Regulation of intracellular free Ca2+ concentration in the outer segments of bovine retinal rods by Na-Ca-K exchange measured with fluo-3. II. Thermodynamic competence of transmembrane Na+ and K+ gradients and inactivation of Na(+)-dependent Ca2+ extrusion.

Regulation of cytosolic free Ca2+ in the physiologically relevant submicromolar range was measured in isolated intact bovine rod outer segments (ROS) with the intracellular Ca(2+)-indicating dye fluo-3. Changes in free Ca2+ were compared with changes in total Ca2+ measured with 45Ca fluxes and a good qualitative correlation was observed. Ca2+ homeostasis in isolated bovine ROS was exclusively mediated via the Na-Ca-K exchanger. Free cytosolic Ca2+ concentration was lowered by an increase in the inward Na+ gradient, was raised by an increase in external K+, and was raised by depolarization of the plasma membrane. The simplest stoichiometry consistent with these qualitative observations is 4Na:(1Ca + 1K). The individual K:Ca, Na:Ca, and K:Na coupling ratios were deduced from quantitative changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ and K+ gradients. The observed changes in free Ca2+ did not agree with changes in free Ca2+ calculated on the basis of the above fixed stoichiometry which may reflect the flexibility in the Ca:K coupling ratio observed before in flux experiments (Schnetkamp, P. P. M., Szerencsei, R. T., and Basu, D. K. (1991) J. Biol. Chem. 266, 198-206). The most dramatic discrepancy was observed for the Na:Ca coupling ratio: the expected very large changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ gradient were not observed. Rapid Na(+)-induced Ca2+ extrusion was unable to lower cytosolic free Ca2+ below 100 nM, even under nonequilibrium conditions and despite the observation that Ca2+ influx via reverse Na-Ca-K exchange readily occurred at a free external Ca2+ concentration of 20 nM. We conclude that the Na(+)-dependent extrusion mode of the Na-Ca-K exchanger occurs in a brief (20-s) burst of high maximal velocity transport followed by a nearly complete inactivation of transport. The importance of our findings for Ca2+ homeostasis in functioning rod photoreceptors is discussed.     

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.     

Ionic regulation of the cardiac sodium-calcium exchanger

The Na(+)-Ca(2+) exchanger (NCX) links transmembrane movements of Ca(2+) ions to the reciprocal movement of Na(+) ions. It normally functions primarily as a Ca(2+) efflux mechanism in excitable tissues such as the heart, but it can also mediate Ca(2+) influx under certain conditions. Na(+) and Ca(2+) ions exert complex regulatory effects on NCX activity. Ca(2+) binds to two regulatory sites in the exchanger's central hydrophilic domain, and this interaction is normally essential for activation of exchange activity. High cytosolic Na(+) concentrations, however, can induce a constitutive activity that by-passes the need for allosteric Ca(2+) activation. Constitutive NCX activity can also be induced by high levels of phopshotidylinositol-4,5-bisphosphate (PIP?) and by mutations affecting the regulatory calcium binding domains. In addition to promoting constitutive activity, high cytosolic Na(+) concentrations also induce an inactivated state of the exchanger (Na(+)-dependent inactivation) that becomes dominant when cytosolic pH and PIP? levels fall. Na(+)-dependent inactivation may provide a means of protecting cells from Ca(2+) overload due to NCX-mediated Ca(2+) influx during ischemia.     

Inhibition of sodium-calcium exchange by ceramide and sphingosine

Na(+)/Ca(2+) exchange activity in Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger was inhibited by the short chain ceramide analogs N-acetylsphingosine and N-hexanoylsphingosine (5-15 micrometer). The sphingolipids reduced exchange-mediated Ba(2+) influx by 50-70% and also inhibited the Ca(2+) efflux mode of exchange activity. The biologically inactive ceramide analog N-acetylsphinganine had only modest effects on exchange activity. Cells expressing the Delta(241-680) and Delta(680-685) deletion mutants of the Na(+)/Ca(2+) exchanger were not inhibited by ceramide; these mutants show defects in both Na(+)-dependent and Ca(2+)-dependent regulatory behavior. Another mutant, which was defective only in Na(+)-dependent regulation, was as sensitive to ceramide inhibition as the wild-type exchanger. Inhibition of exchange activity by ceramide was time-dependent and was accelerated by depletion of internal Ca(2+) stores. Sphingosine (2.5 micrometer) also inhibited the Ca(2+) influx and efflux modes of exchange activity in cells expressing the wild-type exchanger; sphingosine did not affect Ba(2+) influx in the Delta(241-680) mutant. The effects of the exogenous sphingolipids were reproduced by blocking cellular ceramide utilization pathways, suggesting that exchange activity is inhibited by increased levels of endogenous ceramide and/or sphingosine. We propose that sphingolipids impair Ca(2+)-dependent activation of the exchanger and that in cardiac myocytes, this process serves as a feedback mechanism that links exchange activity to the diastolic concentration of cytosolic Ca(2+).     

Unidirectional Na+, Ca2+, and K+ fluxes through the bovine rod outer segment Na-Ca-K exchanger

The properties of the Na-Ca exchanger in the plasma membrane of rod outer segments isolated from bovine retinas (ROS) were studied. Unidirectional Ca2+, Na+, and K+ fluxes were measured with radioisotopes and atomic absorption spectroscopy. We measured K+ fluxes associated with the Ca-Ca self-exchange mode of the Na-Ca exchanger to corroborate our previous conclusion that the ROS Na-Ca exchanger differs from Na-Ca exchangers in other tissues by its ability to transport K+ (Schnetkamp, P. P. M., Basu, D. K. & Szerencsei, R. T. (1989) Am. J. Physiol. 257, C153-C157). The Na-Ca-K exchanger was the only functional cation transporter in the plasma membrane of bovine ROS with an upper limit of a flux of 10(5) cations/ROS/s or a current of 0.01 pA contributed by other cation channels, pumps, or carriers; cation fluxes via the Na-Ca-K exchanger amounted to 5 x 10(6) cations/ROS/s or a current of 1 pA. Ca2+ efflux via the forward mode of the Na-Ca-K exchanger did not operate with a fixed single stoichiometry. 1) The Na/Ca coupling ratio was increased from three to four when ionophores were added that could provide electrical compensation for the inward Na-Ca exchange current. 2) The K/Ca coupling ratio could vary by at least 2-fold as a function of the external Na+ and K+ concentration. The results are interpreted in terms of a model that can account for the variable Ca/K coupling ratio: we conclude that the Ca2+ site of the exchanger can translocate independent of translocation of the K+ site, whereas translocation of the K+ site requires occupation of the Ca2+ site, but not its translocation. The results are discussed with respect to the physiological role of Na-Ca-K exchange in rod photoreceptors.     

Feedback inhibition of sodium/calcium exchange by mitochondrial calcium accumulation

Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger were subjected to two periods of 5 and 3 min, respectively, during which the extracellular Na(+) concentration ([Na(+)](o)) was reduced to 20 mm; these intervals were separated by a 5-min recovery period at 140 mm Na(+)(o). The cytosolic Ca(2+) concentration ([Ca(2+)](i)) increased during both intervals due to Na(+)-dependent Ca(2+) influx by the exchanger. However, the peak rise in [Ca(2+)](i) during the second interval was only 26% of the first. The reduced rise in [Ca(2+)](i) was due to an inhibition of Na(+)/Ca(2+) exchange activity rather than increased Ca(2+) sequestration since the influx of Ba(2+), which is not sequestered by internal organelles, was also inhibited by a prior interval of Ca(2+) influx. Mitochondria accumulated Ca(2+) during the first interval of reduced [Na(+)](o), as determined by an increase in fluorescence of the Ca(2+)-indicating dye rhod-2, which preferentially labels mitochondria. Agents that blocked mitochondrial Ca(2+) accumulation (uncouplers, nocodazole) eliminated the observed inhibition of exchange activity during the second period of low [Na(+)](o). Conversely, diltiazem, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger, increased mitochondrial Ca(2+) accumulation and also increased the inhibition of exchange activity. We conclude that Na(+)/Ca(2+) exchange activity is regulated by a feedback inhibition process linked to mitochondrial Ca(2+) accumulation.     

Mode-specific inhibition of sodium-calcium exchange during protein phosphatase blockade

The effects of the protein phosphatase inhibitors calyculin A and okadaic acid on Na(+)/Ca(2+) exchange activity were examined in transfected Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger. Incubating the cells for 5-10 min with 100 nM calyculin A reduced exchange-mediated (45)Ca(2+) uptake or Ba(2+) influx by 50-75%. Half-maximal inhibition of (45)Ca(2+) uptake was observed at 15 nM calyculin A. The nonselective protein kinase inhibitors K252a and staurosporine provided partial protection against the effects of calyculin A. Okadaic acid, another protein phosphatase inhibitor, nearly completely blocked exchange-mediated Ba(2+) influx. Chinese hamster ovary cells expressing a mutant exchanger in which 420 out of 520 amino acid residues were deleted from the central hydrophilic domain of the exchanger remained sensitive to the inhibitory effects of calyculin A and okadaic acid. Surprisingly, Na(o)(+)-dependent Ca(2+) efflux appeared to be only modestly inhibited, if at all, by calyculin A or okadaic acid. We conclude that protein hyperphosphorylation during protein phosphatase blockade selectively inhibits the Ca(2+) influx mode of Na(+)/Ca(2+) exchange, probably by an indirect mechanism that does not involve phosphorylation of the exchanger itself.     

Characterization of Na/Ca exchange in plasmalemmal vesicles from zona fasciculata cells of the bovine adrenal gland

The presence of an Na/Ca exchange system in fasciculata cells of the bovine adrenal gland was tested using isolated plasmalemmal vesicles. In the presence of an outwardly Na(+) gradient, Ca(2+) uptake was about 2-fold higher than in K(+) condition. Li(+) did not substitute for Na(+) and 5 mM Ni(2+) inhibited Ca(2+) uptake. Ca(2+) efflux from Ca(2+)-loaded vesicles was Na(+)-stimulated and Ni(2+)-inhibited. The saturable part of Na(+)-dependent Ca(2+) uptake displayed Michaelis-Menten kinetics. The relationship of Na(+)-dependent Ca(2+) uptake versus intravesicular Na(+) concentration was sigmoid (apparent K(0.5) approximately 24 mM; Hill number approximately 3) and Na(+) acted on V(max) without significant effect on K(m). Na(+)-stimulated Ca(2+) uptake was temperature-dependent (apparent Q(10) approximately 2.2). The inhibition properties of several divalent cations (Cd(2+), Sr(2+), Ni(2+), Ba(2+), Mn(2+), Mg(2+)) were tested and were similar to those observed in kidney basolateral membrane. The above results indicate the presence of an Na/Ca exchanger located on plasma membrane of zona fasciculata cells of bovine adrenal gland. This exchanger displays similarities with that of renal basolateral cell membrane.     

pH and monovalent cations regulate cytosolic free Ca(2+) in E. coli

The results here show for the first time that pH and monovalent cations can regulate cytosolic free Ca(2+) in E. coli through Ca(2+) influx and efflux, monitored using aequorin. At pH 7.5 the resting cytosolic free Ca(2+) was 0.2-0.5 microM. In the presence of external Ca(2+) (1 mM) at alkaline pH this rose to 4 microM, being reduced to 0.9 microM at acid pH. Removal of external Ca(2+) caused an immediate decrease in cytosolic free Ca(2+) at 50-100 nM s(-1). Efflux rates were the same at pH 5.5, 7.5 and 9.5. Thus, ChaA, a putative Ca(2+)/H(+)exchanger, appeared not to be a major Ca(2+)-efflux pathway. In the absence of added Na(+), but with 1 mM external Ca(2+), cytosolic free Ca(2+) rose to approximately 10 microM. The addition of Na(+)(half maximum 60 mM) largely blocked this increase and immediately stimulated Ca(2+) efflux. However, this effect was not specific, since K(+) also stimulated efflux. In contrast, an increase in osmotic pressure by addition of sucrose did not significantly stimulate Ca(2+) efflux. The results were consistent with H(+) and monovalent cations competing with Ca(2+) for a non-selective ion influx channel. Ca(2+) entry and efflux in chaA and yrbG knockouts were not significantly different from wild type, confirming that neither ChaA nor YrbG appear to play a major role in regulating cytosolic Ca(2+) in Escherichia coli. The number of Ca(2+) ions calculated to move per cell per second ranged from <1 to 100, depending on conditions. Yet a single eukaryote Ca(2+) channel, conductance 100 pS, should conduct >6 million ions per second. This raises fundamental questions about the nature and regulation of Ca(2+) transport in bacteria, and other small living systems such as mitochondria, requiring a new mathematical approach to describe such ion movements. The results have important significance in the adaptation of E. coli to different ionic environments such as the gut, fresh water and in sea water near sewage effluents.     

Differential contribution of plasmalemmal Na/Ca exchange isoforms to sodium-dependent calcium influx and NMDA excitotoxicity in depolarized neurons

Inhibition of Na(+),K(+)-ATPase during NMDA applications greatly increased NMDA-induced excitotoxicity in primary cultures of forebrain neurons (FNs), but not in cerebellar granule cells (CGCs). Because Na(+),K(+)-ATPase inhibition promotes reversal of plasmalemmal Na(+)/Ca(2+) exchangers, we compared the activities of reversed K(+)-independent (NCX) and K(+)-dependent (NCKX) Na(+)/Ca(2+) exchangers in these cultures. To this end, we measured gramicidin-induced and Na(+)-dependent elevation in cytosolic [Ca(2+)] ([Ca(2+)](c)) that represents Ca(2+) influx via reversed NCX and NCKX; NCX activity was dissected out by removing external K(+). The [Ca(2+)](c) elevations mediated by NCX alone, and NCX plus NCKX combined, were 17 and 6 times more rapid in FNs than in CGCs, respectively. Northern blot analysis showed that FNs preferentially express NCX1 whereas CGCs expressed NCX3. Differences in expression of other isoforms (NCX2, NCKX2, NCKX3 and NCKX4) were less pronounced. We tested whether the NCX or NCKX family of exchangers contributes most to the toxic NMDA-induced Ca(2+) influx in depolarized neurons. We found that in FNs, inhibition of NCX alone was sufficient to significantly limit NMDA excitotoxicity, whereas in CGCs, inhibition of both NCX and NCKX was required. The data suggest that the high activity of NCX isoforms expressed in FNs, possibly NCX1, sensitizes these neurons to NMDA excitotoxicity.     

The effect of alkanols on Ca2+ transport in brain mitochondria.

Ethanol stimulates the Na(+)-dependent Ca2+ efflux in brain mitochondria and inhibits the Na(+)-independent Ca(2+)-efflux. Here, we studied the effects of n-alkanols on the various Ca2+ transport processes in brain mitochondria. Only short-chain alcohols (i.e. methanol, ethanol and propanol) stimulated Na+/Ca2+ exchange. The inhibition of H+/Ca2+ exchange was significant only with ethanol. Short-chain alcohols inhibit while long-chain alcohols activate the cyclosporin-sensitive Ca(2+)-efflux. These data suggest that the mechanism of the alkanols' effects on Na+/Ca2+ exchange, H+/Ca2+ exchange and the cyclosporin sensitive pore are entirely different. Alkanols have no effect on the electrogenic Ca2+ uniporter. Ethanol did not affect the apparent K0.5 for Na+ (7.5 mM) of the Na+/Ca2+ exchange. Similarly, the magnitude of the effect of ethanol did not depend on matrix Ca2+ concentration, suggesting that short-chain alkanols do not stimulate the rate of Na+/Ca2+ exchange by increasing the affinity of the carrier to Ca2+in or Na+out. High concentrations of K+, Mg2+ and Ca2+ enhanced the ethanol effect. It is possible that high surface potential attenuates the effect of ethanol. It is suggested that ethanol stimulation of Na+/Ca2+ exchange depends on the modulation of the surface dielectric constant.     

Bile acids induce a cationic current, depolarizing pancreatic acinar cells and increasing the intracellular Na+ concentration

Biliary disease is a major cause of acute pancreatitis. In this study we investigated the electrophysiological effects of bile acids on pancreatic acinar cells. In perforated patch clamp experiments we found that taurolithocholic acid 3-sulfate depolarized pancreatic acinar cells. At low bile acid concentrations this occurred without rise in the cytosolic calcium concentration. Measurements of the intracellular Na(+) concentration with the fluorescent probe Sodium Green revealed a substantial increase upon application of the bile acid. We found that bile acids induce Ca(2+)-dependent and Ca(2+)-independent components of the Na(+) concentration increase. The Ca(2+)-independent component was resolved in conditions when the cytosolic Ca(2+) level was buffered with a high concentration of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The Ca(2+)-dependent component of intracellular Na(+) increase was clearly seen during stimulation with the calcium-releasing agonist acetylcholine. During acetylcholine-induced Ca(2+) oscillations the recovery of cytosolic Na(+) was much slower than the recovery of Ca(2+), creating a possibility for the summation of Na(+) transients. The bile-induced Ca(2+)-independent current was found to be carried primarily by Na(+) and K(+), with only small Ca(2+) and Cl(-) contributions. Measurable activation of such a cationic current could be produced by a very low concentration of taurolithocholic acid 3-sulfate (10 microm). This bile acid induced a cationic current even when applied in sodium- and bicarbonate-free solution. Other bile acids, taurochenodeoxycholic acid, taurocholic acid, and bile itself also induced cationic currents. Bile-induced depolarization of acinar cells should have a profound effect on acinar fluid secretion and, consequently, on transport of secreted zymogens.     

Na(+)/Ca(2+) exchange facilitates Ca(2+)-dependent activation of endothelial nitric-oxide synthase.

Recent evidence suggests the expression of a Na(+)/Ca(2+) exchanger (NCX) in vascular endothelial cells. To elucidate the functional role of endothelial NCX, we studied Ca(2+) signaling and Ca(2+)-dependent activation of endothelial nitric-oxide synthase (eNOS) at normal, physiological Na(+) gradients and after loading of endothelial cells with Na(+) ions using the ionophore monensin. Monensin-induced Na(+) loading markedly reduced Ca(2+) entry and, thus, steady-state levels of intracellular free Ca(2+) ([Ca(2+)](i)) in thapsigargin-stimulated endothelial cells due to membrane depolarization. Despite this reduction of overall [Ca(2+)](i), Ca(2+)-dependent activation of eNOS was facilitated as indicated by a pronounced leftward shift of the Ca(2+) concentration response curve in monensin-treated cells. This facilitation of Ca(2+)-dependent activation of eNOS was strictly dependent on the presence of Na(+) ions during treatment of the cells with monensin. Na(+)-induced facilitation of eNOS activation was not due to a direct effect of Na(+) ions on the Ca(2+) sensitivity of the enzyme. Moreover, the effect of Na(+) was not related to Na(+) entry-induced membrane depolarization or suppression of Ca(2+) entry, since neither elevation of extracellular K(+) nor the Ca(2+) entry blocker 1-(beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazol e hydrochloride (SK&F 96365) mimicked the effects of Na(+) loading. The effects of monensin were completely blocked by 3', 4'-dichlorobenzamil, a potent and selective inhibitor of NCX, whereas the structural analog amiloride, which barely affects Na(+)/Ca(2+) exchange, was ineffective. Consistent with a pivotal role of Na(+)/Ca(2+) exchange in Ca(2+)-dependent activation of eNOS, an NCX protein was detected in caveolin-rich membrane fractions containing both eNOS and caveolin-1. These results demonstrate for the first time a crucial role of cellular Na(+) gradients in regulation of eNOS activity and suggest that a tight functional interaction between endothelial NCX and eNOS may take place in caveolae.     

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.     

The sodium-calcium exchanger of bovine rod photoreceptors: K(+)-dependence of the purified and reconstituted protein

The K(+)-dependence of the rod photoreceptor sodium-calcium exchanger was investigated using the Ca2(+)-sensitive dye arsenazo III after reconstitution of the purified protein into proteoliposomes. The uptake of Ca2+ by Na(+)-loaded liposomes was found to be greatly enhanced by the presence of external K+ (EC50 approximately 1 mM) in a Michaelis-Menten manner, suggesting that one K+ ion is involved in the transport of one Ca2+ ion. We also found a minimal degree of Ca2+ uptake in the total absence of K+. Other alkali cations, notably Rb+ and, to a lesser extent, Cs+, were also able to stimulate Na(+)-Ca2+ exchange. We also investigated the K(+)-dependence of the photoreceptor Na(+)-Ca2+ exchanger by determining the effects of electrochemical K+ gradients on the Na(+)-activated Ca2+ efflux from proteoliposomes. We found that, under conditions of membrane voltage clamp with FCCP, inwardly directed electrochemical K+ gradients (i.e., K0+ greater than Ki+) inhibited, whereas an outwardly directed electrochemical K+ gradient (i.e., Ki+ greater than K0+) enhanced, Na(+)-dependent Ca2+ efflux, consistent with the notion that K+ is cotransported in the same direction as Ca2+. The investigation of the reconstituted exchanger at physiological (i.e. Ki+ = 110 mM, K0+ = 2.5 mM) potassium concentrations revealed that the Na(+)-dependence of Ca2(+)-efflux was highly cooperative (n = 3.01 from Hill plots), indicating that at least three, but possibly four, Na+ ions are exchanged for one Ca2+ ion. Under these conditions the reconstituted exchanger showed a Km for Na+ of 26.1 mM, and a turnover number of 115 Ca2+.s-1 per exchanger molecule. Our results with the purified and reconstituted sodium-calcium exchanger from rod photoreceptors are therefore consistent with previous reports (Cervetto, L., Lagnado, L., Perry, R.J., Robinson, D.W. and McNaughton, P.A. (1989) Nature 337, 740-743; Schnetkamp, P.P.M., Basu, D.K. and Szerencsei, R.T. (1989) Am. J. Physiol. 257, C153-C157) that the sodium-calcium exchanger of rod photoreceptors cotransports K+ under physiological conditions with a stoichiometry of 4 Na+:1 Ca2+, 1K+.     

Conformational changes of the Ca(2+) regulatory site of the Na(+)-Ca(2+) exchanger detected by FRET

The Na(+)-Ca(2+) exchanger is a plasma membrane protein expressed at high levels in cardiomyocytes. It extrudes 1 Ca(2+) for 3 Na(+) ions entering the cell, regulating intracellular Ca(2+) levels and thereby contractility. Na(+)-Ca(2+) exchanger activity is regulated by intracellular Ca(2+), which binds to a region (amino acids 371-508) within the large cytoplasmic loop between transmembrane segments 5 and 6. Regulatory Ca(2+) activates the exchanger and removes Na(+)-dependent inactivation. The physiological role of intracellular Ca(2+) regulation of the exchanger is not yet established. Yellow (YFP) and cyan (CFP) fluorescent proteins were linked to the NH(2)- and CO(2)H-termini of the exchanger Ca(2+) binding domain (CBD) to generate a construct (YFP-CBD-CFP) capable of responding to changes in intracellular Ca(2+) concentrations by FRET efficiency measurements. The two fluorophores linked to the CBD are sufficiently close to generate FRET. FRET efficiency was reduced with increasing Ca(2+) concentrations. Titrations of Ca(2+) concentration versus FRET efficiency indicate a K(D) for Ca(2+) of approximately 140 nM, which increased to approximately 400 nM in the presence of 1 mM Mg(2+). Expression of YFP-CBD-CFP in myocytes, generated changes in FRET associated with contraction, suggesting that NCX is regulated by Ca(2+) on a beat-to-beat basis during excitation-contraction coupling.     

Discovery of a novel potent Na+/Ca2+ exchanger inhibitor: design, synthesis and structure-activity relationships of 3,4-dihydro-2(1H)-quinazolinone derivatives

Design, synthesis and structure-activity relationships for 3,4-dihydro-2(1H)-quinazolinone derivatives with the inhibitory activities of the Na(+)/Ca(2+) exchanger are discussed. These studies based on lead compound 1a lead to the discovery of a structurally novel and highly potent inhibitor against the Na(+)/Ca(2+) exchanger 4f (SM-15811), which directly inhibited the Na(+)-dependent Ca(2+) influx via the Na(+)/Ca(2+) exchanger in cardiomyocytes with a high potency.     

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.