ATP triggers a robust intracellular [Ca2+ ]-mediated signalling pathway in human synovial fibroblasts.

NEW FINDINGS: What is the central question of this study? What are the main [Ca2+ ]i signalling pathways activated by ATP in human synovial fibroblasts? What is the main finding and its importance? In human synovial fibroblasts ATP acts through a linked G-protein (Gq ) and phospholipase C signalling mechanism to produce IP3 , which then markedly enhances release of Ca2+ from the endoplasmic reticulum. These results provide new information for the detection of early pathophysiology of arthritis. ABSTRACT: In human articular joints, synovial fibroblasts (HSFs) have essential physiological functions that include synthesis and secretion of components of the extracellular matrix and essential articular joint lubricants, as well as release of paracrine substances such as ATP. Although the molecular and cellular processes that lead to a rheumatoid arthritis (RA) phenotype are not fully understood, HSF cells exhibit significant changes during this disease progression. The effects of ATP on HSFs were studied by monitoring changes in intracellular Ca2+ ([Ca2+ ]i ), and measuring electrophysiological properties. ATP application to HSF cell populations that had been enzymatically released from 2-D cell culture revealed that ATP (10-100 µm), or its analogues UTP or ADP, consistently produced a large transient increase in [Ca2+ ]i . These changes (i) were initiated by activation of the P2 Y purinergic receptor family, (ii) required Gq -mediated signal transduction, (iii) did not involve a transmembrane Ca2+ influx, but instead (iv) arose almost entirely from activation of endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate (IP3 ) receptors that triggered Ca2+ release from the ER. Corresponding single cell electrophysiological studies revealed that these ATP effects (i) were insensitive to [Ca2+ ]o removal, (ii) involved an IP3 -mediated intracellular Ca2+ release process, and (iii) strongly turned on Ca2+ -activated K+ current(s) that significantly hyperpolarized these cells. Application of histamine produced very similar effects in these HSF cells. Since ATP is a known paracrine agonist and histamine is released early in the inflammatory response, these findings may contribute to identification of early steps/defects in the initiation and progression of RA.

Structure-function relationships of the NCKX2 Na+/Ca2+-K+ exchanger.

K+-dependent Na+/Ca2+ exchangers (NCKX) have been shown to play important roles in physiological processes as diverse as phototransduction in rod photoreceptors, motor learning and memory in mice, and skin pigmentation in humans. Most structure-function studies on NCKX proteins have been carried out on the NCKX2 isoform, but sequence similarity suggests that the results obtained with the NCKX2 isoform are likely to apply to all NCKX1-5 members of the human SLC24 gene family. Here we review our recent work on the NCKX2 protein concerning the topological arrangement of transmembrane segments carrying out cation transport, and concerning residues important for transport function and cation binding.

Potassium-dependent sodium-calcium exchange through the eye of the fly.

In this review, we describe the characterization of a Drosophila sodium/calcium-potassium exchanger, Nckx30C. Sodium/calcium (-potassium) exchangers (NCX and NCKX) are required for the rapid removal of calcium in excitable cells. The deduced protein topology for NCKX30C is similar to that of mammalian NCKX, with 5 hydrophobic domains in the amino terminus separated from 6 at the carboxy-terminal end by a large intracellular loop. NCKX30C functions as a potassium-dependent sodium-calcium exchanger and is expressed in adult neurons and during ventral nerve cord development in the embryo. Nckx30C is expressed in a dorsal/ventral pattern in the eye-antennal disc, suggesting that large fluxes of calcium may be occurring during imaginal disc development in the larvae. NCKX30C may play a critical role in modulating calcium during development as well as in the removal of calcium and maintenance of calcium homeostasis in adults.

Stoichiometry of the retinal cone Na/Ca-K exchanger heterologously expressed in insect cells: comparison with the bovine heart Na/Ca exchanger.

The transport stoichiometry is an essential property of antiporter and symporter transport proteins. In this study, we determined the transport stoichiometry of the retinal cone potassium-dependent Na/Ca exchanger (NCKX) expressed in sodium-loaded cultured insect cells. The Na/Ca and Rb/Ca coupling ratios were obtained by direct measurements of the levels of (86)Rb and (45)Ca uptake and sodium release associated with reverse Na/Ca exchange. Rb/Ca coupling ratios of 0.98 [standard deviation (SD) of 0.12, 15 observations] and 0.92 (SD of 0.12, 13 observations) were obtained for the chicken and human retinal cone NCKX, respectively. Na/Ca coupling ratios of 4.11 (SD of 0.24, 10 observations) and 3.98 (SD of 0.34, 15 observations) were obtained for the chicken and human retinal cone NCKX, respectively, whereas a lower average coupling ratio of 3.11 (SD of 0.34, 10 observations) was obtained with cells expressing the bovine Na/Ca exchanger (NCX1). These results are consistent with a 4Na/1Ca + 1K stoichiometry for retinal cone NCKX. High Five cells expressing full-length dolphin rod NCKX, Caenorhabditis elegans NCKX, or bovine rod NCKX from which the two large hydrophilic loops were removed all showed a significant calcium-dependent (86)Rb uptake, whereas no calcium-dependent (86)Rb uptake was observed in cells expressing bovine NCX1. The calcium dependence of (45)Ca uptake yielded values between 1 and 2.5 microM for the external calcium dissociation constant of the different NCKX proteins studied here.

Molecular cloning and functional expression of the potassium-dependent sodium-calcium exchanger from human and chicken retinal cone photoreceptors.

Light causes a rapid lowering of cytosolic free calcium in the outer segments of both retinal rod and cone photoreceptors. This light-induced lowering of calcium is caused by extrusion via a Na-Ca exchanger located in the rod and cone outer segment plasma membrane and plays a key role in the process of light adaptation. The Na-Ca exchanger in retinal rod outer segment was shown earlier to be a novel Na-Ca+K exchanger (NCKX), and its cDNA was obtained by molecular cloning from several mammalian species. On the other hand, the proper identity of the retinal cone Na-Ca exchanger, in terms of both functional characteristics (e.g., requirement for and transport of potassium) and molecular identity, has not yet been elucidated. Here, we report the molecular cloning, intraretinal localization by in situ hybridization, and initial functional characterization of the chicken and human cone-specific Na-Ca exchangers. In addition we report the chicken rod-specific NCKX. We identified NCKX transcripts in both human and chicken cones and observed strong potassium-dependent Na-Ca exchange activity after heterologous expression of human and chicken cone NCKX cDNAs in cultured insect cells. In situ hybridization in chicken retina showed abundant rod NCKX transcripts only in rod photoreceptors, whereas abundant cone NCKX transcripts were found in most, if not all, cone photoreceptors and also in a subpopulation of retinal ganglion cells. A detailed comparison with the previously described retinal rod and brain NCKX cDNAs is presented.

Minimal domain requirement for cation transport by the potassium-dependent Na/Ca-K exchanger. Comparison with an NCKX paralog from Caenorhabditis elegans.

The retinal rod Na/Ca-K exchanger (NCKX) is a unique calcium extrusion protein utilizing both inward sodium gradient and outward potassium gradient. Three mammalian rod NCKX cDNAs have been cloned to date, but quantitative analysis of NCKX function in heterologous systems has proven difficult. Here, we describe a simple system for quantitative analysis of NCKX function; stable transformation of cultured insect cells with the novel pEA1/153A vector containing NCKX cDNAs was combined with measurements of potassium-dependent (45)Ca uptake in sodium-loaded cells. We carried out structure-function studies on NCKX with the following results: 1) two-thirds of the full-length sequence of bovine NCKX could be deleted without affecting potassium-dependent calcium transport and without affecting key properties of the potassium binding site; 2) the affinity of NCKX for potassium was about 10-fold greater in choline medium when compared with lithium medium; this shift was observed in rod outer segments or in cells expressing full-length rod NCKX, the above deletion mutant, or a distantly related NCKX paralog cloned from Caenorhabditis elegans. We conclude that the potassium binding site is highly conserved among members of the NCKX family and is formed by residues located within the two sets of transmembrane spanning segments in the NCKX sequence.

Cloning and characterization of a potassium-dependent sodium/calcium exchanger in Drosophila.

Sodium/calcium(-potassium) exchangers (NCX and NCKX) are critical for the rapid extrusion of calcium, which follows the stimulation of a variety of excitable cells. To further understand the mechanisms of calcium regulation in signaling, we have cloned a Drosophila sodium/calcium-potassium exchanger, Nckx30C. The overall deduced protein topology for NCKX30C is similar to that of mammalian NCKX, having five membrane-spanning domains in the NH(2) terminus separated from six at the COOH-terminal end by a large intracellular loop. We show that NCKX30C functions as a potassium-dependent sodium/calcium exchanger, and is not only expressed in adult neurons as was expected, but is also expressed during ventral nerve cord development in the embryo and in larval imaginal discs. Nckx30C is expressed in a dorsal-ventral pattern in the eye-antennal disc in a pattern that is similar to, but broader than that of wingless, suggesting that large fluxes of calcium may be occurring during imaginal disc development. Nckx30C may not only function in the removal of calcium and maintenance of calcium homeostasis during signaling in the adult, but may also play a critical role in signaling during development.

cDNA cloning and functional expression of the dolphin retinal rod Na-Ca+K exchanger NCKX1: comparison with the functionally silent bovine NCKX1.

cDNAs of human and bovine retinal rod Na+-Ca2++K+ exchanger (NCKX1) have previously been cloned, but potassium-dependent Na-Ca exchange activity upon heterologous expression has not been demonstrated. We have cloned NCKX1 cDNA from dolphin, examined function upon transfection in HEK293 cells, and compared the dolphin sequence encoded by the cDNA with those of human and bovine. The dolphin NCKX1 cDNA encodes 1013 amino acid residues. Comparison to bovine and human NCKX1 revealed strong conservation in the transmembrane domains (>95%), but relatively low conservation in the large extracellular ( approximately 50%) and cytosolic (<50%) domains. The dolphin cytosolic domain differs from the bovine sequence by the absence of a stretch of 114 amino acids. HEK293 cells transfected with dolphin NCKX1 cDNA showed K+-dependent Na-Ca exchange in >95% of the experiments, whereas transfection with bovine NCKX1 yielded no function. The bovine NCKX1 phenotype was imparted on dolphin NCKX1 when the dolphin cytosolic loop was replaced by that from bovine. Conversely, deletion of 114 amino acids from the bovine sequence to match the dolphin sequence resulted in a mutant bovine NCKX1 which performed K+-dependent Na-Ca exchange. These results suggest that domains within the large cytosolic loop of NCKX1 control functional activity when expressed in heterologous systems.

Inhibition and acceleration of Na+/Ca2+/K+ exchange fluxes by Ag+ in bovine retinal rod outer segments.

The effect of Ag+ on Ca2+ fluxes mediated by the retinal rod Na+/Ca2+/K+ exchanger was investigated in intact bovine rod outer segments (ROS). Intracellular Na+ concentration ([Na+]in)-dependent Ca2+ influx and extracellular Na+ concentration ([Na+]out)-dependent Ca2+ efflux were monitored by changes in cytosolic free Ca2+ measured with the fluorescent Ca(2+)-indicating dye fluo 3. Ag+ was the most effective inhibitor of Na+/Ca2+/K+ exchange fluxes described to date, with half-maximal inhibition observed at 2-8 microM Ag+. Inhibition by Ag+ could be reversed by addition of beta-mercaptoethanol but not by addition of cysteine. Reversal by beta-mercaptoethanol resulted in a marked acceleration of [Na+]out-dependent lowering of cytosolic free Ca2+ but not of [Na+]in-dependent Ca2+ influx. We suggest that Ag+ inhibits and accelerates Na+/Ca2+/K+ exchange fluxes by binding to cysteine residues on the cytosolic surface of the exchanger protein.

Ca2+ influx into bovine retinal rod outer segments mediated by Na+/Ca2+/K+ exchange.

Ca(2+)-depleted rod outer segments (ROS) were purified from bovine retinal rod photoreceptors, and factors influencing Ca2+ influx into ROS via the plasma membrane Na+/Ca2+/K+ exchanger were analyzed. Intracellular alkali cation concentrations were manipulated by 1) previous loading via the ionophore monensin followed by removal of monensin and 2) addition of the channel ionophore gramicidin during Ca(2+)-influx measurements. Ca2+ influx was measured as a rise in cytosolic free Ca2+ with the Ca(2+)-indicating dye fluo 3. An absolute requirement for intracellular Na+ was observed with a Na+ dissociation constant of 30-40 mM, whereas intracellular K+ was a potent inhibitor of Ca2+ influx, apparently by competing with Na+ for a common site on the Na+/Ca2+/K+ exchanger. Half-maximal Ca2+ influx was observed at an external free Ca2+ concentration of 0.9 microM when no external Na+ was present and 3.5 microM when 10 mM external Na+ was present. Our observations are discussed in the context of 1) a three-site model for the Na+/Ca2+/K+ exchanger and 2) earlier experiments on light adaptation in rods, which depended on minimizing Ca2+ fluxes across the ROS plasma membrane.

The glucose transporter in the plasma membrane of the outer segments of bovine retinal rods.

The facilitated diffusion glucose transporter in the plasma membrane of intact outer segments isolated from bovine retinal rods (ROS) was characterized by measurements of: (1) 14C-labeled 3-O-methylglucose fluxes; and (2) glucose-sensitive binding of 3H-labeled cytochalasin B to ROS membranes. Inhibition of 3-O-methylglucose influx into ROS, inhibition of 3-O-methylglucose efflux from ROS and glucose-sensitive binding of cytochalasin B to ROS showed very similar cytochalasin B inhibition/dissociation constants of 0.9 microM, 1.3 microM and 1.3 microM, respectively. D-glucose inhibited both 14C-labeled 3-O-methylglucose transport and cytochalasin B binding. The above results suggest that D-glucose-sensitive cytochalasin B binding reflects specific binding to the ROS glucose transporter and the density of glucose transporter in the ROS plasma membrane was determined to be 800 microns-2, comparable to relatively abundant ROS plasma membrane proteins such as the cGMP-gated channel and the Na-Ca+K exchanger. Displacement of 3H-labeled cytochalasin B by non-transportable hexoses was used to localize the hexose transporters to the ROS plasma membrane and to examine a simple single-site, alternating conformation model for hexose transport. A comparison between the Glut1 glucose transporters of bovine ROS, bovine erythrocytes and human erythrocytes suggests that kinetic and pharmacological characteristics of glucose transporters cannot be predicted in a simple manner from gene type and species.

Intracellular Ca2+ sequestration and release in intact bovine retinal rod outer segments. Role in inactivation of Na-Ca+K exchange.

Intracellular Ca2+ sequestration and Ca2+ release was analyzed in intact rod outer segments (ROS) purified from bovine retinas. Ca2+ influx in Ca(2+)-depleted and fully bleached ROS was mediated exclusively by the Na-Ca+K exchanger and was measured both as a rise in cytosolic free Ca2+ with fluo 3 and as a total transmembrane Ca2+ flux with 45Ca. Ca2+ fluxes across the ROS plasma membrane were not completely reversible, in small part due to inactivation of the Ca2+ extrusion mode of the Na-Ca+K exchanger but mostly due to sequestration of cytosolic Ca2+ into the intradiskal space. Ca2+ release from the intradiskal space into the cytosol could be induced by low concentrations of the Ca2+ ionophore A23187 which gave rise to increases in cytosolic free Ca2+ by several hundred nanomolar (low A23187 concentrations did not affect the Ca2+ permeability of the ROS plasma membrane). We have used intracellular Ca2+ release induced by A23187 to examine inactivation of the rapid Ca2+ efflux mode mediated by the plasma membrane Na-Ca+K exchanger. Inactivation of Na-Ca+K exchange appeared to be dependent on intradiskal Ca2+ as it was abolished by selective Ca2+ permeabilization of the disk membrane by A23187. We discuss possible physiological roles for Ca2+ sequestration and release from ROS disks.

Regulation of free cytosolic Ca2+ concentration in the outer segments of bovine retinal rods by Na-Ca-K exchange measured with fluo-3. I. Efficiency of transport and interactions between cations.

Regulation of free cytosolic Ca2+ concentration in the rod outer segments (ROS) isolated from bovine retinas was examined with the fluorescent Ca(2+)-indicating dye fluo-3. In situ calibration of cytosolic fluo-3 was done in the presence of the Ca2+ ionophore A23187 and yielded a dissociation constant of 500 nM for the Ca(2+)-fluo-3 complex. Ca2+ influx in Ca(2+)-depleted ROS was completely abolished when internal Na+ was removed suggesting that Ca2+ influx exclusively occurred via Na-Ca-K exchange. The most striking observation was that Na-Ca-K exchange could mediate a rapid increase in cytosolic free Ca2+ over the most of the usable indicating range of fluo-3 (from 10 nM to 2 microM), even when exposed to free external Ca2+ concentrations as low as 10 nM. From a comparison between changes in free Ca2+ and changes in total Ca2+, we conclude that physiologically occurring changes in cytosolic free Ca2+ are mediated by exchange fluxes less than 1% of the maximal Na-Ca-K exchange flux. The Na-Ca-K exchanger could mediate both K(+)-dependent and K(+)-independent Ca2+ influx; Li+ caused a complete inhibition of K(+)-independent Ca2+ influx, but had no effect on K(+)-dependent Ca2+ influx. We examined the complex interactions of alkali cations with Ca2+ influx and discuss the results in terms of a three-site model for the Na-Ca-K exchanger (Schnetkamp, P. P. M. and Szerencsei, R. T. (1991) J. Biol. Chem. 266, 189-197). Ca2+ competed with one Mg2+ ion or two Na+ ions for binding to a common site. High K+ concentration greatly diminished the ability of Na+ and Mg2+ to compete with Ca2+ for this common site on the exchanger protein. As a result, high internal K+ induced a conformation of the exchange protein that kinetically favoured Ca2+ extrusion.

Regulation of intracellular free Ca2+ concentration in the outer segments of bovine retinal rods by Na-Ca-K exchange measured with fluo-3. II. Thermodynamic competence of transmembrane Na+ and K+ gradients and inactivation of Na(+)-dependent Ca2+ extrusion.

Regulation of cytosolic free Ca2+ in the physiologically relevant submicromolar range was measured in isolated intact bovine rod outer segments (ROS) with the intracellular Ca(2+)-indicating dye fluo-3. Changes in free Ca2+ were compared with changes in total Ca2+ measured with 45Ca fluxes and a good qualitative correlation was observed. Ca2+ homeostasis in isolated bovine ROS was exclusively mediated via the Na-Ca-K exchanger. Free cytosolic Ca2+ concentration was lowered by an increase in the inward Na+ gradient, was raised by an increase in external K+, and was raised by depolarization of the plasma membrane. The simplest stoichiometry consistent with these qualitative observations is 4Na:(1Ca + 1K). The individual K:Ca, Na:Ca, and K:Na coupling ratios were deduced from quantitative changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ and K+ gradients. The observed changes in free Ca2+ did not agree with changes in free Ca2+ calculated on the basis of the above fixed stoichiometry which may reflect the flexibility in the Ca:K coupling ratio observed before in flux experiments (Schnetkamp, P. P. M., Szerencsei, R. T., and Basu, D. K. (1991) J. Biol. Chem. 266, 198-206). The most dramatic discrepancy was observed for the Na:Ca coupling ratio: the expected very large changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ gradient were not observed. Rapid Na(+)-induced Ca2+ extrusion was unable to lower cytosolic free Ca2+ below 100 nM, even under nonequilibrium conditions and despite the observation that Ca2+ influx via reverse Na-Ca-K exchange readily occurred at a free external Ca2+ concentration of 20 nM. We conclude that the Na(+)-dependent extrusion mode of the Na-Ca-K exchanger occurs in a brief (20-s) burst of high maximal velocity transport followed by a nearly complete inactivation of transport. The importance of our findings for Ca2+ homeostasis in functioning rod photoreceptors is discussed.

Glycolysis and glucose uptake in intact outer segments isolated from bovine retinal rods.

Glucose transport across the plasma membrane of isolated bovine rod outer segments (ROS) was measured by uptake of 14C-labeled 3-O-methylglucose and 2-deoxyglucose and was inferred from deenergization of ROS with 2-deoxyglucose. Glucose transport was mediated by a facilitated diffusion glucose transporter that equilibrated external and internal free hexose concentrations. Glucose transport in ROS displayed two components as judged from kinetic analysis of hexose equilibration and as judged from inhibition by cytochalasin B and phloretin. Transport under exchange conditions was considerably faster as compared with net hexose uptake, similar to that observed for the erythrocyte glucose transporter. Sensitivity to cytochalasin B and affinity to 3-O-methylglucose were similar to those observed for the hepatocyte glucose transporter. The cytochalasin-insensitive component appears unique to ROS and did not reflect leakage transport as judged from a comparison with L-glucose uptake. Glucose transport feeds glycolysis localized to ROS. We suggest that a major role for glycolysis in ROS is phosphorylation of GDP to GTP via pyruvate kinase and PEP, while phosphorylation of ADP to ATP can use the creatine kinase/phosphocreatine pathway as well.

Effect of potassium ions and membrane potential on the Na-Ca-K exchanger in isolated intact bovine rod outer segments.

Two recent studies reported that Na-Ca exchange in the outer segments of tiger salamander rod photoreceptors (Cervetto, L., Lagnado, L., Perry, R. J., Robinson, D. W., and McNaughton, P. A. (1989) Nature 337, 740-743) and of bovine rod photoreceptors (Schnetkamp, P. P. M., Basu, D. K., and Szerencsei, R. T. (1989) Am. J. Physiol. 257, C153-157) requires and transports K+ in a 4Na/(1Ca+1K) stoichiometry. In this study, we have examined the effects of K+ ions and membrane potential on the kinetics of Na-Ca and Ca-Ca exchange in rod outer segments isolated from bovine retinas. The objective was to establish the ion selectivity and voltage dependence of the different cation binding sites on the Na-Ca-K exchange protein. Potassium ions activated Na-Ca exchange when present on the Ca2+ side, although the extent of activation decreased with decreasing Na+ concentration. Potassium ions inhibited Na-Ca exchange when present on the Na+ side; inhibition arose from competition between Na+ and K+ for a common single cation-binding site. Activation of Na-Ca exchange by K+ displayed a different ion selectivity than that observed for inhibition of Na-Ca exchange by K+. The results are interpreted in terms of a three-site model for the rod Na-Ca-K exchanger. The rate of forward Na-Ca exchange decreased by 1.75-fold for a 60 mV depolarization of the plasma membrane but only at lower Na+ concentrations. The rate of Ca-Ca exchange was not affected by changes in membrane potential.

Unidirectional Na+, Ca2+, and K+ fluxes through the bovine rod outer segment Na-Ca-K exchanger.

The properties of the Na-Ca exchanger in the plasma membrane of rod outer segments isolated from bovine retinas (ROS) were studied. Unidirectional Ca2+, Na+, and K+ fluxes were measured with radioisotopes and atomic absorption spectroscopy. We measured K+ fluxes associated with the Ca-Ca self-exchange mode of the Na-Ca exchanger to corroborate our previous conclusion that the ROS Na-Ca exchanger differs from Na-Ca exchangers in other tissues by its ability to transport K+ (Schnetkamp, P. P. M., Basu, D. K. & Szerencsei, R. T. (1989) Am. J. Physiol. 257, C153-C157). The Na-Ca-K exchanger was the only functional cation transporter in the plasma membrane of bovine ROS with an upper limit of a flux of 10(5) cations/ROS/s or a current of 0.01 pA contributed by other cation channels, pumps, or carriers; cation fluxes via the Na-Ca-K exchanger amounted to 5 x 10(6) cations/ROS/s or a current of 1 pA. Ca2+ efflux via the forward mode of the Na-Ca-K exchanger did not operate with a fixed single stoichiometry. 1) The Na/Ca coupling ratio was increased from three to four when ionophores were added that could provide electrical compensation for the inward Na-Ca exchange current. 2) The K/Ca coupling ratio could vary by at least 2-fold as a function of the external Na+ and K+ concentration. The results are interpreted in terms of a model that can account for the variable Ca/K coupling ratio: we conclude that the Ca2+ site of the exchanger can translocate independent of translocation of the K+ site, whereas translocation of the K+ site requires occupation of the Ca2+ site, but not its translocation. The results are discussed with respect to the physiological role of Na-Ca-K exchange in rod photoreceptors.

The stoichiometry of Na-Ca+K exchange in rod outer segments isolated from bovine retinas.

Ca2+ extrusion in the outer segments of retinal rods (ROS) is mediated by a protein that couples both the inward Na+ gradient and the outward K+ gradient to Ca2+ extrusion. Na(+)-stimulated Ca2+ release from ROS requires internal K+ and is accompanied by release of internal K+, whereas a slow component of Na(+)-stimulated Ca2+ release does not require K+. In this paper we discuss our observations on the K+ transport via Na-Ca+ K exchange in bovine ROS, on the electrogenicity and stoichiometry of the ROS Na-Ca+ K exchanger, and on the mechanism on coupling Ca2+ to K+ via this protein. Finally, we discuss briefly the physiological implications of Na-Ca+ K exchange.