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 the protein and cDNA of the cardiac Na+/H+ exchanger.

We examined the myocardial form of the Na+/H+ exchanger. A partial length cDNA clone was isolated from a rabbit cardiac library and it encoded for a Na+/H+ exchange protein. In comparison with the human Na+/H+ exchanger, the sequence of the 5' end of the cDNA was highly conserved, much more than the 3' region, while the deduced amino acid sequence was also highly conserved. To further characterize the myocardial Na+/H+ exchange protein, we examined Western blots of isolated sarcolemma with antibody produced against a fusion protein of the Na+/H+ exchanger. The antibodies reacted with a sarcolemma protein of 50 kDa and with a protein of 70 kDa. The results show that the rabbit myocardium does possess a Na+/H+ exchanger protein homologous to the known human Na+/H+ exchanger.     

Distribution of the Na(+)-Ca2+ exchange protein in mammalian cardiac myocytes: an immunofluorescence and immunocolloidal gold-labeling study

The present study reports on the location of the Na(+)-Ca2+ exchanger in cardiac sarcolemma with immunofluorescence and immunoelectron microscopy. Both polyclonal and monoclonal antibodies to the Na(+)-Ca2+ exchanger were used. The mAb was produced from a hybridoma cell line generated by the fusion of mouse myeloma NS-1 cells with spleen cells from a mouse repeatedly immunized with isolated reconstituted canine cardiac Na(+)-Ca2+ exchanger (Philipson, K. D. S. Longoni, and R. Ward. 1988. Biochim. Biophys. Acta. 945:298-306). The polyclonal antibody has been described previously and reacts with three proteins (70, 120, 160 kD) in cardiac sarcolemma associated with the Na(+)-Ca2+ exchanger (Nicoll, D. A., S. Longoni, and K. D. Philipson. 1990. Science (Wash. DC). 250:562-565). Both the monoclonal and the polyclonal antibodies appear to react with extracellular facing epitopes in the cardiac sarcolemma. Immunofluorescence studies showed labeling of the transverse tubular membrane and patchy labeling of the peripheral sarcolemma. The immunofluorescent labeling clearly delineates the highly interconnected T-tubular system of guinea pig myocytes. This localization of the exchanger to the sarcolemma, with an apparent high density in the transverse tubules, was also seen with immunoelectron microscopy. It is of great interest that the Na(+)-Ca2+ exchanger, as the main efflux route for Ca2+ in heart cells, would be abundantly located in sarcolemma closest to the release of Ca2+.     

Phosphatidyl inositol-4,5-bisphosphate bound to bovine cardiac Na+/Ca2+ exchanger displays a MgATP regulation similar to that of the exchange fluxes.

This work shows the existence of a phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) bound form of the cardiac sarcolemmal Na+/Ca2+ exchanger. That was demonstrated in Western blots and cross-immunoprecipitation by using specific antibodies against the NCX1 exchanger (NCX1) and against PtdIns-4,5-P2. In addition, PtdIns-4,5-P2 bound to the Na+/Ca2+ exchanger and the Na+/Ca2+ exchange fluxes displayed a similar MgATP regulation: (a) both increase by 100-130% when membrane vesicles are incubated (15-20 s at 37 degrees C) with 1 mM MgATP and 1 microM Ca2+ (b) in the presence of 100 microM Ca2+, MgATP fails to stimulate the exchange fluxes and does not modify the levels of PtdIns-4,5-P2 bound to the exchanger. In addition, in the absence of Ca2+, the net synthesis of total membrane PtdIns-4,5-P2 is greatly reduced compared with that in the presence of 1 microM Ca2+. Furthermore, in the absence of Ca2+ there is no effect of MgATP on the levels of PtdIns-4,5-P2 bound to the exchanger. These results indicate that, in bovine heart, MgATP-stimulation of Na+/Ca2+ exchange is associated with intracellular Ca2+-dependent levels of PtdIns-4,5-P2 bound to the exchanger molecule.     

Site density of the sodium-calcium exchange carrier in reconstituted vesicles from bovine cardiac sarcolemma

The site density of the Na2+-Ca2+ exchanger in bovine cardiac sarcolemma was estimated from measurements of the fraction of reconstituted proteoliposomes exhibiting exchange activity. Sarcolemmal vesicles were solubilized with 1% Triton X-100 in the presence of either 100 mM NaCl or 100 mM KCl; after a 20-40-min incubation period on ice, sufficient KCl, NaCl, CaCl2, and soybean phospholipids were added to each extract to give final concentrations of 40 mM NaCl, 120 mM KCl, 0.1 mM CaCl2, and 10 mg/ml phospholipid. These mixtures were then reconstituted into proteoliposomes, and the rate of 45Ca2+ isotopic exchange was measured under equilibrium conditions. Control studies showed that Na+-Ca2+ exchange activity was completely lost if Na+ was not present during solubilization. The difference in 45Ca2+ uptake between vesicles initially solubilized in the presence or absence of NaCl therefore reflected exchange activity and corresponded to 3.1 +/- 0.3% of the total 45Ca2+ uptake by the entire population of vesicles, as measured in the presence of the Ca2+ ionophore A23187. Assuming that each vesicle with exchange activity contained 1 molecule of the Na+-Ca2+ exchange carrier, a site density of 10-20 pmol/mg of protein for the exchanger was calculated. The Vmax for Na+-Ca2+ exchange activity in the proteoliposomes was approximately 20 nmol/mg of protein.s which indicates that the turnover number of the exchange carrier is 1000 s-1 or more. Thus, the Na+-Ca2+ exchanger is a low density, high turnover transport system.     

The Ca2+-pumping ATPase of heart sarcolemma. Characterization, calmodulin dependence, and partial purification

The (Ca2+ + Mg2+) ATPase of dog heart sarcolemma (Caroni, P., and Carafoli, E. (1980) Nature 283, 765-767) has been characterized. The enzyme possesses an apparent Km (Ca2+) of 0.3 +/- 02 microM, a Vmax of Ca2+ transport of 31 nmol of Ca2+/mg of protein/min, and an apparent Km (ATP) of 30 microM. It is only slightly influenced by monovalent cations and is highly sensitive to orthovanadate (Ki = 0.5 +/- 0.1 microM). The high vanadate sensitivity has been used to distinguish the sarcolemmal and the contaminating sarcoplasmic reticulum Ca2+-dependent ATPase in heart microsomal fractions. Calmodulin has been shown to be present in heart sarcolemma. Its depletion results in the transition of the Ca2+-pumping ATPase to a low Ca2+ affinity; readdition of calmodulin reverses this effect. The Na+/Ca2+ exchange system was not affected by calmodulin. The results of calmodulin extraction can be duplicated by using the calmodulin antagonist trifluoperazine. The calmodulin-depleted Ca2+-ATPase has been solubilized from the sarcolemmal membrane and "purified" on a calmodulin affinity chromatography column. One major (Mr = 150,000) and 3 minor protein bands could be eluted from the column with ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). The major protein band (72%) has Ca2+-dependent ATPase activity and can be phosphorylated by [gamma]32P]ATP in a Ca2+-dependent reaction.     

Influence of long-term treatment of imidapril on mortality, cardiac function, and gene expression in congestive heart failure due to myocardial infarction

Although it is generally accepted that the efficacy of imidapril, an angiotensin-converting enzyme inhibitor, in congestive heart failure (CHF) is due to improvement of hemodynamic parameters, the significance of its effect on gene expression for sarcolemma (SL) and sarcoplasmic reticulum (SR) proteins has not been fully understood. In this study, we examined the effects of long-term treatment of imidapril on mortality, cardiac function, and gene expression for SL Na+/K+ ATPase and Na+ -Ca2+ exchanger as well as SR Ca2+ pump ATPase, Ca2+ release channel (ryanodine receptor), phospholamban, and calsequestrin in CHF due to myocardial infarction. Heart failure subsequent to myocardial infarction was induced by occluding the left coronary artery in rats, and treatment with imidapril (1 mg.kg(-1).day(-1)) was started orally at the end of 3 weeks after surgery and continued for 37 weeks. The animals were assessed hemodynamically and the heart and lung were examined morphologically. Some hearts were immediately frozen at -70 degrees C for the isolation of RNA as well as SL and SR membranes. The mortality of imidapril-treated animals due to heart failure was 31% whereas that of the untreated heart failure group was 64%. Imidapril treatment improved cardiac performance, attenuated cardiac remodeling, and reduced morphological changes in the heart and lung. The depressed SL Na+/K+ ATPase and increased SL Na+-Ca2+ exchange activities as well as reduced SR Ca2+ pump and SR Ca2+ release activities in the failing hearts were partially prevented by imidapril. Although changes in gene expression for SL Na+/K+ ATPase isoforms as well as Na+-Ca2+ exchanger and SR phospholamban were attenuated by treatments with imidapril, no alterations in mRNA levels for SR Ca2+ pump proteins and Ca2+ release channels were seen in the untreated or treated rats with heart failure. These results suggest that the beneficial effects of imidapril in CHF may be due to improvements in cardiac performance and changes in SL gene expression.     

Reconstitution, identification, purification, and immunological characterization of the 110-kDa Na+/Ca2+ antiporter from beef heart mitochondria.

The mitochondrial Na+/Ca2+ antiporter plays a key role in the physiological regulation of intramitochondrial Ca2+, which in turn attunes mitochondrial enzymes to the changing demands of the cell for ATP. We have now purified the Na+/Ca2+ antiporter from beef heart mitochondria by assaying detergent-solubilized chromatography fractions for reconstitutive activity. Na+ and Ca2+ transport were assayed using the fluorescent probes, sodium-binding benzofuran isophthalate and Fura-2, respectively. This approach enabled us to identify Na+/Ca2+ exchange activity with a 110-kDa inner membrane protein that catalyzed Na(+)-dependent Ca2+ transport and Ca(2+)-dependent Na+ transport. A new finding was that the Na+/Ca2+ antiporter also catalyzed Na+/Li+ exchange in the absence of Ca2+. All modes of transport were electroneutral and were inhibited by diltiazem and tetraphenylphosphonium cation. Monospecific polyclonal antibodies to the 110-kDa protein inhibited Na+/Ca2+ and Na+/Li+ exchange in the reconstituted system and recognized 110-kDa proteins in mitochondrial membranes isolated from rat heart, liver, and kidney.     

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.     

Amiloride and its analogs as tools in the study of ion transport

Amiloride inhibits most plasma membrane Na+ transport systems. We have reviewed the pharmacology of inhibition of these transporters by amiloride and its analogs. Thorough studies of the Na+ channel, the Na+/H+ exchanger, and the Na+/Ca2+ exchanger, clearly show that appropriate modification of the structure of amiloride will generate analogs with increased affinity and specificity for a particular transport system. Introduction of hydrophobic substituents on the terminal nitrogen of the guanidino moiety enhances activity against the Na+ channel; whereas addition of hydrophobic (or hydrophilic) groups on the 5-amino moiety enhances activity against the Na+/H+ exchanger. Activity against the Na+/Ca2+ exchanger and Ca2+ channel is increased with hydrophobic substituents at either of these sites. Appropriate modification of amiloride has produced analogs that are several hundred-fold more active than amiloride against specific transporters. The availability of radioactive and photoactive amiloride analogs, anti-amiloride antibodies, and analogs coupled to support matrices should prove useful in future studies of amiloride-sensitive transport systems. The use of amiloride and its analogs in the study of ion transport requires a knowledge of the pharmacology of inhibition of transport proteins, as well as effects on enzymes, receptors, and other cellular processes, such as DNA, RNA, and protein synthesis, and cellular metabolism. One must consider whether the effects seen on various cellular processes are direct or due to a cascade of events triggered by an effect on an ion transport system.     

Characterization of the membrane-bound protein kinase C and its substrate proteins in canine cardiac sarcolemma

Cardiac sarcolemma was purified from canine ventricles. Enrichment of the sarcolemmal membranes was demonstrated by the high (Na+ + K+)-ATPase activity of 28.0 +/- 1.5 mumol Pi/mg protein per h and the high concentration of muscarinic receptors with the Bmax of 8.2 +/- 2.5 pmol/mg protein as determined by [3H]QNB binding. The purified sarcolemma also contains significant levels of a membrane-bound Ca2+ and phospholipid-dependent protein kinase (protein kinase C). To elucidate the protein kinase C activity in sarcolemma, a prior incubation of the membranes with EGTA and Triton X-100 was necessary. The specific activity of protein kinase C was found to be 131.4 pmol Pi/mg per min, in the presence of 6.25 micrograms phosphatidylserine and 0.5 mM CaCl2. Treatment of sarcolemma with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBu2) resulted in a concentration-dependent activation of protein kinase C activity. The effect of TPA and PBu2 on protein kinase C in sarcolemma was independent of exogenous Ca2+ and phosphatidylserine. Polymyxin B inhibited phorbol-ester-induced activation of protein kinase C activity. The distribution of protein kinase C in the cytosolic fraction was also examined. The specific activity of the kinase in the cytosolic fraction was 59.7 pmol Pi/mg per min. However, the total protein kinase C activity in the cytosol was 213500 pmol Pi/min, compared to that of 1025 pmol Pi/min in the sarcolemma isolated from approx. 100 g of canine ventricular muscle. Several endogenous proteins in cardiac sarcolemma were phosphorylated in the presence of Ca2+ and phosphatidylserine. The major substrates for protein kinase C were proteins of Mr 94 000, 87 000, 78 000, 51 000, 46 000, 11 500 and 10 000. Most of these substrate proteins have not been identified before. Other proteins of Mr 38 000, 31 000 and 15 000 were markedly phosphorylated in the presence of Ca2+ only. Phosphorylation of phospholamban (Mr 27 000 and 11 000) was also stimulated in the presence of Ca2+ and phosphatidylserine, but the low Mr form of phospholamban was distinct from two other low Mr substrate proteins for protein kinase C. Polymyxin B was more selective in inhibiting the protein kinase C dependent phosphorylation. On the other hand, trifluoperazine selectively inhibited the phosphorylation of phospholamban and Mr 15 000 protein. Although the exact function of this kinase is unknown, based on these observations, we believe that protein kinase C in the cardiac sarcolemma may play an important role in the cell-surface-signal regulated cardiac function.     

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.     

Influence of sterols and phospholipids on sarcolemmal and sarcoplasmic reticular cation transporters

We have examined the influence of different sterols and phospholipids on the activities of the cardiac sarcolemmal Na+-Ca2+ exchanger and Na+,K+-ATPase and the sarcoplasmic reticular Ca2+-ATPase in reconstituted proteoliposomes. When either the solubilized Na+-Ca2+ exchanger or the Na+,K+-ATPase is reconstituted into phosphatidylcholine (PC):phosphatidylserine (30:50 by weight) vesicles, high cholesterol levels (20% by weight) are required for activity to be expressed. This sterol requirement is highly specific for cholesterol. Several cholesterol analogues with minor structural changes are unable to support Na+-Ca2+ exchange or Na+,K+-ATPase activities. When solubilized sarcolemma is reconstituted into PC:cardiolipin vesicles, however, the requirement for cholesterol is lost. Substantial activity can be obtained in the complete absence of cholesterol or in the presence of several cholesterol analogues. Thus, sterol/protein interactions can be highly dependent on the phospholipid environment. In contrast, the skeletal muscle sarcoplasmic reticular Ca2+-ATPase functions equally well in the presence or absence of cholesterol after reconstitution into either PC:phosphatidylserine or PC:cardiolipin proteoliposomes. Phospholipid requirements of the transporters were also examined. The sarcolemmal Na+-Ca2+ exchanger, Na+,K+-ATPase, and the sarcoplasmic reticular Ca2+-ATPase all function optimally in the presence of phosphatidylserine or cardiolipin after reconstitution. Thus, the sarcolemmal cation transporters have similar sterol and phospholipid requirements and may have structural similarities in their hydrophobic regions. The sarcoplasmic reticular Ca2+ pump evolved in a low cholesterol membrane and has different lipid interactions. These findings may have general applicability to other plasma membrane and endoplasmic reticular enzymes.     

Role of cAMP-dependent phosphorylation in passive transport of Ca2+ by myocardial sarcolemma

The myocardial sarcolemma vesicles loaded with Na2+ can accumulate Ca2+ against the concentration gradient in exchange for Na2+; the rate of this process is about 10 nmole of Ca2+ per mg of protein per min. The cAMP-dependent phosphorylation of sarcolemmal preparations has no effect on the Na+/Ca2+ exchange. At the same time the cAMP-dependent phosphorylation of protein components of the sarcolemma causes inhibition of the passive Ca2+ efflux from the vesicles depending on the degree of membrane phosphorylation.     

Immunohistochemical evidence for the Na+/Ca2+ exchanger in squid olfactory neurons

The olfactory organs from the squid Lolliguncula brevis are composed of a pseudostratified epithelium containing five morphological subtypes of chemosensory neurons and ciliated support cells. Physiological recordings have been made from two of the subtypes and only the type 4 neuron has been studied in detail. Odour-stimulated increases in intracellular calcium and rapid activation of an electrogenic Na+/Ca2+ exchanger current in type 4 neurons suggest that the exchanger proteins are localized very close to the transduction machinery. Electrophysiological studies have shown that olfactory signal transduction takes place in the apical ciliary regions of olfactory neurons. Using polyclonal antiserum against squid Na+/Ca2+ proteins, we observed specific staining in the ciliary region of cells that resemble type 2, 3, 4 and 5 neurons. Staining was also observed in axon bundles, and in muscle tissue. Collectively, these data support the model that Na+/Ca2+ exchanger proteins are localized to transduction machinery in cilia of type 4 neurons and suggest that the other olfactory subtypes also use Ca2+ during chemosensory responses.     

Intracellular Ca2+ requirement for activation of the Na+/H+ exchanger in vascular smooth muscle cells

The role for intracellular Ca2+ in modulating activity of the Na+/H+ exchanger was studied in cultured vascular smooth muscle cells. Na+/H+ exchange was activated by four distinct stimuli: 1) phorbol 12-myristate 13-acetate, 2) thrombin, 3) cell shrinkage, and 4) intracellular acid loading. [Ca2+]i was independently varied between 40 and 200 nM by varying the bathing Ca2+ from 10 nM to 5.0 mM. Thrombin-induced intracellular Ca2+ transients were blocked with bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). In the absence of stimulators of Na+/H+ exchange, varying [Ca2+]i above or below the basal level of 140 nM did not activate Na+/H+ exchange spontaneously. However, varying [Ca2+]i did affect stimulus-induced activation of Na+/H+ exchange. Activation of the exchanger by phorbol 12-myristate 13-acetate was blunted by reduced intracellular Ca2+ (half-maximal activity at 50-90 nM [Ca2+]i), consistent with a Ca2+ requirement for protein kinase C (Ca2+/phospholipid-dependent enzyme). Activation of the exchanger by thrombin in protein kinase C-depleted cells was also sensitive to reduced intracellular Ca2+ (half-maximal activity at 90-140 nM [Ca2+]i) and was increased 40% by raising [Ca2+]i to 200 nM. Activation of the exchanger by cell shrinkage or intracellular acid loads was not significantly affected over the range of [Ca2+]i tested. Thus, altered [Ca2+]i does not itself affect Na+/H+ exchange activity in vascular smooth muscle but instead modulates activation of the transporter by particular stimuli.     

Inhibition of Na+/Ca2+ exchanger activity in cardiac and skeletal muscle sarcolemmal vesicles by monoclonal antibody 44D7

Monoclonal antibodies 44D7 and 4F2 inhibited specifically the Na+-dependent Ca2+ fluxes characteristic of the Na+/Ca2+ exchanger in cardiac and skeletal muscle sarcolemmal vesicles. Preincubation of membrane vesicles with monoclonal antibody 44D7 inhibited 90% of the Na+-dependent Ca2+ uptake measured in the first 10 s of the reaction and 50% of that measured after 60 s. Ca2+/calmodulin-dependent ATPase activity and ATP-dependent Ca2+ uptake by sarcolemmal vesicles were not affected by monoclonal antibody 44D7 whereas the Na+-dependent release of accumulated Ca2+ was inhibited. In the presence of the 44D7 antigen isolated from human kidney, monoclonal antibody 44D7 could no longer inhibit Na+-dependent Ca2+ fluxes. The distribution of 4F2 antigenic activity in the isolated muscle membrane fractions correlated with that of Na+/Ca2+ exchanger activity; cardiac and skeletal muscle sarcolemmal vesicles expressed higher levels of the antigen than skeletal muscle transverse tubule membrane, while no antigen could be detected in sarcoplasmic reticulum membranes. Our results suggest that monoclonal antibodies 44D7 and 4F2 interact either directly with the Na+/Ca2+ exchanger molecules or with some other protein(s) responsible for the regulation of this activity in the heart and skeletal muscle.     

Effect of Quin-2 on 45Ca2+ uptake mediated by Na+i/Ca2+o exchange and 45Ca2+ efflux in rat brain synaptosomes: a requirement for [Ca2+]i

The Na+/Ca2+ exchanger of squid axons, barnacle muscle and sarcolemma requires micromolar intracellular calcium for activation in the Na+i/Ca2+o exchange mode ('reverse' Na+/Ca2+ exchange). The requirement for [Ca2+]i has been demonstrated with the use of intracellular calcium buffers, such as Quin-2, to inhibit Na+i/Ca2+o exchange. However, the inhibition of Na+i/Ca2+o exchange in mammalian nerve terminals loaded with Quin-2 has not been observed [7], suggesting a lower sensitivity to low [Ca2+]i for this system. In contrast, the results reported herein indicate that 45Ca2+ uptake in synaptosomes through Na+i/Ca2+o exchange is inhibited by Quin-2 much in the same way as it is in the squid, provided that synaptosomes are preincubated in low Ca2+ medium to avoid saturation of Quin-2. Under these conditions, 45Ca2+ efflux via Ca2+i/Ca2+o exchange is also inhibited. Our results indicate that the Na+i/Ca2+o and Ca2+i/Ca2+o modes of the Na+/Ca2+ exchanger from rat brain synaptosomes require intracellular calcium for activation. However, because no clear relationship between the observed [Ca2+]i values and the inhibition of Na+i/Ca2+o exchange has been found, it is suggested that localised submembrane calcium concentrations not detected by the [Ca2+]i probe might regulate the exchanger.     

Localization of sarcolemmal proteins to lipid rafts in the myocardium

The localization of sarcolemmal proteins within the membrane can have a dramatic effect on excitation-contraction coupling. We examine the localization of the Na+-Ca2+ exchanger, the dihydropyridine receptor, and other proteins involved in excitation-contraction coupling in rat heart using biochemical and immunolocalization techniques. Specifically, we assess the distribution of proteins within the lipid raft fraction of the sarcolemma. We find that the distribution of proteins in lipid raft fractions is very dependent on the solubilization technique. A common technique using sodium carbonate/pH 11 to solubilize non-lipid raft proteins was inappropriate for use with sarcolemmal membranes. Use of Triton X-100 was more efficacious as a solubilization agent. A large majority of the Na+-Ca2+ exchanger, Na+/K+-ATPase, and plasma membrane Ca2+ pump are not present in lipid rafts. In contrast, most adenosine A1 receptors and dihydropyridine receptors were in lipid raft fractions. Most of the adenosine A1 receptors could be co-immunoprecipitated with caveolin indicating a localization to caveolae (a subclass of lipid rafts). In contrast, the dihydropyridine receptors could not be co-immunoprecipitated with caveolin. Most biochemical data were confirmed by high resolution immunolocalization studies. Using correlation analysis, only a small fraction of the Na+-Ca2+ exchangers colocalized with caveolin whereas a substantial fraction of dihydropyridine and adenosine A1 receptors did colocalize with caveolin. The most pertinent findings are that the Na+-Ca2+ exchanger and the dihydropyridine receptor are in separate sarcolemmal subcompartments. These spatial relationships may be relevant for understanding excitation-contraction coupling.     

The Ba2+ sensitivity of the Na+-induced Ca2+ efflux in heart mitochondria: the site of inhibitory action

The Na+-induced Ca2+ release from rat heart mitochondria was measured in the presence of Ruthenium red. Ba2+ effectively inhibited the Na+-induced Ca2+ release. At 10 mM Na+ 50% inhibition was reached by 1.51 +/- 0.48 (S.D., n = 8) microM Ba2+ in the presence of 0.1 mg/ml albumin and by 0.87 +/- 0.25 (S.D., n = 3) microM Ba2+ without albumin. In order to inhibit, it was not required that Ba2+ ions enter the matrix. 140Ba2+ was not accumulated in the mitochondrial matrix space; further, in contrast to liver mitochondria, Ba2+ inhibition was immediate. The Na+-induced Ca2+ release was inhibited by Ba2+ non-competitively, with respect of the extramitochondrial Na+. The double inhibitor titration of the Na+-Ca2+ exchanger with Ba2+ in the presence and absence of extramitochondrial Ca2+ revealed that the exchanger possesses a common binding site for extramitochondrial Ca2+ and Ba2+, presumably the regulatory binding site of the Na+-Ca2+ exchanger, which was described by Hayat and Crompton (Biochem. J. 202 (1982) 509-518). All these observations indicate that Ba2+ acts at the cytoplasmic surface of the inner mitochondrial membrane. The inhibitory properties of Ba2+ on the Na+-dependent Ca2+ release in heart mitochondria are basically different from those found on Na+-independent Ca2+ release in liver mitochondria (Lukács, G.L. and Fonyó, A. (1985) Biochim. Biophys. Acta 809, 160-166).