Maximal Ca2+i stimulation of cardiac Na+/Ca2+ exchange requires simultaneous alkalinization and binding of PtdIns-4,5-P2 to the exchanger

Using bovine heart sarcolemma vesicles we studied the effects of protons and phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) on the affinity of the mammalian Na(+)/Ca(2+) exchanger (NCX1) for intracellular Ca(2+). By following the effects of extravesicular ligands in inside-out vesicles, their interactions with sites of NCX1 facing the intracellular medium were investigated. Two Na(+)-gradient-dependent fluxes were studied: Ca(2+) uptake and Ca(2+) release. PtdIns-4,5-P2 binding to NCX1 was investigated in parallel. Without MgATP (no 'de novo' synthesis of PtdIns-4,5-P2), alkalinization increased the affinity for Ca(2+) and the PtdIns-4,5-P2 bound to NCX1. Vesicles depleted of phosphoinositides were insensitive to alkalinization, but became responsive following addition of exogenous PtdIns-4,5-P2 or PtdIns plus MgATP. Acidification reduced the affinity for Ca(2+)(ev); this was only partially reversed by MgATP, despite the increase in bound PtdIns-4,5-P2 to levels observed with alkalinization. Inhibition of Ca(2+) uptake by increasing extravesicular [Na(+)] indicates that it is related to H(+)(i) and Na(+)(i) synergistic inhibition of the Ca(2+)(i) regulatory site. Therefore, the affinity of the NCX1 Ca(2+)(i) regulatory site for Ca(2+) was maximal when both intracellular alkalinization and an increase in PtdIns-4,5-P2 bound to NCX1 (not just of the total membrane PtdIns-4,5-P2) occurred simultaneously. In addition, protons influenced the distribution, or the exposure, of PtdIns-4,5-P2 molecules in the surroundings and/or on the exchanger protein.     

Optimal metabolic regulation of the mammalian heart Na(+)/Ca(2+) exchanger requires a spacial arrangements with a PtdIns(4)-5kinase

In inside-out bovine heart sarcolemmal vesicles, p-chloromercuribenzenesulfonate (PCMBS) and n-ethylmaleimide (NEM) fully inhibited MgATP up-regulation of the Na⁺/Ca²⁺ exchanger (NCX1) and abolished the MgATP-dependent PtdIns-4,5P2 increase in the NCX1-PtdIns-4,5P2 complex; in addition, these compounds markedly reduced the activity of the PtdIns(4)-5kinase. After PCMBS or NEM treatment, addition of dithiothreitol (DTT) restored a large fraction of the MgATP stimulation of the exchange fluxes and almost fully restored PtdIns(4)-5kinase activity; however, in contrast to PCMBS, the effects of NEM did not seem related to the alkylation of protein SH groups. By itself DTT had no effect on the synthesis of PtdIns-4,5P2 but affected MgATP stimulation of NCX1: moderate inhibition at 1 mM MgATP and 1 μM Ca²⁺ and full inhibition at 0.25 mM MgATP and 0.2 μM Ca²⁺. In addition, DDT prevented coimmunoprecipitation of NCX1 and PtdIns(4)-5kinase. These results indicate that, for a proper MgATP up-regulation of NCX1, the enzyme responsible for PtdIns-4,5P2 synthesis must be (i) functionally competent and (ii) set in the NCX1 microenvironment closely associated to the exchanger. This kind of supramolecular structure is needed to optimize binding of the newly synthesized PtdIns-4,5P2 to its target region in the exchanger protein.     

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.     

Key role of a PtdIns-4,5P2 micro domain in ionic regulation of the mammalian heart Na/Ca exchanger

Phosphatidylinositol biphosphate (PtdIns-4,5P₂) plays a key role in the regulation of the mammalian heart Na⁺/Ca²⁺ exchanger (NCX1) by protecting the intracellular Ca²⁺ regulatory site against H⁺_i and (H⁺_i + Na⁺_i) synergic inhibition. MgATP and MgATP-γ-S up-regulation of NCX1 takes place via the production of this phosphoinositide. In microsomes containing PtdIns-4,5P₂ incubated in the absence of MgATP and at normal [Na⁺]_i, alkalinization increases the affinity for Ca²⁺_i to the values seen in the presence of the nucleotide at normal pH; under this condition, addition of MgATP does not increase the affinity for Ca²⁺_i any further. On the other hand, prevention of Na⁺_i inhibition by alkalinization in the absence of MgATP does not take place when the microsomes are depleted of PtdIns-4,5P₂. Experiments on NCX1–PtdIns-4,5P₂ cross-coimmunoprecipitation show that the relevant PtdIns-4,5P₂ is not the overall membrane component but specifically that tightly attached to NCX1. Consequently, the highest affinity of the Ca²⁺_i regulatory site is seen in the deprotonated and PtdIns-4,5P₂-bound NCX1. Confirming these results, a PtdIns-5-kinase also cross-coimmunoprecipitates with NCX1 without losing its functional competence. These observations indicate, for the first time, the existence of a PtdIns-5-kinase in the NCX1 microdomain.     

Molecular and cellular characterization of a water-channel protein,AQP-h3, specifically expressed in the frog ventral skin

The Ca2+ content of pancreatic juice is closely regulated by yet unknown mechanisms. One aim of the present study was to find whether rat pancreatic ducts have a Na+/Ca2+ exchanger, as found in some Ca2+ transporting epithelia. Another aim was to establish whether the exchanger is regulated by hormones/agonists affecting pancreatic secretion. Whole pancreas, pure pancreatic acini and ducts were obtained from rats and used for RT-PCR and Western blot analysis, immunohistochemistry and intracellular Ca2+ measurements using Fura-2. RT-PCR analysis indicated Na+/Ca2+-exchanger isoforms NCX1.3 and NCX1.7 in acini and pancreas. Western blot with NCX1 antibody identified bands of 70, 120 and 150 kDa in isolated ducts, acini and pancreas. Immunofluorescence experiments showed the Na+/Ca2+ exchanger on the basolateral membrane of acini and small intercalated/intralobular ducts, but in larger intralobular/extralobular ducts the exchanger was predominantly on the luminal membrane. Na+/Ca2+ exchange in ducts was monitored by changes in intracellular Ca2+ activity upon reversal of the Na+ gradient. Secretin (1 nM) and carbachol (1 mM) reduced Na+/Ca2+ exchange by 40% and 51%, respectively. Insulin (1 nM) increased Na+/Ca2+ exchange by 230% within 5 min. The present study shows that pancreatic ducts express the Na+/Ca2+ exchanger. Its distinct localization along the ductal tree and regulation by secretin, carbachol and insulin indicate that ducts might be involved in regulation of Ca2+ concentrations in pancreatic juice.     

Characterization of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases in human neutrophils

Phosphodiesteric cleavage of phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) is required for transmembrane signaling by chemoattractants in human polymorphonuclear leukocytes (PMN). Considering the importance of PtdIns-4,5-P2 as a reservoir for second messenger substances, we have characterized the enzyme system that synthesizes this phospholipid in human PMN, consisting of kinases for phosphatidylinositol (PtdIns) and phosphatidylinositol-4-phosphate (PtdIns-4-P). The preferred phosphate donor for both enzymes was ATP as compared with GTP. The respective Km for ATP for PtdIns kinase and PtdIns-P kinase were 0.049 +/- 0.013 and 0.062 +/- 0.005 mM and for GTP were 0.242 +/- 0.016 and 0.186 +/- 0.037 mM. PtdIns stimulated the activity of PtdIns kinase to a greater extent than PtdIns-4-P kinase. PtdIns-4-P inhibited the activity of detergent-solubilized PtdIns kinase and stimulated particulate PtdIns-4-P kinase, whereas both enzymes exhibited substrate inhibition to PtdIns-4,5-P2. Mg2+ was the preferred cation for both enzymes, but the apparent Km values (4.1 +/- 0.9 mM for PtdIns kinase and 1.0 +/- 0.7 mM for PtdIns-4-P kinase) were significantly different (p less than 0.005). Mn2+ partially substituted for Mg2+, and both enzymes were inhibited by Ca2+. The polyamine spermine stimulated PtdIns-4-P kinase activity to a greater extent and at lower concentrations than PtdIns kinase. PtdIns kinase was easily solubilized in both Triton X-100 and Nonidet P-40, whereas PtdIns-4-P kinase remained in a detergent-nonextractable membrane fraction. These findings demonstrate that the enzyme system in human PMN that forms PtdIns-4,5-P2 is composed of two distinct enzymes with similar characteristics.     

Electrophysiological demonstration of Na+/Ca2+ exchange in bovine articular chondrocytes

Altered fluxes of Ca2+ across the chondrocyte membrane have been proposed as one pathway by which mechanical load can modulate cartilage turnover. In many cells, Na+/Ca2+ exchange (NCX) plays a key role in Ca2+ homeostasis, and recent studies have suggested it is operative in articular chondrocytes. In this study, an electrophysiological characterisation of NCX in articular bovine chondrocytes has been performed, using the whole-cell patch clamp technique, and the effects of inhibitors and the transmembrane electrochemical gradients of Na+ and Ca2+ on NCX function have been assessed. A Ni2+-sensitive current (I(NCX)) which exhibited outward rectification, was elicited by a voltage ramp protocol. The current was also attenuated by the NCX inhibitors benzamil and KBR7943, without significant differences between the effect of these two compounds upon outward and inward currents. The Ni2+-sensitive current was modulated by changes in extracellular and pipette Na+ and Ca2+ in a manner characteristic of I(NCX). Measured values for the reversal potential differed significantly from those predicted for an exchanger stoichiometry of 3Na+ : 1Ca2+, implying that accumulation of intracellular Ca2+ (from influx or release from stores) or more than one transport mode is occurring. These results demonstrate the operation of NCX in articular chondrocytes and suggest that changes in its turnover rate, as might occur in response to mechanical load, may modify cell composition and thereby dictate cartilage turnover.     

Identification and characterization of a sodium/calcium exchanger, NCX-1, in osteoclasts and its role in bone resorption

We provide the first demonstration for a Na+/Ca2+ exchanger, NCX-1, in the osteoclast. We speculate that by using Na+ exchange, NCX-1 couples H+ extrusion with Ca2+ fluxes during bone resorption. Microspectrofluorimetry of fura-2-loaded osteoclasts revealed a rapid and sustained, but reversible, cytosolic Ca2+ elevation upon Na+ withdrawal. This elevation was abolished by the cytosolic introduction (by gentle permeabilization) of a highly specific Na+/Ca2+ exchange inhibitor peptide, XIP, but not its inactive analogue, sXIP. Confocal microscopy revealed intense plasma membrane immunofluorescence with an isoform-specific monoclonal anti-NCX-1 antibody applied to gently permeabilized osteoclasts. Electrophysiological studies using excised outside-in membrane patches showed a low-conductance, Na+-selective, dichlorobenzamil-sensitive, amiloride-insensitive channel that we tentatively assigned as being an NCX. Finally, to examine for physiological relevance, an osteoclast resorption (pit) assay was performed. There was a dramatic reduction of bone resorption following NCX-1 inhibition by dichlorobenzamil and XIP (but not with S-XIP). Together, the results suggest that a functional NCX, likely NCX-1, is involved in the regulation of osteoclast cytosolic Ca2+ and bone resorption.     

Exporting calcium from cells

All eukaryotic cells import Ca2+ through a number of variously gated plasma membrane channels. Once inside cells, Ca2+ transmits information to a large number of (enzyme) targets. Eventually, it must be exported again, to prevent the overloading of the cytosol with Ca2+. Two systems export Ca2+ from cells: a high affinity, low capacity Ca2+-ATPase, and a lower affinity, but much larger capacity, Na+/Ca2+ exchanger. The ATPase (commonly called the Ca2+ pump) is the fine-tuner of cell Ca2+, as it functions well even if the concentration of the ion drops below the microM level. It is a large enzyme, with 10 transmembrane domains and a C-terminal cytosolic tail that contains regulatory sites, including a calmodulin-binding domain. Four distinct gene products plus a large number of splice variants have been described. Some are tissue specific, the isoform 2 being specifically expressed in the sensorial cells of the Corti organ in the inner-ear. Its genetic absence causes deafness in mice. Two different families of the Na+/Ca2+ exchanger exist, one of which, originally described in photoreceptors, transports K+ and Ca2+ in exchange for Na+. The exchanger is particularly active in excitable cells, e.g., heart, where the necessity cyclically arises to rapidly eject large amounts of Ca2+. In addition to heart, the exchanger is particularly important to neurons: the cleavage of the most important neuronal isoform (NCX3) by calpains activated by excitotoxic treatments generates Ca2+ overload and eventually cell death.     

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.     

Vascular Na+/Ca2+ exchanger: implications for the pathogenesis and therapy of salt-dependent hypertension

The Na+/Ca2+ exchanger is an ion transporter that exchanges Na+ and Ca2+ in either Ca2+ efflux or Ca2+ influx mode, depending on membrane potential and transmembrane ion gradients. In arterial smooth muscle cells, the Na+/Ca2+ exchanger is thought to participate in the maintenance of vascular tone by regulating cytosolic Ca2+ concentration. Recent pharmacological and genetic engineering studies have revealed that the Ca2+ influx mode of vascular Na+/Ca2+ exchanger type-1 (NCX1) is involved in the pathogenesis of salt-dependent hypertension. SEA0400, a specific Na+/Ca2+ exchange inhibitor that preferentially blocks the Ca2+ influx mode, lowers arterial blood pressure in salt-dependent hypertensive models, but not in normotensive rats or other types of hypertensive rats. Furthermore, heterozygous mice with reduced expression of NCX1 are resistant to development of salt-dependent hypertension, whereas transgenic mice with vascular smooth muscle-specific overexpression of NCX1 readily develop hypertension after high-salt loading. SEA0400 reverses the cytosolic Ca2+ elevation and vasoconstriction induced by nanomolar ouabain, as well as humoral factors in salt-loaded animals. One possibility is that circulating endogenous cardiotonic steroids may be necessary for NCX1-mediated hypertension. These findings help to explain how arterial smooth muscle cells in blood vessels contribute to salt-elicited blood pressure elevation and suggest that NCX1 inhibitors might be therapeutically useful for salt-dependent hypertension.     

Ca2+ ionophores affect phosphoinositide metabolism differently than thyrotropin-releasing hormone in GH3 pituitary cells

Thyrotropin-releasing hormone (TRH) stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) by a phospholipase C (or phosphodiesterase) and elevates cytoplasmic-free Ca2+ concentration ([Ca2+]i) in GH3 pituitary cells. To explore whether hydrolysis of PtdIns-4,5-P2 is secondary to the elevation of [Ca2+]i, we studied the effects of Ca2+ ionophores, A23187 and ionomycin. In cells prelabeled with [3H]myoinositol, A23187 caused a rapid decrease in the levels of [3H]PtdIns-4,5-P2, [3H]PtdIns-4-P, and [3H]PtdIns to 88 +/- 2%, 88 +/- 4%, and 86 +/- 1% of control, respectively, and increased [3H]inositol bisphosphate to 200 +/- 20% at 0.5 min. There was no increase in [3H] Ins-P3; the lack of a measurable increase in [3H]Ins-P3 was not due to its rapid dephosphorylation. In cells prelabeled with [14C]stearic acid, A23187 increased [14C]diacylglycerol and [14C]phosphatidic acid to 166 +/- 20% and 174 +/- 17% of control, respectively. In cells prelabeled with [3H]arachidonic acid, A23187, but not TRH, increased unesterified [3H]arachidonic acid to 166 +/- 8% of control. Similar effects were observed with ionomycin. Hence, Ca2+ ionophores stimulate phosphodiesteratic hydrolysis of PtdIns-4-P but not of PtdIns-4,5-P2 and elevate the level of unesterified arachidonic acid in GH3 cells. These data demonstrate that Ca2+ ionophores affect phosphoinositide metabolism differently than TRH and suggest that TRH stimulation of PtdIns-4,5-P2 hydrolysis is not secondary to the elevation of [Ca2+]i.     

In depolarized and glucose-deprived neurons,Na+ influx reverses plasmalemmal K+-dependent and K+-independent Na+/Ca2+ exchangers and contributes to NMDA excitotoxicity

Cerebellar granule cells (CGCs) express K+-dependent (NCKX) and K+-independent (NCX) plasmalemmal Na+/Ca2+ exchangers which, under plasma membrane-depolarizing conditions and high cytosolic [Na+], may reverse and mediate potentially toxic Ca2+ influx. To examine this possibility, we inhibited NCX or NCKX with KB-R7943 or K+-free medium, respectively, and studied how gramicidin affects cytosolic [Ca2+] and 45Ca2+ accumulation. Gramicidin forms pores permeable to alkali cations but not Ca2+. Therefore, gramicidin-induced Ca2+ influx is indirect; it results from fluxes of monovalent cations. In the presence of Na+, but not Li+ or Cs+, gramicidin induced Ca2+ influx that was inhibited by simultaneous application of KB-R7943 and K+-free medium. The data indicate that gramicidin-induced Na+ influx reverses NCX and NCKX. To test the role of NCX and/or NCKX in excitotoxicity, we studied how NMDA affects the viability of glucose-deprived and depolarized CGCs. To assure depolarization of the plasma membrane, we inhibited Na+,K+-ATPase with ouabain. Although inhibition of NCX or NCKX reversal failed to significantly limit 45Ca2+ accumulation and excitotoxicity, simultaneously inhibiting NCX and NCKX reversal was neuroprotective and significantly decreased NMDA-induced 45Ca2+ accumulation. Our data suggest that NMDA-induced Na+ influx reverses NCX and NCKX and leads to the death of depolarized and glucose-deprived neurons.     

Optical measurements of Na-Ca-K exchange currents in intact outer segments isolated from bovine retinal rods

The properties of Na-Ca-K exchange current through the plasma membrane of intact rod outer segments (ROS) isolated from bovine retinas were studied with the optical probe neutral red. Small cellular organelles such as bovine ROS do not offer an adequate collecting area to measure Na-Ca-K exchange currents with electrophysiological techniques. This study demonstrates that Na-Ca-K exchange current in bovine ROS can be measured with the dye neutral red and dual-wavelength spectrophotometry. The binding of neutral red is sensitive to transport of cations across the plasma membrane of ROS by the effect of the translocated cations on the surface potential of the intracellular disk membranes (1985. J. Membr. Biol. 88: 249-262). Electrogenic Na+ fluxes through the ROS plasma membrane were measured with a resolution of 10(5) Na+ ions/ROS per s, equivalent to a current of approximately 0.01 pA; maximal electrogenic Na-Ca-K exchange flux in bovine ROS was equivalent to a maximal exchange current of 1-2 pA. Electrogenic Na+ fluxes were identified as Na-Ca-K exchange current based on a comparison between electrogenic Na+ flux and Na(+)-stimulated Ca2+ release with respect to flux rate, Na+ dependence, and ion selectivity. Neutral red monitored the net entry of a single positive charge carried by Na+ for each Ca2+ ion released (i.e., monitored the Na-Ca-K exchange current). Na-Ca-K exchange in the plasma membrane of bovine ROS had the following properties: (a) Inward Na-Ca-K exchange current required internal Ca2+ (half-maximal stimulation at a free Ca2+ concentration of 0.9 microM), whereas outward Na-Ca-K exchange current required both external Ca2+ (half-maximal stimulation at a free Ca2+ concentration of 1.1 microM) and external K+. (b) Inward Na-Ca-K exchange current depended in a sigmoidal manner on the external Na+ concentration, identical to Na(+)-stimulated Ca2+ release measured with Ca(2+)-indicating dyes. (c) The neutral red method was modified to measure Ca(2+)-activated K+ fluxes (half-maximal stimulation at 2.7 microM free Ca2+) via the Na-Ca-K exchanger in support of the notion that the rod Na-Ca exchanger is in effect a Na-Ca-K exchanger. (d) Competitive interactions between Ca2+ and Na+ ions on the exchanger protein are described.     

Unitary cardiac Na+, Ca2+ exchange current magnitudes determined from channel-like noise and charge movements of ion transport.

The cardiac Na+, Ca2+ exchanger (NCX1) is thought to achieve a high turnover rate, but all estimates to date are indirect. Two new strategies demonstrate that maximum unitary exchange currents are about 1 fA (6000 unitary charges per s) and that they fluctuate between on and off levels similar to ion channel currents. First, exchange current noise has been identified in small cardiac patches with properties expected for a gated transport process. Noise power density spectra correlate well with exchanger inactivation kinetics, and the noise has a predicted bell-shaped dependence on the activation states of the exchanger. From the magnitudes of exchange current noise, maximum unitary exchange currents are estimated to be 0.6-1.3 fA. Second, charge movements with rates of approximately 5000 s-1 have been isolated for the transport of both Na+ and Ca2+ in giant membrane patches using nonsaturating ion concentrations. The Na+ transport reactions are disabled or "immobilized" by exchanger inactivation reactions, thus confirming that inactivation generates fully inactive exchanger states.     

Characterization of zebrafish (Danio rerio) NCX4: a novel NCX with distinct electrophysiological properties

Members of the Na+/Ca2+ exchanger (NCX) family are important regulators of cytosolic Ca2+ in myriad tissues and are highly conserved across a wide range of species. Three distinct NCX genes and numerous splice variants exist in mammals, many of which have been characterized in a variety of heterologous expression systems. Recently, however, we discovered a fourth NCX gene (NCX4), which is found exclusively in teleost, amphibian, and reptilian genomes. Zebrafish (Danio rerio) NCX4a encodes for a protein of 939 amino acids and shows a high degree of identity with known NCXs. Although knockdown of NCX4a activity in zebrafish embryos has been shown to alter left-right patterning, it has not been demonstrated that NCX4a functions as a NCX. In this study, we 1) demonstrated, for the first time, that this gene encodes for a novel NCX; 2) characterized the tissue distribution of zebrafish NCX4a; and 3) evaluated its kinetic and transport properties. While ubiquitously expressed, the highest levels of NCX4a expression occurred in the brain and eyes. NCX4a exhibits modest levels of Na+-dependent inactivation and requires much higher levels of regulatory Ca2+ to activate outward exchange currents. NCX4a also exhibited extremely fast recovery from Na+-dependent inactivation of outward currents, faster than any previously characterized wild-type exchanger. While this result suggests that the Na+-dependent inactive state of NCX4a is far less stable than in other NCX family members, this exchanger was still strongly inhibited by 2 microM exchanger inhibitory peptide. We demonstrated that a new putative member of the NCX gene family, NCX4a, encodes for a NCX with unique functional properties. These data will be useful in understanding the role that NCX4a plays in embryological development as well as in the adult, where it is expressed ubiquitously.     

Cells expressing unique Na+/Ca2+ exchange (NCX1) splice variants exhibit different susceptibilities to Ca2+ overload

The Na+/Ca2+ exchanger (NCX) NCX1 exhibits tissue-specific alternative splicing. Such NCX splice variants as NCX1.1 and NCX1.3 are also differentially regulated by Na+ and Ca2+, although the physiological implications of these regulatory characteristics are unclear. On the basis of their distinct regulatory profiles, we hypothesized that cells expressing these different splice variants might exhibit unique responses to conditions promoting Ca2+ overload, such as during exposure to cardiac glycosides or simulated ischemia. NCX1.1 or NCX1.3 was expressed in human embryonic kidney (HEK)-293 cells or rat neonatal ventricular cardiomyocytes (NVC), and expression was confirmed by Western blotting and immunocytochemical analyses. HEK-293 cells lacked NCX1 protein before transfection. With use of adenoviral vectors, neonatal cardiomyocytes were induced to overexpress the NCX1.1 splice variant by nearly twofold, whereas the NCX1.3 isoform was expressed on the endogenous NCX1.1 background. Total expression was comparable for NCX1.1 and NCX1.3. Exposure of NVC to ouabain induced a significant increase in cellular Ca2+, an effect that was exaggerated in cells overexpressing NCX1.1, but not NCX1.3. The increase in intracellular Ca2+ was inhibited by 5 microM KB-R7943. Cardiomyocytes overexpressing NCX1.1 also exhibited a greater accumulation of intracellular Ca2+ in response to simulated ischemia than did cells expressing NCX1.3. Similar responses were observed in HEK-293 cells where NCX1.1 was expressed. We conclude that expression of the NCX1.3 splice variant protects against severe Ca2+ overload, whereas NCX1.1 promotes Ca2+ overload in response to cardiac glycosides and ischemic challenges. These results highlight the importance of ionic regulation in controlling NCX1 activity under conditions that promote Ca2+ overload.     

Inhibition of Na+/Ca2+ exchange by KB-R7943: transport mode selectivity and antiarrhythmic consequences

The Na+/Ca2+ exchanger plays a prominent role in regulating intracellular Ca2+ levels in cardiac myocytes and can serve as both a Ca2+ influx and efflux pathway. A novel inhibitor, KB-R7943, has been reported to selectively inhibit the reverse mode (i.e., Ca2+ entry) of Na+/Ca2+ exchange transport, although many aspects of its inhibitory properties remain controversial. We evaluated the inhibitory effects of KB-R7943 on Na+/Ca2+ exchange currents using the giant excised patch-clamp technique. Membrane patches were obtained from Xenopus laevis oocytes expressing the cloned cardiac Na+/Ca2+ exchanger NCX1.1, and outward, inward, and combined inward-outward currents were studied. KB-R7943 preferentially inhibited outward (i.e., reverse) Na+/Ca2+ exchange currents. The inhibitory mechanism consists of direct effects on the transport machinery of the exchanger, with additional influences on ionic regulatory properties. Competitive interactions between KB-R7943 and the transported ions were not observed. The antiarrhythmic effects of KB-R7943 were then evaluated in an ischemia-reperfusion model of cardiac injury in Langendorff-perfused whole rabbit hearts using electrocardiography and measurements of left ventricular pressure. When 3 microM KB-R7943 was applied for 10 min before a 30-min global ischemic period, ventricular arrhythmias (tachycardia and fibrillation) associated with both ischemia and reperfusion were almost completely suppressed. The observed electrophysiological profile of KB-R7943 and its protective effects on ischemia-reperfusion-induced ventricular arrhythmias support the notion of a prominent role of Ca2+ entry via reverse Na+/Ca2+ exchange in this process.     

The role of the Na+/H+ exchange system in cardiac cells in relation to the control of the internal Na+ concentration. A molecular basis for the antagonistic effect of ouabain and amiloride on the heart

Cardiac cells in culture (from rat and chick heart) have a membrane Na+/H+ exchange system that is inhibited by amiloride (K0.5 = 5 microM) and by its more potent N-5-disubstituted derivatives dimethylamiloride (K0.5 = 300 nM) and ethylisopropylamiloride (K0.5 = 30 nM). The properties of the cardiac Na+/H+ exchange system are similar to those found for the Na+/H+ exchanger in other cellular types. The Na+/H+ exchange system is a major pathway for Na+ uptake by cardiac cells. Ouabain which inhibits the (Na+,K+)-ATPase, a major pathway for Na+ efflux, is known to provoke Na+ accumulation and to stimulate 45Ca2+ entry via the Na+/Ca2+ exchange mechanism, thereby producing an inotropic effect. N-5-Disubstituted amiloride derivatives, by blocking Na+ entry into cardiac cells, antagonize both ouabain-induced intracellular Na+ accumulation and the ouabain-induced acceleration of 45Ca2+ uptake.