Low-sodium contractures indicating sarcolemmal Na/Ca-exchange in the frog heart

1. In the frog heart, Ca2+ enters the cell by the slow inward current (Isi) and by an electrogenic, carrier-mediated, and passive Na-out/Ca-in-exchange. 2. The latter reverses to Na-in/Ca-out-exchange during depolarization and thereby controls relaxation. 3. The exchange ratio is 3 Na+ for 1 Ca2+. 4. The Na/Ca-exchange is not inhibited by organic Ca-antagonists in frog myocardium, indicating that the initiation of the heart beat may mainly depend on Isi. 5. This is not necessarily in contradiction with the Na-Ca-antagonism, since there also exists an antagonism between Na+ and Ca2+ in the slow channel. 6. However, the contractures caused by a decrease of NaO+ are mediated by the Na/Ca-exchange.     

Generation of twitch tension in frog atrial fibers by Na/Ca exchange

Electrical and mechanical responses of frog atrial trabeculae were studied simultaneously using the double-sucrose gap method. Action potentials and twitch tension could be successively generated in fibers in which the slow inward calcium channel current was not observed. As a rule, this could be obtained in the course of a long experiment (3 to 4 hours). Peak tension was shown to increase monotonically with membrane potential in these preparations. In preparations with the slow inward current the total peak tension could be separated into two components. The first component (tonic) monotonically increased with the membrane potential and was probably related to Na/Ca exchange (Horackova 1984). The potential dependency of the second (phasic) component correlated with that of the slow inward calcium current. Only the tonic but not the phasic component could be observed in preparations without the presence of the slow inward calcium current. The tonic component prevailed when both the slow inward current and phasic tension were greatly reduced by nifedipine. Long experiments, long depolarizing clamp pulses, a metabolic inhibitor 2,4-dinitrophenol, inhibitors of Na/K pump ouabain and AR-L57, toxins promoting intracellular sodium accumulation (aconitine, scorpion toxin) were all shown to increase the tonic tension, but not the slow inward current; they induced a transition from biphasic tension-voltage curve into a monotonically increasing one. We concluded that these procedures and agents greatly stimulate Ca influx via Na/Ca exchange. These results show that Na/Ca exchange can function as a reserve system of Ca2+ used for contraction, thus supporting the heart function, especially under unfavourable metabolic 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.     

Na+-Ca2+ exchange activity in rat skeletal myotubes: effect of lithium ions

The whole-cell patch-clamp technique coupled with intracellular [Ca2+] measurements was used to investigate the sodium-calcium exchange mechanism in rat skeletal muscle cells in primary culture. Replacing external Na+ ions with Li+ or N-methyl-D-glucamine (NMDG+) ions generated outward currents which were correlated with significant increases of free cytosolic-calcium concentration. These results strongly argue for a functional Na+-Ca2+ exchange mechanism working in its reverse mode. Moreover, the outward currents were sensitive to the new compound KB-R7943 (10 microM), which has been shown to be a potent inhibitor of the sodium-calcium exchanger. Outward Na+-Ca2+ exchange current densities were reduced in the presence of external Li+ as compared to those measured in the presence of NMDG+. After replacing internal sodium by lithium ions, rapid changes of external lithium concentrations generated sarcolemmal currents which were accompanied by subsequent variations of intracellular calcium activity. The currents were dependent on extracellular Li+ with a half-maximal activation at 67 mM and a Hill coefficient of 2.9. This work shows that the Na+-Ca2+ exchanger is able to significantly influence the myoplasmic calcium concentration of cultured rat myotubes. On the other hand, our results suggest that Li+ ions may substitute Na+ ions to catalyse an electrogenic Li+/Ca2+ counter transport.     

Evidence for electrogenic Na+/Ca2+ exchange in Limulus ventral photoreceptors

The Ca2+ indicator photoprotein, aequorin, was used to estimate and monitor intracellular Ca2+ levels in Limulus ventral photoreceptors during procedures designed to affect Na+/Ca2+ exchange. Dark levels of [Ca2+]i were estimated at 0.66 +/- 0.09 microM. Removal of extracellular Na+ caused [Ca2+]i to rise transiently from an estimated 0.5-0.6 microM in a typical cell to approximately 21 microM; [Ca2+]i approached a plateau level in 0-Na+ saline of approximately 5.5 microM; restoration of normal [Na+]o lowered [Ca2+]i to baseline with a time course of 1 log10 unit per 9 s. The apparent rate of Nao+-dependent [Ca2+]i decline decreased with decreasing [Ca2+]i. Reintroduction of Ca2+ to 0-Na+, 0-Ca2+ saline in a typical cell caused a transient rise in [Ca2+]i from an estimated 0.36 microM (or lower) to approximately 16.5 microM. This was followed by a decline in [Ca2+]i approaching a plateau of approximately 5 microM; subsequent removal of Cao2+ caused [Ca2+]i to decline slowly (1 log unit in approximately 110 s). Intracellular injection of Na+ in the absence of extracellular Na+ caused a transient rise in [Ca2+]i in the presence of normal [Ca2+]o; in 0-Ca2+ saline, however, no such rise in [Ca2+]i was detected. Under constant voltage clamp (-80 mV) inward currents were measured after the addition of Nao+ to 0-Na+ 0-Ca2+ saline and outward currents were measured after the addition of Cao2+ to 0-Na+ 0-Ca2+ saline. The results suggest the presence of an electrogenic Na+/Ca2+ exchange process in the plasma membrane of Limulus ventral photoreceptors that can operate in forward (Nao+-dependent Ca2+ extrusion) or reverse (Nai+-dependent Ca2+ influx) directions.     

Characterization of contractions in enzymatically isolated rat ventricular myocytes: effects of ouabain and rubidium

The role of sarcolemma and especially sodium pump activity in the control of phasic contractile activity of Ca2+ tolerant myocytes was studied using ouabain and rubidium as sodium pump inhibitors. Initially, ouabain increased both the amplitude of shortening and the frequency of phasic contractions. Later, the amplitude began to decline whereas the frequency of beating continued to rise, often terminating in a steady contracture of the myocyte. Rubidium caused a rapid rise of beating frequency, which reached its full effect within 1-5 min and remained steady after that. The stimulation of contraction frequency and the inhibition of Na+-K+ ATPase were correlated in the case of ouabain but not in the case of rubidium. The results suggest that the stimulation of phasic contractions may be caused by increased uptake of cellular calcium through Na+-Ca+ exchange as a consequence of sodium pump inhibition and (or) depolarization of the sarcolemma by ouabain and rubidium.     

Stimulation of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by phospholipase D

Treatment of canine cardiac sarcolemmal vesicles with phospholipase D resulted in a large stimulation (up to 400%) of Na+-Ca2+ exchange activity. The phospholipase D treatment decreased the apparent Km (Ca2+) for the initial rate of Nai+-dependent Ca2+ uptake from 18.2 +/- 2.6 to 6.3 +/- 0.3 microM. The Vmax increased from 18.0 +/- 3.6 to 31.5 +/- 3.6 nmol of Ca2+/mg of protein/s. The effect was specific for Na+-Ca2+ exchange; other sarcolemmal transport enzymes ((Na+, K+)-ATPase; ATP-dependent Ca2+ transport) are inhibited by incubation with phospholipase D. Phospholipase D had little effect on the passive Ca2+ permeability of the sarcolemmal vesicles. After treatment with 0.4 unit/ml of phospholipase D (20 min, 37 degrees C), the sarcolemmal content of phosphatidic acid rose from 0.9 +/- 0.2 to 8.9 +/- 0.4%; simultaneously, Na+-Ca2+ exchange activity increased 327 +/- 87%. It is probable that the elevated phosphatidic acid level is responsible for the enhanced Na+-Ca2+ exchange activity. In a previous study (Philipson, K. D., Frank, J. S., and Nishimoto, A. Y. (1983) J. Biol. Chem. 258, 5905-5910), we hypothesized that negatively charged phospholipids were important in Na+-Ca2+ exchange, and the present results are consistent with this hypothesis. Stimulation of Na+-Ca2+ exchange by phosphatidic acid may be important in explaining the Ca2+ influx which accompanies the phosphatidylinositol turnover response which occurs in a wide variety of tissues.     

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.     

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.     

Na+-Ca2+ exchange and calcium permeability in canine basolateral membrane vesicles: the effects of dibutyryl cAMP and specific inhibitors

The role of dibutyryl 3',5'-cyclic adenosine monophosphate (dibutyryl cAMP) as putative second messenger for parathyroid hormone (PTH) in regulating canine proximal tubular basolateral membrane Na+-Ca2+ exchange and passive calcium permeability was assessed, as was the nature of this passive calcium permeability. Dibutyryl cAMP (50 mg) infused in vivo over 30 min increased fractional phosphate excretion from 4.9 +/- 1.8% to 20.5 +/- 4.6%, P less than 0.05, n = 6, but had no effect on either passive Ca2+ efflux or sodium-stimulated Ca2+ efflux from Ca2+-preloaded basolateral membrane vesicles (BLMV). Both of these mechanisms have been previously shown to be stimulated by PTH. Further studies were performed to investigate the mechanism of the passive calcium flux. Calcium uptake by BLMV was blocked by lanthanum (La3+) but not by the calcium-channel blocker verapamil. La3+ blocked efflux of Ca2+ from preloaded vesicles when it was placed in the external solution. This La3+-blockable efflux was larger in potassium equivalent BLMV prepared from normal dogs than in BLMV prepared from thyroparathyroidectomized dogs. Benzamil produced 50% inhibition of sodium-stimulated Ca2+ uptake at 250 microM whereas neither amiloride nor diltiazem achieved 50% inhibition at the maximal doses studied. Benzamil, 1 mM, had no effect on passive calcium efflux and neither did the substitution of sucrose for potassium, which has been shown to affect Ca2+-Ca2+ exchange by the Na+-Ca2+ exchanger. This suggests that the calcium flux under potassium equivalent conditions was not mediated by Ca2+-Ca2+ exchange by the Na+-Ca2+ exchanger. These results demonstrate that the basolateral membrane of proximal tubular cells possesses both a Na+-Ca2+ exchanger inhibitable by benzamil and a passive calcium permeability not inhibited by benzamil nor by verapamil but by La3+. Neither of these two mechanisms of calcium flux was affected by dibutyryl cAMP whereas both have been shown to be stimulated by PTH.     

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.     

Inhibition of Na+-Ca2+ exchange in heart sarcolemmal vesicles by phosphatidylethanolamine N-methylation

The effect of phosphatidylethanolamine N-methylation on Na+-Ca2+ exchange was studied in sarcolemmal vesicles isolated from rat heart. Phosphatidylethanolamine N-methylation following incubation of membranes with S-adenosyl-L-methionine, a methyl donor for the enzymatic N-methylation, inhibited Nai+-dependent Ca2+ uptake by about 50%. The N-methylation reaction did not alter the passive permeability of the sarcolemmal vesicles to Na+ and Ca2+ and did not modify the electrogenic characteristics of the exchanger. The depressant effect of phosphatidylethanolamine N-methylation on Nai+-dependent Ca2+ uptake was prevented by S-adenosyl-L-homocysteine, an inhibitor of the N-methylation. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino-group-blocking agent, also prevented methylation-induced inhibition of Ca2+ uptake. In the presence of exogenous phospholipid substrate, the phospholipid N-methylation process in methyl-acetimidate-treated sarcolemmal vesicles was restored and the inhibitory effect on Ca2+ uptake was evident. These results suggest that phosphatidylethanolamine N-methylation influences the heart sarcolemmal Na+-Ca2+ exchange system.     

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.     

In dialyzed squid axons Ca2+i activates Ca2+o-Na+i and Na+o-Na+i exchanges in the absence of Ca chelating agents

We used internally dialyzed squid axons to explore whether the reported activatory effect of Ca2+i on the partial reactions of the Na+-Ca2+ exchange (essential activator) is secondary to the presence of Ca2+ chelating agents in the internal medium. The effect of Ca2+i pulses on both the reverse (Ca2+o-dependent Na+ efflux) and Na+-Na+ exchange (Na+o-dependent Na+ efflux) modes of the Na+-Ca2+ exchange was studied in axons dialyzed without EGTA. For these experiments a substantial inhibition of the Ca2+ buffer capacity of the axoplasm was achieved by the use of Ruthenium red (10-20 microM), cyanide (1 mM) and vanadate (1 mM) in the dialysis solution. Our results indicate that the Ca2+i requirement of the reverse and Na+-Na+ exchange can not be explained by a direct inhibition of the Na+-Ca2+ exchanger by EGTA. In fact, both modes of operation of the exchanger can be activated by internal Ca2+ ions in the complete absence of Ca2+ chelating agents thus indicating that the 'catalytic' effect of Ca2+i on the Na+-Ca2+ exchanger is a real phenomenon.     

Possible role of Na(+)-Ca2+ exchange in the regulation of contractility in isolated adult ventricular myocytes from rat and guinea pig

Action potentials and developed contractions of externally unloaded single ventricular myocytes isolated from adult rat and guinea pig hearts were recorded by means of an optical system for recording contractile activity during regular stimulation by microelectrodes. Under control conditions, the shortenings (twitches) in the rat myocytes were fully inhibited by 0.1 microM ryanodine, but they were rather insensitive to the Ca2+ blocker 0.2-0.5 microM nifedipine. In contrast, the contractions of the isolated guinea pig ventricular myocytes were greatly suppressed by 0.2-0.5 microM nifedipine (to less than 30%), while they were only slightly reduced by 1 microM ryanodine. When the Na+ gradient was decreased by reducing [Na]o or by elevating [Na]i in the presence of veratridine, the twitch contractions were increased in both species. The effect of reduced [Na]o on twitch contractions was not affected by ryanodine in either type of myocytes, while nifedipine still fully abolished the twitches in the guinea pig cells, indicating a strong dependence of guinea pig contractions on Ca2+ influx. On the other hand, the effect of a reduced Na gradient by veratridine was more complex; the usual twitch (phasic component) was increased and it was followed by a second (tonic) component which relaxed only after the repolarization of the action potential. While the phasic component was decreased by nifedipine and ryanodine in the usual way (as in the controls), the sustained contractions (lasting up to several seconds) were ryanodine and nifedipine insensitive. Furthermore, the cardiomyocytes of both species exposed to strontium in place of external calcium still exhibited all the effects observed when reducing the Na+ gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+-Ca2+ exchange in rat brain microsomal membranes pretreated with pronase and/or SDS

The effects of pronase and/or SDS pretreatment on Na+-Ca2+ exchange were studied in rat brain microsomal membranes. Pronase in concentrations that liberated 11% of the membrane proteins stimulated the Na+-Ca2+ exchange. When about 24% of the proteins were split off, the results did not differ from those in control experiments. When 40% or more of the proteins were solubilized, Na+-Ca2+ exchange was abolished. Pronase pretreatment did not change the Km value for Ca2+, it increased Vmax only. The effect of pronase was partially blocked by Trasylol. Neuraminidase had no effect on Na+-Ca2+ exchange. SDS pretreatment of the membranes inhibited Na+-Ca2+ exchange: when 25% of membrane proteins were solubilized with SDS, the Na+-Ca2+ exchange was abolished while the same amount of proteins split off with pronase did not change the rate of Na+-Ca2+ exchange as related to membrane proteins. Ischaemia lasting for 2-4 h or complete hypoxia which should stimulate endogenous proteinases due to the rise of free intracellular calcium did not influence the Na+-Ca2+ exchange. A decrease in Na+-Ca2+ exchange rate was observed when proteins with molecular weight between 45,000 and 20,000 were split off from the membranes. It is assumed that the Na+-Ca2+ antiporter is a polypeptide from the group of proteins within the above molecular weights.     

Purification of the cardiac Na+-Ca2+ exchange protein

We have used fractionation procedures to enrich solubilized cardiac sarcolemma in the Na+-Ca2+ exchange protein. Sarcolemma is extracted with an alkaline medium to remove peripheral proteins and is then solubilized with decylmaltoside. Next, the exchanger is applied to DEAE-Sepharose and eluted with high salt. The DEAE fraction is applied to WGA-agarose, and a small fraction of protein, enriched in the exchanger, can be eluted by changing the detergent to Triton X-100. This fraction is reconstituted into asolectin proteoliposomes for measurement of Na+-Ca2+ exchange activity and gel electrophoresis. The purified fraction has a Na+-Ca2+ exchange activity of 600 nmol Ca2+/mg of protein per s at 10 microM Ca2+ and a purification factor of about 30 as compared with control reconstituted sarcolemmal vesicles. Ca2+-Ca2+ exchange and Na+-Ca2+ exchange activities were both present in the same final reconstituted vesicles indicating that the same protein is responsible for both transport activities. SDS-PAGE reveals two prominent protein bands at 70 and 120 kDa. After mild chymotrypsin treatment (1 microgram/ml), there is no loss of exchange activity, but the 120 kDa band disappears and the 70 kDa band becomes more dense. This suggests that the 70 kDa band is due to an active proteolytic fragment of the 120 kDa protein. Under non-reducing gel conditions, only a single protein band is seen with an apparent molecular weight of 160 kDa. Antibodies to the purified exchanger preparation are able to immunoprecipitate exchange activity and confirm that the 70 kDa protein derives from the 120 kDa protein. We propose that both the 70 and 120 kDa proteins are associated with the Na+-Ca2+ exchanger.     

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.     

Na+-Ca2+ exchanger in the soluble fraction of crayfish striated muscle

Proteins with Na+-Ca2+ exchange activity from the soluble fraction of crayfish striated muscle were inserted into asolectin proteoliposomes. A pH dependent calcium uptake with an optimum at the alkaline side and inhibition in the presence of sodium or strontium ions in the external medium was observed. When expressed per tissue wet weight the capacity for Na+-Ca2+ exchange of proteoliposomes with inserted soluble proteins was by one half higher than that of the membrane fraction and more than twice higher in comparison with the reconstituted membrane bound exchanger. Using polyacrylamide gel electrophoresis two most prominent proteins with Mr over 200 and 43 kDa could be detected in proteoliposomes with the highest Na+-Ca2+ exchange. It is assumed that protein(s) with Mr 43 kDa could represent the soluble Na+-Ca2+ exchanger in crayfish striated muscle soluble fraction.     

Na+-dependent alkaline earth metal uptake in cardiac sarcolemmal vesicles

The ability of alkaline earth metals (M2+) to substitute for Ca2+ in Na+-Ca2+ exchange was examined in sarcolemmal vesicles isolated from the canine heart. 85Sr2+ and 133Ba2+, in addition to 45Ca2+, were used to determine the characteristics of Na+-M2+ exchange. The Na+i-dependent M2+ uptake was measured as a function of time, with t ranging from 0.5 to 360 s, [Na+]i = 140 mM and [M2+]o = 40 microM. This function was linear for Ca2+ and Sr2+ uptake for approx. 6 s and for Ba2+ for about 60 s. Plateau levels were achieved within 120 s for Ca2+ and Sr2+ but Ba2+ took considerably longer. The Km values for Na+-M2+ exchange, derived from Eadie-Hofstee plots, were 30, 58, and 73 microM for Ca2+, Sr2+ and Ba2+, respectively. The Na+i-dependent uptake of all three ions was stimulated in the presence of 0.36 microM valinomycin. Na+-Ca2+ exchange was also measured in the presence of either 20 microM Sr2+ or 100 microM Ba2+. Both of these ions behaved (at these concentrations) as competitive inhibitors of Na+-Ca2+ exchange with the KI being 32 microM for Sr2+ and 92 microM for Ba2+. Passive efflux was determined by first allowing Na+-M2+ exchange to continue to plateau values and then diluting the loaded vesicles in the presence of EGTA. The rate constants for the passive efflux were 8.4, 6.3 and 4.4 min-1 for Ca2+, Sr2+ and Ba2+, respectively.