Topology of a functionally important region of the cardiac Na+/Ca2+ exchanger

The cardiac Na+/Ca2+ exchanger, NCX1, has been modeled to consist of 11 transmembrane segments and a large cytoplasmic loop (loop f). Cysteine mutagenesis and sulfhydryl modification experiments demonstrate that the loop connecting transmembrane segments 1 and 2 (loop b) is located on the cytoplasmic side of the membrane, as previously modeled. A mutation in loop b, asparagine 101 to cysteine (N101C), renders the exchanger insensitive to regulation by cytoplasmic Na+ and Ca2+. Nearby mutations at residue threonine 103 (T103C or T103V) increase the apparent affinity of the exchanger for cytoplasmic Na+ and also produce a significant Li+ transport capacity. The evidence suggests that the region at the interface of cytoplasmic loop b and transmembrane segment 2 is important in Na+ transport and also in secondary regulation. Thus, this region may form part of the link between the ion translocation pathway formed by the transmembrane segments and regulatory sites that have previously been localized to loop f.     

Catecholamine secretion from bovine adrenal chromaffin cells: the role of the Na+/Ca2+ exchanger and the intracellular Ca2+ pool

The role of the Na+/Ca2+ exchanger and intracellular nonmitochondrial Ca2+ pool in the regulation of cytosolic free calcium concentration ([Ca2+]i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca2+]i were simultaneously monitored in a single chromaffin cell. After high-K+ stimulation, control cells and cells in which the Na+/Ca2+ exchange activity was inhibited showed similar rates of [Ca2+]i elevation. However, the recovery of [Ca2+]i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca2+ pump of the intracellular Ca2+ pool with thapsigargin caused a significant delay in the recovery of [Ca2+]i and greatly enhanced the secretory events. These data suggest that both the Na+/Ca2+ exchanger and the thapsigargin-sensitive Ca2+ pool are important in the regulation of [Ca2+]i and, by modulating the time course of secretion, are important in determining the extent of secretion.     

Immunohistochemical localization of Na+/Ca2+ exchanger in human retina and retinal pigment epithelium

OBJECTIVE: Validation a self-administered form used by patients to record their food intake and compare the recorded data with the observed intake. DESIGN: Data were obtained from an unselected cross-sectional group of hospitalized patients. SUBJECTS: Forty-five adult men and women volunteered to participate. Five of these dropped out. METHODS: Observed intake at breakfast, lunch and dinner was obtained by recording the servings of food before they were served to the patients and subtracting weighed leftovers. At meal times the patients recorded food items eaten in fractions of amount served to the nearest 25%. SETTING: Inpatients from five different wards at Rikshospitalet, Oslo. RESULTS: There was a significant under-reporting of the number of foods served (P < 0.005) resulting in a significant underestimation of energy 231 kJ (P < 0.02). There was good agreement between the patients and the observers for the portions of most foods (Kappa 0.44-0.92, P < 0.00001). The differences in amount had little influence on the difference in total energy. The difference in number of foods correlated with the difference in energy (r = 0.68, P < 0.001) and with the difference in protein (r = 0.50, P < 0.01). Patients with an underestimation of energy above 20% had forgotten seven or more food items. CONCLUSIONS: For most patients, the self-administered form adapted to the hospital menu appears to have acceptable validity, but for some patients it was unacceptable, mainly owing to food items being omitted and not because of incorrect estimate of amounts of food.     

Localization and functional significance of the Na+/Ca2+ exchanger in presynaptic boutons of hippocampal cells in culture

Immunocytochemical evidence for localized distribution of the Na+/Ca2+ exchange protein in nerve terminals of cultured hippocampal cells is presented together with results on the functional relevance of the exchanger in the control of [Ca2+]i and of synaptic vesicle recycling. The monoclonal antibody R3F1, directed against an epitope on the intracellular loop of the protein, revealed higher densities of expression in synaptic regions than in other parts of the neurons. Removal of extracellular Na+ produced enhanced and prolonged elevation of [Ca2+]i in nerve terminals during and after electrical stimulation of the cells. Correspondingly, initial rates of exocytosis, measured by fluorescence changes of FM 1-43 during stimulation, were faster in LiCl-containing solution than in NaCl-containing solution. By contrast, endocytosis at 20 s was the same in both solutions.     

Zinc inhibits Ca2+ transport by rat brain NA+/Ca2+ exchanger

Calcium transport by the Na+/Ca2+ exchanger was measured in plasma membranes vesicles purified from rat brain and in primary rat cortical cell culture. Sodium-loaded vesicles rapidly accumulate Ca2+ via Na+/Ca2+ exchange (Na+(i)-dependent Ca2+ uptake). Extravesicular zinc inhibited Na+/Ca2+ exchange as evidenced by a reduction of the initial velocity of Ca2+ uptake. Significant inhibition of Ca2+ uptake was seen at concentrations of zinc as low as 3 microM. Lineweaver-Burk analysis of the data was consistent with noncompetitive inhibition with respect to extravesicular Ca2+ concentration. The Ki for zinc inhibition of Ca2+ uptake determined from a Dixon plot was 14.5 microM. This is within the range of zinc concentrations thought to be obtained extracellularly after excitation. When vesicles were preloaded with Ca2+, extravesicular zinc also inhibited reversal of Na+/Ca2+ exchange (Na+(i)-dependent Ca2+ release) although its potency was much less: concentrations of > or = 30 microM zinc were required. Zinc inhibition of Ca2+ release was not Na+ dependent. Na+(i)-dependent calcium uptake by rat cortical cells in primary culture also was inhibited by zinc. The extent of inhibition was similar to that seen for inhibition of Na+(i)-dependent Ca2+ uptake in membrane vesicles, but the potency was less. The results suggest that Ca2+ transport by the Na+/Ca2+ exchanger is inhibited by concentrations of zinc thought to be attained extracellularly after excitation.     

The low-affinity dihydropyridine receptor and Na+/Ca2+ exchanger are associated in adrenal medullary mitochondria

The effect of Ca2+ channel-acting drugs on bovine adrenal mitochondria Ca2+ movements was investigated. Mitochondrial Ca2+ uptake is performed by an energy-driven Ca2+ uniporter with a Km of 20.9 +/- 3.2 microM and Vmax of 148.1 +/- 7.2 nmol 45Ca2+ min-1 mg-1. Ca2+ release is performed through an Na+/Ca2+ antiporter with a Km for Na+ of 4.2 +/- 0.5 mM, a Vmax of 7.5 +/- 0.4 nmol 45Ca2+ min-1 mg-1, and a Hill coefficient of 1.4 +/- 0.2 Ca2+ efflux through the mitochondrial Na+/Ca2+ exchanger was inhibited by several dihydropyridines (nitrendipine, felodipine, nimodipine, (+)isradipine) and by the benzothiazepine diltiazem with similar potencies. In contrast, neither CGP 28392, Bay-K-8644, amlodipine, nor verapamil had any effect on Ca2+ efflux. Nitrendipine at 20 microM modified neither the Km nor the Hill coefficient for Na+, whereas the Vmax was reduced to 2.9 nmol 45Ca2+ min-1 mg-1, thus demonstrating noncompetitive modulation of the Na+/Ca2+ exchanger. None of the Ca2+ channel-acting drugs assayed at 100 microM affected Ca2+ influx through the uniporter. Ca2+ channel blockers inhibited the Na+/Ca2+ antiporter and displaced the specific binding of [3H]nitrendipine to intact mitochondria with Ki values similar to the IC50s obtained for the inhibition of the Ca2+ efflux. Ca2+ channel-acting drugs that did not inhibit the Na+/Ca2+ exchanger (amlodipine, CGP 28392, Bay-K-9644, and verapamil, at concentrations of 100 microM or higher) had no effect on [3H]nitrendipine binding. These results suggest that the adrenomedullary mitochondrial dihydropyridine receptor is associated with the Na+/Ca2+ exchanger.     

Interaction of the Na(+)-Ca2+ exchanger with small molecules on cell Ca2+ signaling

BACKGROUND: Evaluation of outcome after CPR in severe hypothermic patients. DESIGN: Perspective study from October 1995 to April 1996. SETTING: First aid team of Italian Red Cross, Busto Arsizio (Varese), Italy. METHODS: A population of 22 patients in cardiac arrest in which CPR was performed immediately after rescue team's arrival is studied. ECG, core temperature, SpO2 and MAP were monitored whereas vital parameters were present during Basic Life Support. Outcome after CPR was evaluated with GOS scale. RESULTS: It has been observed that severe hypothermia and time of cardiac arrest impact on the clinical outcome after CPR. The high mortality rate after CPR with BLS standard is worsened by a core temperature < or = 33 degrees C. CONCLUSIONS: Severe hypothermia seems to have a dangerous effect upon outcome after cardiopulmonary resuscitation; heating systems for body temperature could prevent this situation improving CPR results.     

Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.     

Contribution of Na+/Ca2+ exchanger in maintaining [Ca2+]c at a stable state in rat pancreatic islets

The effects of lowering extracellular Na+ concentration [Na+]o, on cytosolic Ca2+ concentration, [Ca2+]c were examined by a microfluorimetric method using fura-2 in perifused preparations of isolated rat pancreatic islets. The total replacement of extracellular Na+ (Na+o) by equimolar N-methyl-D-(--)-glucamine caused a rapid rise in [Ca2+]c, and partial replacement of Na+o resulted in correlative rises in [Ca2+]c in accordance with the magnitude of reduced [Na+]o. The rise in [Ca2+]c induced by Na+o removal was strongly inhibited in the Ca2+o-deficient environment or by Ni2+. The [Ca2+]c rise, however, remained almost unchanged in the presence of nifedipine or SK&F 96365, and was enhanced by the addition of ouabain. The electrochemical gradients for Ca2+ (delta mu Ca2+) and Na+ (delta mu Na+) were calculated to be 39.08 and 12.8 kJ/mol, respectively, in this study, indicating a stoichiometry of 3Na+: 1 Ca2+. These results indicate that, in rat pancreatic islets, the rise in [Ca2+]c induced by lowering [Na+]o is mainly due to Ca2+ entry medicated by the Na+/Ca2+ exchanger operating with the stoichiometry of 3Na+:1 Ca2+, and that the Na+/Ca2+ exchanger plays an important role in maintaining stable-state [Ca2+]c.     

Alkalinization prolongs recovery from glutamate-induced increases in intracellular Ca2+ concentration by enhancing Ca2+ efflux through the mitochondrial Na+/Ca2+ exchanger in cultured rat forebrain neurons

Increasing extracellular pH from 7.4 to 8.5 caused a dramatic increase in the time required to recover from a glutamate (3 microM, for 15 s)-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in indo-1-loaded cultured cortical neurons. Recovery time in pH 7.4 HEPES-buffered saline solution (HBSS) was 126 +/- 30 s, whereas recovery time was 216 +/- 19 s when the pH was increased to 8.5. Removal of extracellular Ca2+ did not inhibit the prolongation of recovery caused by increasing pH. Extracellular alkalinization caused rapid intracellular alkalinization following glutamate exposure, suggesting that pH 8.5 HBSS may delay Ca2+ recovery by affecting intraneuronal Ca2+ buffering mechanisms, rather than an exclusively extracellular effect. The effect of pH 8.5 HBSS on Ca2+ recovery was similar to the effect of the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxyphenyl)hydrazone (FCCP; 750 nM). However, pH 8.5 HBSS did not have a quantitative effect on mitochondrial membrane potential comparable to that of FCCP in neurons loaded with a potential-sensitive fluorescent indicator, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide (JC-1). We found that the effect of pH 8.5 HBSS on Ca2+ recovery was completely inhibited by the mitochondrial Na+/Ca2+ exchange inhibitor CGP-37157 (25 microM). This suggests that increased mitochondrial Ca2+ efflux via the mitochondrial Na+/Ca2+ exchanger is responsible for the prolongation of [Ca2+]i recovery caused by alkaline pH following glutamate exposure.     

Effect of angiotensin II on Ca2+ efflux from freshly isolated adult rat cardiomyocytes: possible involvement of Na+/Ca2+ exchanger

Facial immersion testing in cold water (< 4 degrees C) was performed to study the responses of sinus cycle length to increased parasympathetic tone before and 5 min after exercise testing in 27 children. There were no episodes of sinus arrest or extrasystole during the facial immersion testing. The resting sinus cycle lengths were significantly shorter after (539 +/- 68 msec) than before (597 +/- 96 msec) exercise testing (p < 0.001). The maximal sinus cycle lengths before and after exercise testing during cold water facial immersion testing did not differ significantly (928 +/- 167 msec and 909 +/- 128 msec, respectively). Vagal chronotropic responses were calculated from the control sinus cycle lengths and the maximal sinus cycle lengths during facial immersion testing. Facial immersion caused greater prolongation of sinus cycle length after than before exercise (73 +/- 27% and 54 +/- 26%, respectively; p < 0.005). We speculate that this augmentation of vagal activity represents accentuated antagonism in these children, i.e., the same parasympathetic stimulus causes a greater response in the presence of a stronger background sympathetic activity.     

Identification of a mitochondrial Na+/H+ exchanger

The electroneutral exchange of protons for Na+ and K+ across the mitochondrial inner membrane contributes to organellar volume and Ca2+ homeostasis. The molecular nature of these transporters remains unknown. In this report, we characterize a novel gene (YDR456w; renamed NHA2) in Saccharomyces cerevisiae whose deduced protein sequence is homologous to members of the mammalian Na+/H+ exchanger gene family. Fluorescence microscopy showed that a Nha2-green fluorescent protein chimera colocalizes with 4',6-diamidino-2-phenylindole staining of mitochondrial DNA. To assess the function of Nha2, we deleted the NHA2 gene by homologous disruption and found that benzamil-inhibitable, acid-activated 22Na+ uptake into mitochondria was abolished in the mutant strain. It also showed retarded growth on nonfermentable carbon sources and severely reduced survival during the stationary phase of the cell cycle compared with the parental strain, consistent with a defect in aerobic metabolism. Sequence comparisons revealed that Nha2 has highest identity to a putative Na+/H+ exchanger homologue (KIAA0267; renamed NHE6) in humans. Northern blot analysis demonstrated that NHE6 is ubiquitously expressed but is most abundant in mitochondrion-rich tissues such as brain, skeletal muscle, and heart. Fluorescence microscopy showed that a NHE6-green fluorescent protein chimera also accumulates in mitochondria of transfected HeLa cells. These data indicate that NHA2 and NHE6 encode homologous Na+/H+ exchangers and suggest they may be important for mitochondrial function in lower and higher eukaryotes, respectively.     

Human esophageal epithelial cells possess an Na+/H+ exchanger for H+ extrusion

Chronic progressive renal function loss is a main cause of long-term graft loss after initially successful renal transplantation. Transplanted kidneys share some risk factors for renal function loss, such as hypertension or proteinuria, with diseased native kidneys. Recently, it has been shown that renal function loss is influenced by the angiotensin-converting enzyme (ACE) (insertion/deletion [I/D]) genotype in renal disease in diseased native kidneys. This study examines whether donor or recipient ACE (I/D) genotype is a risk factor for graft loss after renal transplantation. To avoid bias by acute events, graft survival was studied, with patients dying with a functioning graft censored, starting at 12 mo after transplantation in a cohort of 367 patients transplanted between 1987 and 1994 with at least 2 yr of follow-up. Mean follow-up was 58 mo. ACE (I/D) genotype was determined by PCR on stored donor and recipient lymphocytes. Neither donor nor recipient ACE (I/D) genotype was associated with graft survival. However, Cox proportional hazards analysis identified recipient, but not donor, ACE (I/D) genotype D-allele to be independently associated with a shorter time to graft loss in subgroups of patients at high risk for graft loss defined by a creatinine clearance <50 ml/min (n = 108, P = 0.017) or proteinuria > or =0.5 g/24 h at 12 mo (n = 97, P = 0.0051) after transplantation. In conclusion, recipient ACE (I/D) genotype was associated with time to graft loss in a specific high-risk subgroup of the study population. This suggests that the effect of ACE (I/D) genotype on graft survival only becomes apparent when other risk factors are simultaneously present.     

Activation of Ca2+-sensitive Cl- current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

We investigated how Ca2+-sensitive transient outward current, Ito(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na+ (Na+i) using the whole-cell patch-clamp technique at 36 degreesC. In cells dialysed with Na+-free solutions, the application of nicardipine (5 microM) to block L-type Ca2+ current (ICa) completely inhibited Ito(Ca). In cells dialysed with a [Na+]i>/=5 mM, however, Ito(Ca) could be observed after blockade of ICa, indicating the activity of an ICa-independent component. The amplitude of ICa-independent Ito(Ca) increased with voltage in a [Na+]i-dependent manner. The block of Ca2+ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked ICa-independent Ito(Ca). In Ca2+-free bath solution Ito(Ca) was completely abolished. The application of 2 mM Ni2+ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na+/Ca2+ exchange, or perfusion with pipette solution containing XIP (10 microM), a selective blocker of the exchanger, blocked ICa-independent Ito(Ca). From these results we conclude that, in the presence of Na+i, Ito(Ca) can be activated via Ca2+-induced Ca2+ release triggered by Na+/Ca2+ exchange operating in the reverse mode after blockade of ICa.     

Mutation of calmodulin-binding site renders the Na+/H+ exchanger (NHE1) highly H(+)-sensitive and Ca2+ regulation-defective

The ubiquitous plasma membrane Na+/H+ exchanger (NHE1) is rapidly activated in response to various extracellular signals. To understand how the intracellular Ca2+ is involved in this activation process, we investigated the effect of Ca2+ ionophore ionomycin on activity of the wild-type or mutant NHE1 expressed in the exchanger-deficient fibroblasts (PS120). In wild-type transfectants, a short (up to 1 min) incubation with ionomycin induced a significant alkaline shift (approximately 0.2 pH unit) in the intracellular pH (pHi) dependence of the rate of 5-(N-ethyl-N-isopropyl) amiloride-sensitive 22Na+ uptake, without changes in the cell volume and phosphorylation state of NHE1. Mutations that prevented calmodulin (CaM) binding to a high affinity binding region (region A, amino acids 636-656) rendered NHE1 constitutively active by inducing a similar alkaline shift in pHi dependence of Na+/H+ exchange. These same mutations abolished the ionomycin-induced NHE1 activation. These data suggest that CaM-binding region A functions as an "autoinhibitory domain" and that Ca2+/CaM activates NHE1 by binding to region A and thus abolishing its inhibitory effect. Furthermore, we found that a short stimulation with thrombin and ionomycin had apparently no additive effects on the alkaline shift in the pHi dependence of Na+/H+ exchange and that deletion of region A also abolished such an alkaline shift induced by a short thrombin stimulation. The results strongly suggest that the early thrombin response and the ionomycin response share the same activation mechanism. Based on these data and the results shown in the accompanying paper (Bertrand, B., Wakabayashi, S., Ikeda, T., Pouysségur, J., and Shigekawa, M. (1994) J. Biol. Chem. 269, 13703-13709), we propose that CaM is one of the major "signal transducers" that mediate distinct extracellular signals to the "pHi sensor" of NHE1.     

Protein kinase C-dependent regulation of Na+/Ca2+ exchanger isoforms NCX1 and NCX3 does not require their direct phosphorylation

We compared the phosphorylation-dependent regulation of three mammalian Na+/Ca2+ exchanger isoforms (NCX1-NCX3) expressed in CCL39 fibroblasts that have little endogenous activity. Na+i-dependent 45Ca2+ uptake into NCX1- or NCX3-expressing cells, but not that into NCX2-expressing cells, was significantly enhanced by phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor-BB, which was abolished by pretreatment of cells with calphostin C or a prior long exposure to PMA. This suggests that NCX1 or NCX3, but not NCX2, is stimulated by a pathway involving protein kinase C (PKC). Immunoprecipitation experiments using [32P]orthophosphate-labeled cells revealed that both NCX2 and NCX3 proteins were phosphorylated to a much lesser extent than the NCX1 protein in unstimulated cells and that the extent of phosphorylation was not increased by treatment with PKC activators, although NCX1 phosphorylation was enhanced significantly. Using site-directed mutagenesis, we identified three phosphorylation sites in the NCX1 protein in the PMA-stimulated cells to be Ser-249, Ser-250, and Ser-357 with Ser-250 being predominantly phosphorylated. We found that the NCX1 mutant with these serine residues substituted with alanine still maintained a normal response to PMA. In contrast, the NCX1 or NCX3 mutant, with the large central cytoplasmic loop deleted, lost the responsiveness to PMA. These results suggest that the PKC-dependent regulation of NCX1 or NCX3 requires the central cytoplasmic loop but does not require the direct phosphorylation of the exchanger.     

Effect of long-term hyperosmolality on the Na+/H+ exchanger isoform NHE-3 in LLC-PK1 cells

Communication is an important cornerstone to the physician-patient relationship when considering advance directives. Discussing advance directives with patients is a process best initiated in routine, well-adult care that can be made more daunting when the patient is critically ill; yet, when patients are afflicted with cancer, communication on advance directives can be optimized when the primary care physician and oncologist together work with the patient. The need to counsel patients on advance directives regardless of the venue (whether inpatient or outpatient) highlights that an ongoing alliance between the oncologist and the primary care physician can help facilitate consent to, and allow periodic review of, advance directives by cancer patients. This process ensures that the patient's preferences are respected at life's end.     

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.