca2+ exchange in catfish retina horizontal cells: regulation of intracellular Ca2+ store function

The role of theNa⁺/Ca²⁺exchanger in intracellular Ca²⁺regulation was investigated in freshly dissociated catfish retinalhorizontal cells (HC).Ca²⁺-permeable glutamate receptorsand L-type Ca²⁺ channels as wellas inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitiveintracellular Ca²⁺ stores regulateintracellular Ca²⁺ in these cells.We used the Ca²⁺-sensitive dyefluo 3 to measure changes in intracellularCa²⁺ concentration([Ca²⁺]_i)under conditions in whichNa⁺/Ca²⁺exchange was altered. In addition, the role of theNa⁺/Ca²⁺exchanger in the refilling of the caffeine-sensitiveCa²⁺ store followingcaffeine-stimulated Ca²⁺ releasewas assessed. Brief applications of caffeine (1-10 s) producedrapid and transient changes in[Ca²⁺]_i.Repeated applications of caffeine produced smallerCa²⁺ transients until no furtherCa²⁺ was released. Store refillingoccurred within 1-2 min and required extracellularCa²⁺. Ouabain-induced increases inintracellular Na⁺ concentration([Na⁺]_i)increased both basal free[Ca²⁺]_iand caffeine-stimulated Ca²⁺release. Reduction of external Na⁺concentration([Na⁺]_o)further and reversibly increased[Ca²⁺]_iin ouabain-treated HC. This effect was not abolished by the Ca²⁺ channel blocker nifedipine,suggesting that increases in[Na⁺]_ipromote net extracellular Ca²⁺influx through aNa⁺/Ca²⁺exchanger. Moreover, when[Na⁺]_owas replaced by Li⁺, caffeine didnot stimulate release of Ca²⁺ fromthe caffeine-sensitive store afterCa²⁺ depletion. TheNa⁺/Ca²⁺exchanger inhibitor 2',4'-dimethylbenzamil significantlyreduced the caffeine-evoked Ca²⁺response 1 and 2 min after store depletion.

     

Na+/Ca2+ countertransport in plasma membrane of rat pancreatic acinar cells

Summary The presence of a coupled Na⁺/Ca²⁺ exchange system has been demonstrated in plasma membrane vesicles from rat pancreatic acinar cells. Na⁺/Ca²⁺ exchange was investigated by measuring⁴⁵Ca²⁺ uptake and⁴⁵Ca²⁺ efflux in the presence of sodium gradients and at different electrical potential differences across the membrane (=) in the presence of sodium. Plasma membranes were prepared by a MgCl₂ precipitation method and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the plasma membrane, (Na⁺+K⁺)-ATPase was enriched by 23-fold. Markers for the endoplasmic reticulum, such as RNA and NADPH cytochromec reductase, as well as for mitochondria, the cytochromec oxidase, were reduced by twofold, threefold and 10-fold, respectively. For the Na⁺/Ca²⁺ countertransport system, the Ca²⁺ uptake after 1 min of incubation was half-maximal at 0.62 mol/liter Ca²⁺ and at 20 mmol/liter Na⁺ concentration and maximal at 10 mol/liter Ca²⁺ and 150 mmol/liter Na⁺ concentration, respecitively. When Na⁺ was replaced by Li⁺, maximal Ca²⁺ uptake was 75% as compared to that in the presence of Na⁺. Amiloride (10^–3 mol/liter) at 200 mmol/liter Na⁺ did not inhibit Na⁺/Ca²⁺ countertransport, whereas at low Na⁺ concentration (25 mmol/liter) amiloride exhibited dose-dependent inhibition to be 62% at 10^–2 mol/liter. CFCCP (10^–5 mol/liter) did not influence Na⁺/Ca²⁺ countertransport. Monensin inhibited dose dependently; at a concentration of 5×10^–6 mol/liter inhibition was 80%. A SCN^– or K⁺ diffusion potential (=), being positive at the vesicle inside, stimulated calcium uptake in the presence of sodium suggesting that Na⁺/Ca²⁺ countertransport operates electrogenically, i.e. with a stoichiometry higher than 2 Na⁺ for 1 Ca²⁺. In the absence of Na⁺, did not promote Ca²⁺ uptake. We conclude that in addition to ATP-dependent Ca²⁺ outward transport as characterized previously (E. Bayerdörffer, L. Eckhardt, W. Haase & 1. Schulz, 1985,J. Membrane Biol. 84:45–60) the Na⁺/Ca²⁺ countertransport system, as characterized in this study, represents a second transport system for the extrusion of calcium from the cell. Furthermore, the high affinity for calcium suggests that this system might participate in the regulation of the cytosolic free Ca²⁺ level.