Occurrence of Na(+)-Ca2+ exchange in the ciliate Euplotes crassus and its role in Ca2+ homeostasis.

Ciliates possess diverse Ca2+ homeostasis systems, but little is known about the occurrence of a Na(+)-Ca2+ exchanger. We studied Na(+)-Ca2+ exchange in the ciliate Euplotes crassus by digital imaging. Cells were loaded with fura-2/AM or SBF1/AM for fluorescence measurements of cytosolic Ca2+ and Na+ respectively. Ouabain pre-treatment and Na+o substitution in fura-2/AM-loaded cells elicited a bepridil-sensitive [Ca2+]i rise followed by partial recovery, indicating the occurrence of Na(+)-Ca2+ exchanger working in reverse mode. In experiments on prolonged effects, ouabain, Na+o substitution, and bepridil all caused Ca2+o-dependent [Ca2+]i increase, showing a role for Na(+)-Ca2+ exchange in Ca2+ homeostasis. In addition, by comparing the effect of orthovanadate (affecting not only Ca2+ ATPase, but also Na(+)-K+ ATPase and, hence, Na(+)-Ca2+ exchange) to that of bepridil on [Ca2+]i, it was shown that Na(+)-Ca2+ exchange contributes to Ca2+ homeostasis. In electrophysiological experiments, no membrane potential variation was observed after bepridil treatment suggesting compensatory mechanisms for ion effects on cell membrane voltage, which also agrees with membrane potential stability after ouabain treatment. In conclusion, data indicate the presence of a Na(+)-Ca2+ exchanger in the plasma membrane of E. crassus, which is essential for Ca2+ homeostasis, but could also promote Ca2+ entry under specific conditions.     

Roles of Na(+)-Ca2+ exchange and of mitochondria in the regulation of presynaptic Ca2+ and spontaneous glutamate release

The release of neurotransmitter from presynaptic terminals depends on an increase in the intracellular Ca2+ concentration ([Ca2+]i). In addition to the opening of presynaptic Ca2+ channels during excitation, other Ca2+ transport systems may be involved in changes in [Ca2+]i. We have studied the regulation of [Ca2+]i in nerve terminals of hippocampal cells in culture by the Na(+)-Ca2+ exchanger and by mitochondria. In addition, we have measured changes in the frequency of spontaneous excitatory postsynaptic currents (sEPSC) before and after the inhibition of the exchanger and of mitochondrial metabolism. We found rather heterogeneous [Ca2+]i responses of individual presynaptic terminals after inhibition of Na(+)-Ca2+ exchange. The increase in [Ca2+]i became more uniform and much larger after additional treatment of the cells with mitochondrial inhibitors. Correspondingly, sEPSC frequencies changed very little when only Na(+)-Ca2+ exchange was inhibited, but increased dramatically after additional inhibition of mitochondria. Our results provide evidence for prominent roles of Na(+)-Ca2+ exchange and mitochondria in presynaptic Ca2+ regulation and spontaneous glutamate release.     

Expression of the cardiac Na(+)-Ca2+ exchanger in insect cells using a baculovirus vector.

We constructed a recombinant baculovirus containing cardiac Na(+)-Ca2+ exchanger cDNA under control of the polyhedrin promoter. When either Sf9 or Sf21 insect cells are infected with the recombinant baculovirus, both Na(+)-Ca2+ exchanger protein and Na(+)-Ca2+ exchange activity are expressed at high level. The exchanger protein can be detected either by immunoblot or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole cell lysate. At maximal expression, the exchanger protein comprises about 3-5% of total cell protein. The Na(+)-Ca2+ exchanger can be purified by alkaline extraction of infected cells followed by elution from a Bio-Rad Prep Cell. The expressed exchanger, in contrast to the native sarcolemmal exchanger, is not glycosylated. Sf9 cells expressing the exchanger are intensely stained by anti-exchanger antibodies as observed by immunofluorescence. The expressed exchanger is predominantly in the cell plasma membrane since it is susceptible to extracellular trypsin. In 45Ca2+ flux experiments, the expressed Na(+)-Ca2+ exchange activity is about 4-fold higher than that in cultured neonatal rat heart cells. The expressed exchanger was also analyzed electrophysiologically using whole cell patch clamp techniques. The characteristics of inward exchange currents in infected Sf21 cells are very similar to those of ventricular myocytes, although of a larger magnitude.     

Voltage-dependent modulation of ion binding and translocation in the cardiac Na(+)-Ca2+ exchange system

The transport of Na+ and Ca2+ ions in the cardiac Na(+)-Ca2+ exchanger can be described as separate events (Khananshvili, D. (1990) Biochemistry 29, 2437-2442). Thus, the Na(+)-Na+ and Ca(2+)-Ca2+ exchange reactions reflect reversible partial reactions of the transport cycle. The effect of diffusion potentials (K(+)-valinomycin) on different modes of the Na(+)-Ca2+ exchanger (Na(+)-Ca2+, Ca(2+)-Ca2+, and Na(+)-Na+ exchanges) were tested in reconstituted proteoliposomes, obtained from the Triton X-100 extracts of the cardiac sarcolemmal membranes. The initial rates of the Nai-dependent 45Ca-uptake (t = 1 s) were measured in EGTA-entrapped proteoliposomes at different voltages. At the fixed values of voltage [45 Ca]o was varied from 4 to 122 microM, and [Na]i was saturating (150 mM). Upon varying delta psi from -94 to +91 mV, the Vmax values were increased from 9.5 +/- 0.5 to 26.5 +/- 1.5 nmol.mg-1.s-1 and the Km from 17.8 +/- 2.5 to 39.1 +/- 5.2 microM, while the Vmax/Km values ranged from only 0.53 +/- 0.08 to 0.73 +/- 0.17 nmol.mg-1.s-1.microM-1. The equilibrium Ca(2+)-Ca2+ exchange was voltage sensitive at very low [Ca]o = [Ca]i = 2 microM, while at saturating [Ca]o = [Ca]i = 200 microM the Ca(2+)-Ca2+ exchange became voltage-insensitive. The rates of the equilibrium Na(+)-Na+ exchange appears to be voltage insensitive at saturating [Na]o = [Na]i = 160 mM. Under the saturating ionic conditions, the rates of the Na(+)-Na+ exchange were at least 2-3-fold slower than the Ca(2+)-Ca2+ exchange. The following conclusions can be drawn. (a) The near constancy of the Vmax/Km for Na(+)-Ca2+ exchange at different voltages is compatible with the ping-pong model proposed previously. (b) The effects of voltage on Vmax of Na(+)-Ca2+ exchange are consistent with the existence of a single charge carrying transport step. (c) It is not yet possible to clearly assign this step to the Na+ or Ca2+ transport half of the cycle although it is more likely that 3Na(+)-transport is a charge carrying step. Thus, the unloaded ion-binding domain contains either -2 or -3 charges (presumably carboxyl groups). (d) The binding of Na+ and Ca2+ appears to be weakly voltage-sensitive. The Ca(2+)-binding site may form a small ion-well (less than 2-3 A).     

The effect of alteration of extracellular Na+ or Ca2+ and inhibition of Ca2+ entry, Na(+)-H+ exchange, and Na(+)-Ca2+ exchange by diltiazem, amiloride, and dichlorobenzamil on the response of cardiac cell aggregates to epidermal growth factor

The purpose of this study was to examine the effect of epidermal growth factor (EGF) on cardiac function and to explore ionic mechanisms as potential explanations for EGF-induced changes in cardiac contractile frequency. Cardiac cell aggregates were prepared from 7-day-old chick embryo hearts and were maintained in culture. EGF over a concentration range of 5 to 20 ng/ml produced a dose-dependent increase in cardiac contractile frequency. Inhibition of Na(+)-H+ exchange by amiloride antagonized the action of EGF. Inhibition of Na(+)-Ca2+ exchange by dichlorobenzamil prevented the effects of EGF. Inhibition of voltage-dependent calcium influx by diltiazem also antagonized the effect of EGF. The positive chronotropic action of EGF was significantly enhanced when the concentration of Na+ or Ca2+ was increased in the medium. These data indicate that EGF has a definite dose-dependent effect on the cardiac contractile frequency that is operative through ionic transport mechanisms that include increased calcium entry through voltage-dependent calcium channels and stimulation of Na(+)-H+ and Na(+)-Ca2+ exchange. The similarity in the effects of inhibition of these three ionic mechanisms suggests they are interrelated so that interference at any step in the process inhibits the action of EGF on cardiac myocytes.     

Rapid charge translocation by the cardiac Na(+)-Ca2+ exchanger after a Ca2+ concentration jump.

The kinetics of Na(+)-Ca2+ exchange current after a cytoplasmic Ca2+ concentration jump (achieved by photolysis of DM-nitrophen) was measured in excised giant membrane patches from guinea pig or rat heart. Increasing the cytoplasmic Ca2+ concentration from 0.5 microM in the presence of 100 mM extracellular Na+ elicits an inward current that rises with a time constant tau 1 < 50 microseconds and decays to a plateau with a time constant tau 2 = 0.65 +/- 0.18 ms (n = 101) at 21 degrees C. These current signals are suppressed by Ni2+ and dichlorobenzamil. No stationary current, but a transient inward current that rises with tau 1 < 50 microseconds and decays with tau 2 = 0.28 +/- 0.06 ms (n = 53, T = 21 degrees C) is observed if the Ca2+ concentration jump is performed under conditions that promote Ca(2+)-Ca2+ exchange (i.e., no extracellular Na+, 5 mM extracellular Ca2+). The transient and stationary inward current is not observed in the absence of extracellular Ca2+ and Na+. The application of alpha-chymotrypsin reveals the influence of the cytoplasmic regulatory Ca2+ binding site on Ca(2+)-Ca2+ and forward Na(+)-Ca2+ exchange and shows that this site regulates both the transient and stationary current. The temperature dependence of the stationary current exhibits an activation energy of 70 kj/mol for temperatures between 21 degrees C and 38 degrees C, and 138 kj/mol between 10 degrees C and 21 degrees C. For the decay time constant an activation energy of 70 kj/mol is observed in the Na(+)-Ca2+ and the Ca(2+)-Ca2+ exchange mode between 13 degrees C and 35 degrees C. The data indicate that partial reactions of the Na(+)-Ca2+ exchanger associated with Ca2+ binding and translocation are very fast at 35 degrees C, with relaxation time constants of about 6700 s-1 in the forward Na(+)-Ca2+ exchange and about 12,500 s-1 in the Ca(2+)-Ca2+ exchange mode and that net negative charge is moved during Ca2+ translocation. According to model calculations, the turnover number, however, has to be at least 2-4 times smaller than the decay rate of the transient current, and Na+ inward translocation appears to be slower than Ca2+ outward movement.     

Allosteric activation of Na+-Ca2+ exchange by L-type Ca2+ current augments the trigger flux for SR Ca2+ release in ventricular myocytes

The possible contribution of Na(+)-Ca(2+) exchange to the triggering of Ca(2+) release from the sarcoplasmic reticulum in ventricular cells remains unresolved. To gain insight into this issue, we measured the "trigger flux" of Ca(2+) crossing the cell membrane in rabbit ventricular myocytes with Ca(2+) release disabled pharmacologically. Under conditions that promote Ca(2+) entry via Na(+)-Ca(2+) exchange, internal [Na(+)] (10 mM), and positive membrane potential, the Ca(2+) trigger flux (measured using a fluorescent Ca(2+) indicator) was much greater than the Ca(2+) flux through the L-type Ca(2+) channel, indicating a significant contribution from Na(+)-Ca(2+) exchange to the trigger flux. The difference between total trigger flux and flux through L-type Ca(2+) channels was assessed by whole-cell patch-clamp recordings of Ca(2+) current and complementary experiments in which internal [Na(+)] was reduced. However, Ca(2+) entry via Na(+)-Ca(2+) exchange measured in the absence of L-type Ca(2+) current was considerably smaller than the amount inferred from the trigger flux measurements. From these results, we surmise that openings of L-type Ca(2+) channels increase [Ca(2+)] near Na(+)-Ca(2+) exchanger molecules and activate this protein. These results help to resolve seemingly contradictory results obtained previously and have implications for our understanding of the triggering of Ca(2+) release in heart cells under various conditions.     

Na+-Ca2+ exchange in squid optic nerve membrane vesicles is activated by internal calcium

The role of intracellular Ca2+ as essential activator of the Na+-Ca2+ exchange carrier was explored in membrane vesicles containing 67% right-side-out and 10% inside-out vesicles, isolated from squid optic nerves. Vesicles containing 100 microM free calcium exhibited a 2-fold increase in the initial rate of Na+i-dependent Ca2+ uptake as compared with vesicles where intravesicular calcium was chelated by 2 mM EGTA or 10 mM HEDTA. The activatory effect exerted by intravesicular Ca2+ on the reverse mode of Na+-Ca2+ exchange (i.e. Na+i-Ca2+o exchange) is saturated at about 100 microM Ca2+i and displays an apparent K 1/2 of 12 microM. Intravesicular Ca2+ produced activation of Na+i-Ca2+i exchange activity rather than an increase in Ca2+ uptake due to Ca2+-Ca2+ exchange. The presence of Ca2+i was essential for the Na+i-dependent Na+ influx, a partial reaction of the Na+-Ca2+ exchanger. In fact, the Na+ influx levels in vesicles loaded with 2 mM EGTA were close to those expected from diffusional leak while in vesicles containing Ca2+i an additional Na+-Na+ exchange was measured. The results suggest that in nerve membrane vesicles Ca2+ at the inner aspect of the membrane acts as an activator of the Na+-Ca2+ exchange system.     

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.     

Effect of amiloride on diaphragmatic contractility: evidence of a role for Na(+)-Ca2+ exchange

Extracellular Ca2+ has been shown to be important for the normal function of the diaphragm. In this study we have examined the potential importance of Na(+)-Ca2+ exchange as a mechanism for Ca2+ influx during the contractile process by studying the effect of inhibition or stimulation of Na(+)-Ca2+ exchange. Blockade of Na(+)-Ca2+ exchange with amiloride attenuated the twitch response, altered the force-frequency response curve, and enhanced the development of fatigue. The effect of amiloride could be partially reversed by increasing the extracellular Ca2+ concentration. The ability of amiloride to decrease force was associated with decreased Ca2+ uptake by the diaphragm. Enhancing intracellular Na(+)-extracellular Ca2+ exchange by inhibiting the Na(+)-K+ pump significantly decreased the rate of the development of muscle fatigue (89%). The maximal inhibition of diaphragmatic force produced by the amiloride analogue benzamil, which possesses 10-fold greater selectivity for Na(+)-Ca2+ exchange, was not significantly different from that produced by amiloride (76.2 +/- 1.1%), with a concentration that decreased maximum force by 50% equal to 46 microM compared with 460 microM for amiloride. Both agents slowed the maximal rate of relaxation up to 90%. Benzamil elevated resting tension during continuous stimulation of the diaphragm at 0.15 Hz. The results suggest that Na(+)-Ca2+ exchange may have a role in the normal function of the diaphragm.     

The effect of opiate agonists and antagonists on Na(+)-Ca2+ exchange in cardiac sarcolemma vesicles.

Opiate agonists and antagonists inhibit Na(+)-Ca2+ exchange in the isolated cardiac sarcolemma vesicles. Non-opioid stereoisomers (dextrorphan, Mr 1542MS, WIN 44,441-3) display effects similar to their opioid isomers (levorphanol, Mr 1543MS, WIN 44,441-2) suggesting that inhibition is not mediated by opiate receptors. Naloxone (permeable) and methylnaloxone (impermeable) inhibit the Na(+)-Ca2+ exchange similarly, suggesting an extravesicular location of inhibitory site. The inhibitory potency of naloxone is pH-independent in the range of 7.4-9.1, suggesting that the charge-carrying properties of drug-protein interactions are not altered under the tested conditions. Opiates display similar dose-response relationships for Na(+)-Ca2+ exchange and its partial reaction, the Ca(2+)-Ca2+ exchange. The opiate-induced inhibition is complete and noncompetitive in regard to extravesicular calcium. These data suggest that opiates do not bind to the Ca(2+)-binding domain (A-site), but they may interest either with the Na(+)-binding site (B-site) or with a putative opiate-binding site, presumably located outside of the ion-binding vicinity. Further studies on structure-activity relationship might lead to the discovery of potent and more specific inhibitors of cardiac Na(+)-Ca2+ exchanger. A possible relevance of these findings to some non-opioid pharmacological effects of naloxone on the cardiac muscle is suggested.     

Effects of Na+/Ca2+ exchange induced by SR Ca2+ release on action potentials and afterdepolarizations in guinea pig ventricular myocytes

In cardiac cells, evoked Ca2+ releases or spontaneous Ca2+ waves activate the inward Na+/Ca2+ exchange current (INaCa), which may modulate membrane excitability and arrhythmogenesis. In this study, we examined changes in membrane potential due to INaCa elicited by sarcoplasmic reticulum (SR) Ca2+ release in guinea pig ventricular myocytes using whole cell current clamp, fluorescence, and confocal microscopy. Inhibition of INaCa by Na+-free, Li+-containing Tyrode solution reversibly abbreviated the action potential duration at 90% repolarization (APD90) by 50% and caused SR Ca2+ overload. APD90 was similarly abbreviated in myocytes exposed to the Na+/Ca2+ exchange inhibitor KB-R7943 (5 microM) or after inhibition of SR Ca2+ release with ryanodine (20 microM). In the absence of extracellular Na+, spontaneous SR Ca2+ releases caused minimal changes in resting membrane potential. After the myocytes were returned to Na+-containing solution, the potentiated intracellular Ca2+ concentration ([Ca2+]i) transients dramatically prolonged APD90 and [Ca2+]i oscillations caused delayed and early afterdepolarizations (DADs and EADs). Laser-flash photolysis of caged Ca2+ mimicked the effects of spontaneous [Ca2+]i oscillations, confirming that APD prolongation, DADs, and EADs could be ascribed to intracellular Ca2+ release. These results suggest that Na+/Ca2+ exchange is a major physiological determinant of APD and that INaCa activation by spontaneous SR Ca2+ release/oscillations, depending on the timing, can account for both DADs and EADs during SR Ca2+ overload.     

Na(+)-Ca2+ exchange in locust striated muscles

High Na+ + Ca2+ exchange rates comparable with those reported for crayfish striated muscle, rat heart and rat brain, were observed in locust striated muscle homogenates and membrane preparations. The Na(+)-Ca2+ exchange followed the 1st order kinetics with a Km value of 18 mumol.l-1 for Ca, the pH optimum was at 8, the temperature optimum at 30 degrees C, and the exchange was inhibited in the presence of sodium in the incubation medium, with a KiNa of approx. 25 mmol.l-1. The present results suggest a high Na(+)-Ca2+ exchange in locust striated muscles which operate on the calcium electrogenesis principle.     

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.     

Identification of a peptide inhibitor of the cardiac sarcolemmal Na(+)-Ca2+ exchanger

The deduced amino acid sequence of the cardiac sarcolemmal Na(+)-Ca2+ exchanger has a region which could represent a calmodulin binding site. As calmodulin binding regions of proteins often have an autoinhibitory role, a synthetic peptide with this sequence was tested for functional effects on Na(+)-Ca2+ exchange activity. The peptide inhibits the Na(+)-dependent Ca2+ uptake (KI approximately 1.5 microM) and the Nao(+)-dependent Ca2+ efflux of sarcolemmal vesicles in a noncompetitive manner with respect to both Na+ and Ca2+. The peptide is also a potent inhibitor (KI approximately 0.1 microM) of the Na(+)-Ca2+ exchange current of excised sarcolemmal patches. The binding site for the peptide on the exchanger is on the cytoplasmic surface of the membrane. The exchanger inhibitory peptide binds calmodulin with a moderately high affinity. From the characteristics of the inhibition of the exchange of sarcolemmal vesicles, we deduce that only inside-out sarcolemmal vesicles participate in the usual Na(+)-Ca2+ exchange assay. This contrasts with the common assumption that both inside-out and right-side-out vesicles exhibit exchange activity.     

Altered contractility and [Ca2+]i homeostasis in phospholemman-deficient murine myocytes: role of Na+/Ca2+ exchange

Phospholemman (PLM) regulates contractility and Ca(2+) homeostasis in cardiac myocytes. We characterized excitation-contraction coupling in myocytes isolated from PLM-deficient mice backbred to a pure congenic C57BL/6 background. Cell length, cell width, and whole cell capacitance were not different between wild-type and PLM-null myocytes. Compared with wild-type myocytes, Western blots indicated total absence of PLM but no changes in Na(+)/Ca(2+) exchanger, sarcoplasmic reticulum (SR) Ca(2+)-ATPase, alpha(1)-subunit of Na(+)-K(+)-ATPase, and calsequestrin levels in PLM-null myocytes. At 5 mM extracellular Ca(2+) concentration ([Ca(2+)](o)), contraction and cytosolic [Ca(2+)] ([Ca(2+)](i)) transient amplitudes and SR Ca(2+) contents in PLM-null myocytes were significantly (P < 0.0004) higher than wild-type myocytes, whereas the converse was true at 0.6 mM [Ca(2+)](o). This pattern of contractile and [Ca(2+)](i) transient abnormalities in PLM-null myocytes mimics that observed in adult rat myocytes overexpressing the cardiac Na(+)/Ca(2+) exchanger. Indeed, we have previously reported that Na(+)/Ca(2+) exchange currents were higher in PLM-null myocytes. Activation of protein kinase A resulted in increased inotropy such that there were no longer any contractility differences between the stimulated wild-type and PLM-null myocytes. Protein kinase C stimulation resulted in decreased contractility in both wild-type and PLM-null myocytes. Resting membrane potential and action potential amplitudes were similar, but action potential duration was much prolonged (P < 0.04) in PLM-null myocytes. Whole cell Ca(2+) current densities were similar between wild-type and PLM-null myocytes, as were the fast- and slow-inactivation time constants. We conclude that a major function of PLM is regulation of cardiac contractility and Ca(2+) fluxes, likely by modulating Na(+)/Ca(2+) exchange activity.     

Na+-Ca2+ exchange and Ca2+ efflux in transfected Chinese hamster ovary cells

The objective of this study was to assess the contribution of Na+-Ca2+ exchange activity to Ca2+ efflux at various cytosolic Ca2+ concentrations ([Ca2+]i) in transfected Chinese hamster cells expressing the bovine cardiac Na+-Ca2+ exchanger. Ionomycin was added to fura-2 loaded cells and the resulting [Ca2+]i transient was monitored in Ca2+-free media with or without extracellular Na+. The presence of Na+ reduced both the amplitude and duration of the [Ca2+]i transient. Na+ had similar effects when the peak of the [Ca2+]i transient was buffered to 100 nM by cytosolic EGTA, or when Ca2+ was slowly released from internal stores with thapsigargin. Ca2+ efflux following ionomycin addition was directly measured with extracellular fura-2 and followed a biphasic time course (t(1/2) approximately = 10 s and 90s). The proportion of total efflux owing to the rapid phase was increased by Na+ and reduced by EGTA-loading. Na+ accelerated the initial rate of Ca2+ efflux by 65% in unloaded cells but only by 16% in EGTA-loaded cells. In both cases, the stimulation by Na+ was less than expected, given the pronounced effects of Na+ on the [Ca2+]i transient. We conclude that the exchanger contributes importantly to Ca2+ efflux activity at all [Ca2+]i values above 40 nM. We also suggest that Ca2+ efflux pathways may involve non-cytosolic or local routes of Ca2+ traffic.     

Regulation of in vivo cardiac contractility by phospholemman: role of Na+/Ca2+ exchange

Phospholemman (PLM), when phosphorylated at serine 68, relieves its inhibition on Na(+)-K(+)-ATPase but inhibits Na(+)/Ca(2+) exchanger 1 (NCX1) in cardiac myocytes. Under stress when catecholamine levels are high, enhanced Na(+)-K(+)-ATPase activity by phosphorylated PLM attenuates intracellular Na(+) concentration ([Na(+)](i)) overload. To evaluate the effects of PLM on NCX1 on in vivo cardiac contractility, we injected recombinant adeno-associated virus (serotype 9) expressing either the phosphomimetic PLM S68E mutant or green fluorescent protein (GFP) directly into left ventricles (LVs) of PLM-knockout (KO) mice. Five weeks after virus injection, ～40% of isolated LV myocytes exhibited GFP fluorescence. Expression of S68E mutant was confirmed with PLM antibody. There were no differences in protein levels of α(1)- and α(2)-subunits of Na(+)-K(+)-ATPase, NCX1, and sarco(endo)plasmic reticulum Ca(2+)-ATPase between KO-GFP and KO-S68E LV homogenates. Compared with KO-GFP myocytes, Na(+)/Ca(2+) exchange current was suppressed, but resting [Na(+)](i), Na(+)-K(+)-ATPase current, and action potential amplitudes were similar in KO-S68E myocytes. Resting membrane potential was slightly lower and action potential duration at 90% repolarization (APD(90)) was shortened in KO-S68E myocytes. Isoproterenol (Iso; 1 μM) increased APD(90) in both groups of myocytes. After Iso, [Na(+)](i) increased monotonically in paced (2 Hz) KO-GFP but reached a plateau in KO-S68E myocytes. Both systolic and diastolic [Ca(2+)](i) were higher in Iso-stimulated KO-S68E myocytes paced at 2 Hz. Echocardiography demonstrated similar resting heart rate, ejection fraction, and LV mass between KO-GFP and KO-S68E mice. In vivo closed-chest catheterization demonstrated enhanced contractility in KO-S68E compared with KO-GFP hearts stimulated with Iso. We conclude that under catecholamine stress when [Na(+)](i) is high, PLM minimizes [Na(+)](i) overload by relieving its inhibition of Na(+)-K(+)-ATPase and preserves inotropy by simultaneously inhibiting Na(+)/Ca(2+) exchanger.     

Dependence of Na+-Ca2+ exchange and Ca2+-Ca2+ exchange on monovalent cations

Two mechanisms of passive Ca2+ transport, Na+-Ca2+ exchange and Ca2+-Ca2+ exchange, were studied using highly-purified dog heart sarcolemmal vesicles. About 80% of the Ca2+ accumulated by Na+-Ca2+ exchange or Ca2+-Ca2+ exchange could be released as free Ca2+, while up to 20% was probably bound. Na+-Ca2+ exchange was simultaneous, coupled countertransport of Na+ and Ca2+. The movement of anions during Na+-Ca2+ exchange did not limit the initial rate of Na+-Ca2+ exchange. Na+-Ca2+ exchange was electrogenic, with a reversal potential of about -105 mV. The apparent flux ratio of Na+-Ca2+ exchange was 4 Na+:1 Ca2+. Coupled cation countertransport by the Na+-Ca2+ exchange mechanism required a monovalent cation gradient with the following sequence of ion activation: Na+ much greater than Li+ greater than Cs+ greater than K+ greater than Rb+. In contrast to Na+-Ca2+ exchange, Ca2+-Ca2+ exchange did not require a monovalent cation gradient, but required the presence of Ca2+ plus a monovalent cation on both sides of the vesicle membrane. The sequence of ion activation of Ca2+-Ca2+ exchange was: K+ much greater than Rb+ greater than Na+ greater than Li+ greater than Cs+. Na+ inhibited Ca2+-Ca2+ exchange when Ca2+-Ca2+ exchange was supported by another monovalent cation. Both Na+-Ca2+ exchange and Ca2+-Ca2+ exchange were inhibited, but with different sensitivities, by external MgCl2, quinidine, or verapamil.