Light-dependent Na(+)-Ca2+ exchange in retinal rod discs

Experiments are described demonstrating that Na(+)-Ca2+ exchange of retinal rod disc membrane is highly sensitive to light. The Na(+)-Ca2+ exchanger was shown to possess two types of binding sites with different affinities for calcium. The low affinity binding sites (KCaD = 5.8 mumol/l) are light-insensitive. After bleaching, KD of the high affinity Ca2(+)-binding sites an Ki for Na+ changed from 0.2 to 0.3 mumol/l and from 3.2 to 0.7 nmol/l, respectively. Light inhibits the steady-state Ca2+ uptake by a factor of 1.5. Photocontrol of the Na(+)-Ca2+ exchanger affinity is observed at the physiological level of rhodopsin bleaching.     

Electrophysiological characterization of ionic transport by the retinal exchanger expressed in human embryonic kidney cells.

The retinal Na+:Ca2+, K+exchanger cDNA was transiently expressed in human embryonic kidney (HEK 293) cells by transfection with plasmid DNA. The correct targeting of the expressed protein to the plasma membrane was confirmed by immunocytochemistry. The reverse exchange offrent (Ca2+ imported per Na+ extruded) was measured in whole-cell voltage-clamp experiments after intracellular perfusion with Na+ (Na+i, 128 mM) and extracellular perfusion with Ca2+ (Ca2o+, 1 mM) and Ko+ (20 mM). As expected, the exchange current was suppressed by removing Ca2o+. Surprisingly, however, it was also abolished by increasing Na+o to almost abolish the Na+ gradient, and it was almost unaffected by the removal of Ko+. Apparently, then, at variance with the exchanger in the rod outer segment, the retinal exchanger expressed in 293 cells acts essentially as a Na+:Ca2+ exchanger and does not require K+ for its electrogenic activity.     

Symmetry properties of the Na+-Ca2+ exchange mechanism in cardiac sarcolemmal vesicles

The Na+-Ca2+ exchange mechanism in cardiac sarcolemmal vesicles can catalyze the exchange of Ca2+ on either side of the sarcolemmal membrane for Na+ on the opposing side. Little is known regarding the relative affinities of Na+ and Ca2+ for exchanger binding sites on the intra- and extracellular membrane surfaces. We have previously reported (Philipson, K.D. and Nishimoto, A.Y. (1982) J. Biol. Chem. 257, 5111-5117) a method for measuring the Na+-Ca2+ exchange of only the inside-out vesicles in a mixed population of sarcolemmal vesicles (predominantly right-side-out). We concluded that the apparent Km(Ca2+) for Na+i-dependent Ca2+ uptake was similar for inside-out and right-side-out vesicles. In the present study, we examine in detail Na+o-dependent Ca2+ efflux from both the inside-out and the total population of vesicles. To load vesicles with Ca2+ prior to measurement of Ca2+ efflux, four methods are used: 1, Na+-Ca2+ exchange; 2, passive Ca2+ diffusion; 3, ATP-dependent Ca2+ uptake; 4, exchange of Ca2+ for Na+ which has been actively transported into vesicles by the Na+ pump. The first two methods load all sarcolemmal vesicles with Ca2+, while the latter two methods selectively load inside-out vesicles with Ca2+. We are able to conclude that the dependence of Ca2+ efflux on the external Na+ concentration is similar in inside-out and right-side-out vesicles. Thus the apparent Km(Na+) values (approximately equal to 30 mM) of the Na+-Ca2+ exchanger are similar on the two surfaces of the sarcolemmal membrane. In other experiments, external Na+ inhibited the Na+i-dependent Ca2+ uptake of the total population of vesicles much more potently than that of the inside-out vesicles. Apparently Na+ can compete for the Ca2+ binding site more effectively on the external surface of right-side-out than on the external surface of inside-out vesicles. Thus, although affinities for Na+ or Ca2+ (in the absence of the other ion) appear symmetrical, the interactions between Na+ and Ca2+ at the two sides of the exchanger are not the same. The Na+-Ca2+ exchanger is not a completely symmetrical transport protein.     

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).     

ATP-dependent calcium pump and Na+-Ca2+ exchange in plasma membrane vesicles from squid optic nerve

Purified plasma membrane vesicles from the optic nerve of the squid Sepiotheutis sepioidea accumulate calcium in the presence of Mg2+ and ATP. Addition of the Ca2+ ionophore A23187 to vesicles which have reached a steady state of calcium-active uptake induces complete discharge of the accumulated cation. Kinetic analysis of the data indicates that the apparent Km for free Ca2+ and ATP are 0.2 muM and 21 muM, respectively. The average Vmax is 1 nmol Ca2+/min per mg protein at 25 degrees C. This active transport is inhibited by orthovanadate in the micromolar range. An Na+-Ca2+ exchange mechanism is also present in the squid optic nerve membrane. When an outwardly directed Na+ gradient is imposed on the vesicles, they accumulate calcium in the absence of Mg2+ and/or ATP. This ability to accumulate Ca2+ is absolutely dependent on the Na+ gradient: replacement of Na+ by K+, or passive dissipation of the Na+ gradient, abolishes transport activity. The apparent Km for Ca2+ of the Na+-Ca2+ exchange is more than 10-fold higher than that of the ATP-driven pump (app. Km=7.5 muM). While the apparent Km for Na+ is 74 mM, the Vmax of the exchanger is 27 nmol Ca2+/min per mg protein at 25 degrees C. These characteristics are comparable to those displayed by the uncoupled Ca pump and Na+-Ca2+ exchange previously described in dialyzed squid axons.     

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.     

Effect of temperature on sodium-calcium exchange in sarcolemma from mammalian and amphibian hearts

We have investigated temperature dependence of Ca2+ uptake by the cardiac sarcolemmal Na(+)-Ca2+ exchanger from dog, rabbit and bullfrog. In native rabbit sarcolemmal vesicles, Ca2+ affinity of the Na(+)-Ca2+ exchanger is unchanged from 7 to 37 degrees C; however, the initial velocity of Ca2+ uptake declines much more steeply below 22 degrees C than above 22 degrees C. In native dog sarcolemma, the temperature dependence of Na(+)-Ca2+ exchange velocity is similar to that of native rabbit. However, in frog heart the velocity of Na(+)-Ca2+ exchange declines much more slowly with decreasing temperature at both temperature ranges. Reconstitution of the Na(+)-Ca2+ exchanger into artificial lipid vesicles consisting of either asolectin or phosphatidylserine, phosphatidylcholine, and cholesterol has little effect on temperature dependence of Na(+)-Ca2+ exchange velocity in any of the three species. We conclude that the lesser temperature sensitivity of the cardiac sarcolemmal Na(+)-Ca2+ exchanger of a poikilothermic species is at least partly an intrinsic property of the transport protein.     

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.     

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.     

Identification of the sodium-calcium exchanger as the major ricin-binding glycoprotein of bovine rod outer segments and its localization to the plasma membrane

After neuraminidase treatment the Na+/Ca2+ exchanger of bovine rod outer segments was found to specifically bind Ricinus communis agglutinin. SDS gel electrophoresis and Western blotting of ricin-binding proteins purified from rod outer segment membranes by lectin affinity chromatography revealed the existence of two major polypeptides of Mr 215K and 103K, the former of which was found to specifically react with PMe 1B3, a monoclonal antibody specific for the 230-kDa non-neuraminidase-treated Na+/Ca2+ exchanger. Reconstitution of the ricin affinity-purified exchanger into calcium-containing liposomes revealed that neuraminidase treatment had no significant effect on the kinetics of Na+/Ca2+ exchange activation by sodium. We further investigated the density of the Na+/Ca2+ exchanger in disk and plasma membrane preparations using Western blotting, radioimmunoassays, immunoelectron microscopy, and reconstitution procedures. The results indicate that the Na+/Ca2+ exchanger is localized in the rod photoreceptor plasma membrane and is absent or present in extremely low concentrations in disk membranes, as we have previously shown to be the case for the cGMP-gated cation channel. Previous reports describing the existence of Na+/Ca2+ exchange activity in rod outer segment disk membrane preparations may be due to the fusion of plasma membrane components and/or the presence of contaminating plasma membrane vesicles.     

A (Ca2+, Mg2+)-ATPase activity in plasma membrane fragments isolated from squid nerves

A (Ca2+, Mg2+)-ATPase activity and a (Ca2+, Mg2+)-dependent phosphorylation from ATP have been found in plasma membrane fragments from squid optical nerves under conditions where contamination by intracellular organelles is unlikely. The properties of this (Ca2+, Mg2+)-ATPase activity are almost identical to those of the ATP-dependent uncoupled Ca2+ efflux observed in dialyzed squid giant axons. This gives further support to the notion that the mechanism responsible for maintaining the low levels of ionized Ca concentration in nerves at rest is not a Na+-Ca2+ exchange system but an ATP-driven uncoupled Ca2+ pump.     

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.     

Role of phosphatidylinositol in cardiac sarcolemmal membrane sodium-calcium exchange

The purpose of this investigation was to study the effects of a distinct type of phospholipase C on sarcolemmal Na+-Ca2+ exchange. With this phospholipase C (Staphylococcus aureus), treatment of cardiac sarcolemmal vesicles resulted in a specific hydrolysis of membrane phosphatidylinositol. This hydrolysis of phosphatidylinositol also released two proteins (110 and 36 kDa) from the sarcolemmal membrane. Phospholipase C pretreatment of the sarcolemma resulted in an unexpected stimulation of Na+-Ca2+ exchange. The Vmax of Na+-Ca2+ exchange was increased but the Km for Ca2+ was not altered. This stimulation was specific to the Na+-Ca2+ exchange pathway. ATP-dependent Ca2+ uptake was depressed after phospholipase C treatment, but passive membrane permeability to Ca2+ was unaffected. Sarcolemmal Na+,K+-ATPase activity was not altered, whereas passive Ca2+ binding was modestly decreased after phospholipase C pretreatment. The stimulation of Na+-Ca2+ exchange after phosphatidylinositol hydrolysis was greater in inside-out vesicles than in a total population of vesicles of mixed orientation. This finding suggests that the cardiac sarcolemmal Na+-Ca2+ exchanger is functionally asymmetrical. The results also suggest that membrane phosphatidylinositol is inhibitory to the Na+-Ca2+ exchanger or, alternatively, this phospholipid may anchor an endogenous inhibitory protein in the sarcolemmal membrane. The observation that a transsarcolemmal Ca2+ flux pathway may be stimulated solely by phosphatidylinositol hydrolysis independently of phosphoinositide metabolic products like inositol triphosphate is novel.     

Phospholipid composition modulates the Na+-Ca2+ exchange activity of cardiac sarcolemma in reconstituted vesicles

Na+-Ca2+ exchange activity in cardiac sarcolemmal vesicles is known to be sensitive to charged, membrane lipid components. To examine the interactions between membrane components and the exchanger in more detail, we have solubilized and reconstituted the Na+-Ca2+ exchanger into membranes of defined lipid composition. Our results indicate that optimal Na+-Ca2+ exchange activity requires the presence of certain anionic phospholipids. In particular, phosphatidylserine (PS), cardiolipin, or phosphatidic acid at 50% by weight results in high Na+-Ca2+ exchange activity, whereas phosphatidylinositol and phosphatidylglycerol provide a poor environment for exchange. In addition, incorporation of cholesterol at 20% by weight greatly facilitates Na+-Ca2+ exchange activity. Thus, for example, an optimal lipid environment for Na+-Ca2+ exchange is phosphatidylcholine (PC, 30%)/PS (50%)/cholesterol (20%). Na+-Ca2+ exchange activity is also high when cardiac sarcolemma is solubilized and then reconstituted into asolectin liposomes. We fractionated the lipids of asolectin into subclasses for further reconstitution studies. When sarcolemma is reconstituted into vesicles formed from the phospholipid component of asolectin, Na+-Ca2+ exchange activity is low. When the neutral lipid fraction of asolectin (including sterols) is also included in the reconstitution medium, Na+-Ca2+ exchange activity is greatly stimulated. This result is consistent with the requirement for cholesterol described above. Proteinase treatment, high pH, intravesicular Ca2+ and dodecyl sulfate all stimulate Na+-Ca2+ exchange in native sarcolemmal vesicles. We examined the effects of these interventions on exchange activity in reconstituted vesicles of varying lipid composition. In general, Na+-Ca2+ exchange could be stimulated only when reconstituted into vesicles of a suboptimal lipid composition. That is, when reconstituted into asolectin or PC/PS/cholesterol (30:50:20), the exchanger is already in an activated state and can no longer be stimulated. The one exception was that the Na+-Ca2+ exchanger responded to altered pH in an identical manner, independent of vesicle lipid composition. The mechanism of action of altered pH on the exchanger thus appears to be different from other interventions.     

Characterization of the ATP-promoted aspect of Na+-Ca2+ exchange present in squid retinal nerve axolemma

Using an in vitro system which consists of an axolemma-rich vesicle fraction prepared from squid retinal nerve fibers, an Na+-Ca2+ exchange process has been characterized and appears identical with that reported in squid giant axon. This exchange is absolutely dependent on the establishment of an Na+ gradient, shows monovalent and divalent cation specificity and is highly sensitive to monensin, A23187 and valinomycin but not to ouabain, digitoxigenin, vanadate, pentylenetetrazole, tetrodotoxin or tetraethylammonium. Furthermore, it was found that the exchange process is enhanced by the addition of ATP. This ATP-promoted aspects of Na+-Ca2+ exchange shares many similar characteristics with Na+-Ca2+ ATP hydrolysis and may indicate a common mechanism for both activities via a protein phosphorylation-dephosphorylation event.     

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.     

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.     

Stimulation of sodium-calcium exchange by cholesterol incorporation into isolated cardiac sarcolemmal vesicles

Modification of the cholesterol content of highly purified cardiac sarcolemma from dog ventricles was accomplished by incubation with phosphatidylcholine liposomes containing various amounts of cholesterol. The degree of cholesterol enrichment could be varied by changing the liposomal cholesterol/phospholipid ratio or varying the liposome-membrane incubation time. Na+-Ca2+ exchange measured in cholesterol-enriched sarcolemmal vesicles was increased up to 48% over control values. The stimulation of Na+-Ca2+ exchange was associated with an increased affinity of the exchanger for Ca2+ (Km = 17 microM compared with Km = 22 microM for control preparations). Na+-Ca2+ exchange measured in cholesterol-depleted membrane preparations was decreased by 15%. This depressed activity was associated with a decreased affinity of the exchanger for Ca2+ (Km = 27 microM). These changes were not due to either a change in membrane permeability to Ca2+ or an increase in the amount of Ca2+ bound to sarcolemmal vesicles. The stimulating effect of cholesterol enrichment was specific to the Na+-Ca2+ exchange process since sarcolemmal Ca2+-Mg2+ ATPase activity was depressed 40% by cholesterol enrichment. Further, K+-p-nitrophenylphosphatase and Na+-K+ ATPase activities were depressed in both cholesterol-depleted and cholesterol-enriched sarcolemmal vesicles. In situ oxidation of membrane cholesterol completely eliminated Na+-Ca2+ exchange. These results suggest that cholesterol is intimately associated with Na+-Ca2+ exchange and may interact with the exchange protein and modulate its activity.