Kinetics, stoichiometry and role of the Na-Ca exchange mechanism in isolated cardiac myocytes

Compelling evidence has existed for more than a decade for a sodium/calcium (Na-Ca) exchange mechanism in the surface membrane of mammalian heart muscle cells which exchanges about three sodium ions for each calcium ion. Although it is known that cardiac muscle contraction is regulated by a transient increase in intracellular calcium ([Ca2+]i) triggered by the action potential, the contribution of the Na-Ca exchanger to the [Ca2+]i transient and to calcium extrusion during rest is unclear. To clarify these questions, changes in [Ca2+]i were measured with indo-1 in single cardiac myocytes which were voltage clamped and dialysed with a physiological level of sodium. We find that Ca entry through the Na-Ca exchanger is too slow to affect markedly the rate of rise of the normal [Ca2+]i transient. On repolarization, Ca extrusion by the exchanger causes [Ca2+]i to decline with a time constant of 0.5 s at -80 mV. The rate of decline can be slowed e-fold with a 77-mV depolarization. Calcium extrusion by the exchanger can account for about 15% of the rate of decline of the [Ca2+]i transient (the remainder being calcium resequestration by the sarcoplasmic reticulum (SR]. The ability of the cell to extrude calcium was greatly reduced on inhibiting the exchanger by removing external sodium, which itself led to an increase in resting [Ca2+]i. This finding is in contrast to the suggestion that calcium extrusion at rest is mediated mainly by a sarcolemmal Ca-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of Na-Ca exchange current in single cardiac myocytes

In cardiac muscle the exchange of intracellular Ca2+ for extracellular Na+ is an important transport mechanism for regulation of the intracellular free Ca2+ concentration [( Ca]i) and hence the contractile strength of the heart. Due to its stoichiometry of greater than or equal to 3:1 Na+/Ca2+ (refs 3,5), Na-Ca exchange is supposed to generate a current across the cell membrane. It is thought that such a current may contribute to cardiac action potential and physiological or pathological pacemaker activity. Although the occurrence of Na-Ca exchange is well documented, a membrane current generated by this transport has not been identified unequivocally. Previous attempts to detect such a current in multicellular preparations, for example, by measuring small current differences after varying the extracellular ionic composition, although providing evidence, did not rule out other possible interpretations. Here we demonstrate that a transient rise in [Ca]i caused by release of Ca from sarcoplasmic reticulum (SR) generates a membrane current in cardiac myocytes. The dependence of this current on the transmembrane gradients for Na+ and Ca2+ and on membrane potential meets the criteria for a current produced by electrogenic Na-Ca exchange. Cyclic activation of this current by release of Ca from the SR can cause maintained spontaneous activity, suggesting that Na-Ca exchange contributes to certain forms of cardiac pacemaking.     

Electrogenic Na-Ca exchange in retinal rod outer segment

Previous work has suggested that a Na-Ca exchanger may have a key role in visual transduction in retinal rods. This exchanger is thought to maintain a low internal free Ca2+ concentration in darkness and to contribute to the rod's recovery after light by removing any internally released Ca2+. Little else is known about this transport mechanism in rods. We describe here an inward membrane current recorded from single isolated rods which appears to be associated with such external Na+-dependent Ca2+ efflux activity. External Na+, but not Li+, could generate this current; high external K+ inhibited it while small amounts of La3+ (10 microM) completely abolished it. The exchanger can also transport Sr2+, but not Ba2+ or other divalent cations. The exchange ratio was estimated to be 3Na+:1Ca2+. As well as demonstrating clearly the Na-Ca exchanger in the rod outer segment, our experiments also cast serious doubt on the commonly held view that light simply releases internal Ca2+ to bind to and block the light-sensitive conductance.     

Identification of a secretory granule-binding protein as caldesmon

Stimulation of adrenal chromaffin cells results in a rise in the concentration of intracellular free calcium which initiates catecholamine secretion by exocytosis. An understanding of the molecular basis of exocytosis will require knowledge of the sites of action of calcium. A role for calmodulin has been implicated in secretion from chromaffin cells, and isolated granule membranes bind both calmodulin and a series of cytosolic proteins in a calcium-dependent fashion. Here, we demonstrate that one of the cytosolic granule-binding proteins with a relative molecular mass (Mr) of 70,000 (70K) is a form of the calmodulin-regulated actin-binding protein caldesmon, first isolated from smooth muscle. Cytoplasmic gels assembled from an adrenal medullary extract in the absence of Ca2+ contained actin and the 70K protein. The association of both of these proteins with the cytoplasmic gel was inhibited by a micromolar concentration of Ca2+. In addition, we have demonstrated that the 70K protein is localized at the periphery of chromaffin cells. These results are consistent with the notion that 70K protein (caldesmon) has a role in regulating the organization of actin filaments of the cell periphery during the secretory process.     

Na-Ca exchange current in mammalian heart cells

Electrogenic Na-Ca exchange has been known to act in the cardiac sarcolemma as a major mechanism for extruding Ca ions. Ionic flux measurements in cardiac vesicles have recently suggested that the exchange ratio is probably 3 Na:1 Ca, although a membrane current generated by such a process has not been isolated. Using the intracellular perfusion technique combined with the whole-cell voltage clamp, we were able to load Na+ inside and Ca2+ outside the single ventricular cells of the guinea pig and have succeeded in recording an outward Na-Ca exchange current while blocking most other membrane currents. The current is voltage-dependent, blocked by La3+ and does not develop in the absence of intracellular free Ca2+. This report presents the first direct measurement of the cardiac Na-Ca exchange current, and should facilitate the study of Ca2+ fluxes during cardiac activity, together with various electrical changes attributable to the Na-Ca exchange and the testing of proposed models.     

Light-induced reduction of cytoplasmic free calcium in retinal rod outer segment

The response of retinal rod photoreceptors to light consists of a membrane hyperpolarization resulting from the decrease of a light-sensitive conductance in the outer segment. According to the calcium hypothesis, this conductance is blocked by a rise in intracellular free Ca triggered by light, a notion supported by the findings that an induced rise in internal Ca leads to blockage of the light-sensitive conductance and that light triggers a net Ca efflux from the outer segment via a Na-Ca exchanger, suggesting a rise in internal free Ca in the light. We have now measured both Ca influx and efflux through the outer segment plasma membrane and find that, contrary to the calcium hypothesis, light seems to decrease rather than increase the free Ca concentration in the rod outer segment. This result implies that Ca does not mediate visual excitation but it probably has a role in light adaptation.     

Membrane depolarization evokes neurotransmitter release in the absence of calcium entry

The discovery that Ca2+ is necessary for the release of neurotransmitter, the primary means by which nerve cells communicate, led to the calcium hypothesis of neutransmitter release, in which release is initiated after an action potential only by an increase in intracellular Ca2+ concentration near the release sites and is terminated (1-2 ms) by the rapid removal of Ca2+. Since then, the calcium-voltage hypothesis has been proposed, in which the depolarization of the presynaptic terminals has two functions. First, in common with the calcium hypothesis, the Ca2+ conductance is increased, thereby permitting Ca2+ entry. Second, a conformational change is induced in a membrane molecule that renders it sensitive to Ca2+, and then binding of Ca2+ to this active form triggers release of neurotransmitter. When the membrane is repolarized, the molecule is inactivated and release is terminated, regardless of the local Ca2+ concentration at that moment. This hypothesis, in contrast to the calcium hypothesis, accounts for the insensitivity of the time course of release to experimental manipulations of intracellular Ca2+ concentration. Furthermore, it explains rapid termination of release after depolarization, even though Ca2+ concentration may still be high. Here we describe experiments that distinguish between these two hypotheses and find that our results support the calcium voltage hypothesis.     

CaT1 manifests the pore properties of the calcium-release-activated calcium channel

The calcium-release-activated Ca2+channel, ICRAC, is a highly Ca2+-selective ion channel that is activated on depletion of either intracellular Ca2+ levels or intracellular Ca2+ stores. The unique gating of ICRAC has made it a favourite target of investigation for new signal transduction mechanisms; however, without molecular identification of the channel protein, such studies have been inconclusive. Here we show that the protein CaT1 (ref. 4), which has six membrane-spanning domains, exhibits the unique biophysical properties of ICRAC when expressed in mammalian cells. Like ICRAC, expressed CaT1 protein is Ca2+ selective, activated by a reduction in intracellular Ca2+ concentration, and inactivated by higher intracellular concentrations of Ca2+. The channel is indistinguishable from ICRAC in the following features: sequence of selectivity to divalent cations; an anomalous mole fraction effect; whole-cell current kinetics; block by lanthanum; loss of selectivity in the absence of divalent cations; and single-channel conductance to Na+ in divalent-ion-free conditions. CaT1 is activated by both passive and active depletion of calcium stores. We propose that CaT1 comprises all or part of the ICRAC pore.     

Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca(2+)-permeable channel.

Receptor-mediated increases in the cytosolic free calcium ion concentration in most mammalian cells result from mobilization of Ca2+ from intracellular stores as well as transmembrane Ca2+ influx. Inositol 1,4,5-trisphosphate (InsP3) releases calcium from intracellular stores by opening a Ca(2+)-permeable channel in the endoplasmic reticulum. But the mechanism and regulation of Ca2+ entry into nonexcitable cells has remained elusive because the entry pathway has not been defined. Here we characterize a novel inositol 1,3,4,5-tetrakisphosphate (InsP4) and Ca(2+)-sensitive Ca(2+)-permeable channel in endothelial cells. We find that InsP4, which induces Ca2+ influx into acinar cells, enhances the activity of the Ca(2+)-permeable channel when exposed to the intracellular surface of endothelial cell inside-out patches. Our results suggest a molecular mechanism which is likely to be important for receptor-mediated Ca2+ entry.     

Distribution of two distinct Ca2+-ATPase-like proteins and their relationships to the agonist-sensitive calcium store in adrenal chromaffin cells

Many cellular functions are regulated by activation of cell-surface receptors that mobilize calcium from internal stores sensitive to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The nature of these internal calcium stores and their localization in cells is not clear and has been a subject of debate. It was originally suggested that the Ins(1,4,5)P3-sensitive store is the endoplasmic reticulum, but a new organelle, the calciosome, identified by its possession of the calcium-binding protein, calsequestrin, and a Ca2+-ATPase-like protein of relative molecular mass 100,000 (100K), has been described as a potential Ins(1,4,5)P3-sensitive calcium store. Direct evidence on whether the calciosome is the Ins(1,4,5)P3-sensitive store is lacking. Using monoclonal antibodies raised against the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, we show that bovine adrenal chromaffin cells contain two Ca2+-ATPase-like proteins with distinct subcellular distributions. A 100K Ca2+-ATPase-like protein is diffusely distributed, whereas a 140K Ca2+-ATPase-like protein is restricted to a region in close proximity to the nucleus. In addition, Ins(1,4,5)P3-generating agonists result in a highly localized rise in cytosolic calcium concentration ([Ca2+]i) initiated in a region close to the nucleus, whereas caffeine results in a rise in [Ca2+]i throughout the cytoplasm. Our results indicate that chromaffin cells possess two calcium stores with distinct Ca2+-ATPases and that the organelle with the 100K Ca2+-ATPase is not the Ins(1,4,5)P3-sensitive store.     

Recruitment of cytosolic proteins to a secretory granule membrane depends on Ca2+-calmodulin

An increase in free calcium triggers catecholamine secretion from chromaffin cells and calmodulin is strongly implicated as the intracellular Ca2+ receptor. In our recent studies of calmodulin action in the chromaffin cell, micromolar Ca2+ concentrations resulted in calmodulin and cytosolic proteins becoming bound to the chromaffin granule membranes. We now report that calmodulin is bound with high affinity to granule membrane proteins of molecular weights (Mrs) 25,000 and 22,000 (25K and 22K) at low Ca2+ (less than 10(-8) M) and to proteins with Mrs 69K and 50K at high Ca2+ (greater than 1 microM). Other cytosolic components (Mrs 70K, 36K, 34K and 32K) require calmodulin for their interfraction with membrane. These proteins separately bound to calmodulin-Sepharose at high Ca2+ concentrations. Although the functions of these adrenal proteins have not been established, the 34K and 32K Mr components co-migrate with clathrin light chains isolated from medullary coated vesicles and the Mr 34K components from both sources share the same one-dimensional peptide map. These interactions were observed at micromolar Ca2+ levels at 'intracellular' conditions of pH and ionic strength and would be expected to occur during secretion from the chromaffin cell.     

Inhibition of mitosis by an antibody to the mitotic calcium transport system

Calcium ions are important in the regulation of mitotic apparatus assembly and in the control of chromosome movement. Changes in intracellular free calcium concentration, [Ca2+]i are achieved by an intracellular calcium-transport system which is highly conserved in different cell types. A membrane-bound protein of relative molecular mass (Mr) 46,000 (46K) is part of this transport system and has been implicated in the regulation of the [Ca2+]i changes associated with the course of mitosis. A monoclonal antibody against this 46K protein inhibits Ca2+-uptake into isolated Ca2+-sequestering membranes and specifically labels membranes associated with the mitotic apparatus of sea urchin embryos. Here we investigate the relationship between the intracellular calcium transport system and mitosis by injection of this monoclonal antibody into living mitotic sea urchin embryos. We find that after injection the intracellular free calcium increases up to 10(-6) M, the mitotic apparatus is rapidly destroyed and the cell is irreversibly blocked in its development.     

Heterotrimeric G protein participated in modulation of cytoplasmic calcium concentration in pollen cells

Cytoplasmic free calcium concentration([Ca2+]c) in pollen cells of Lilium daviddi is measured with confocal laser scanning microscopy to investigate the effect of heterotrimeric G protein (G protein) on [Ca2+]c and the possible signal transduction pathway of G protein triggering cellular calcium signal. After application, cholera toxin (CTX), an agonist of G protein, triggers a transient increase of [Ca2+]c in pollen cells, and evokes a spatial-temporal characteristic calcium dynamics; while pertussis toxin (PTX), a G protein antagonist, leads to the decrease of [Ca2+]c. Both L-type Ca2+ channel blocker verapamil and inhibitor of IP3 receptor heparin inhibit CTX-induced [Ca2+]c increase. The results show that G protein may play a role in the modulation of [Ca2+]c through enhancing the extracellular Ca2+ influx and releasing of Ca2+ from intracellular stores.     

Study of transmembrane La3+ movement in rat ventricular myocytes by the patch-clamp technique

We have studied transmembrane La3+ movement in rat ventricular myocytes for the first time by using the whole-cell patch-clamp recording mode. La3+ (0.01-5.0 mmol/L) could not bring out inward currents through the L-type calcium channel in rat ventricular myocytes, while it could enter the cells by the same way carried by 1μmol/L ionomycin. When the outward Na+ concentration gradient is formed, La3+ can enter the cells via Na-Ca exchange, and the exchange currentsincrease with the increase of external La3+ concentrations. But compared with Na-Ca exchange currents in the same concentration, the former is only 14%-38% of the latter. The patch-clamp experiment indicates that La3+ normally can not enter ventricular myocytes through L-type calcium channel, but it can enter the cells via Na-Ca exchange.     

Exo1 and Exo2 proteins stimulate calcium-dependent exocytosis in permeabilized adrenal chromaffin cells.

In many cell types an increase in cytosolic calcium is the main signal for the exocytotic release of stored secretory components such as hormones and neurotransmitters. The site of action of calcium in exocytosis is not known, neither are the participating molecules. In the case of the intracellular membrane fusions that occur during transport through early stages of the secretory pathway, several cytosolic and peripheral membrane proteins are necessary. Permeabilized cells have been useful in understanding the requirements for calcium and nucleotides in regulated exocytosis and under certain conditions there is leakage of soluble protein components and run-down of the exocytotic response. This system can be used to identify the soluble proteins involved in exocytosis, one candidate in chromaffin cells being annexin II (calpactin). Here we use this assay to identify two other cytosolic protein factors that regulate exocytosis in permeabilized adrenal chromaffin cells, which we term Exo1 and Exo2. Exo1 from brain cytosol resolves on electrophoresis in SDS-polyacrylamide gels as a group of polypeptides of relative molecular mass approximately 30,000 and shares sequence homology with the 14-3-3 family of proteins. The ability of Exo1 to reactivate exocytosis is potentiated by protein kinase C activation and therefore Exo1 may influence the protein kinase C-mediated control of Ca(2+)-dependent exocytosis.     

Role for microsomal Ca storage in mammalian neurones?

Alterations in the intracellular concentration of calcium ions [( Ca2+]i) are increasingly being found to be associated with regulatory functions in cells of all kinds. In muscle, an elevation of [Ca2+]i is the final link in excitation-contraction coupling while at nerve endings and in secretory cells, similar rises in [Ca2+]i are thought to mediate exocytosis. The discovery of calcium-activated ion channels indicated a role for intracellular calcium in the regulation of membrane excitability. Calcium transients associated with either intracellular release or the inward movement of Ca2+ across the membrane have been recorded in molluscan neurons and more recently in neurones of bullfrog sympathetic ganglia. Here, we report the first recordings of calcium transients in single mammalian neurones. In these experiments we have found that the methylxanthine, caffeine, causes the release of calcium from a labile intracellular store which can be refilled by Ca2+ entering the cell during action potentials.     

Activation of Ca2+ entry into acinar cells by a non-phosphorylatable inositol trisphosphate.

In many cell types, receptor activation of phosphoinositidase C results in an initial release of intracellular Ca2+ stores followed by sustained Ca2+ entry across the plasma membrane. Inositol 1,4,5-trisphosphate is the mediator of the initial Ca2+ release, although its role in the mechanism underlying Ca2+ entry remains controversial. We have now used two techniques to introduce inositol phosphates into mouse lacrimal acinar cells and measure their effects on Ca2+ entry: microinjection into cells loaded with Fura-2, a fluorescent dye which allows the measurement of intracellular free calcium concentration by microspectrofluorimetry, and perfusion of patch clamp pipettes in the whole-cell configuration while monitoring the activity of Ca(2+)-activated K+ channels as an indicator of intracellular Ca2+. We report here that inositol 1,4,5-trisphosphate serves as a signal that is both necessary and sufficient for receptor activation of Ca2+ entry across the plasma membrane in these cells.     

Augmentation of cardiac calcium current by flash photolysis of intracellular caged-Ca2+ molecules

The entry of calcium ions into cells through voltage-activated Ca2+ channels in the plasma membrane triggers many important cellular processes. The activity of these channels is regulated by several hormones and neurotransmitters, as well as intracellular messengers such as Ca2+ itself (for examples, see refs 1-9). In cardiac muscle, myoplasmic Ca2+ has been proposed to potentiate Ca2+ influx, although a direct effect of Ca2+ on these channels has not yet been demonstrated. Photosensitive 'caged-Ca2+' molecules such as nitr-5, however, provide powerful tools for investigating possible regulatory roles of Ca2+ on the functioning of Ca2+ channels. Because its affinity for Ca2+ is reduced by irradiation, nitr-5 can be loaded into cells and induced to release Ca2+ with a flash of light. By using this technique we found that the elevation of intracellular Ca2+ concentration directly augmented Ca2+-channel currents in isolated cardiac muscle cells from both frog and guinea pig. The time course of the current potentiation was similar to that seen with beta-adrenergic stimulation. Thus Ca2+ may work through a similar pathway, involving phosphorylation of a regulatory Ca2+-channel protein. This mechanism is probably important for the accumulation of Ca2+ and the amplification of the contractile response in cardiac muscle, and may have a role in other excitable cells.     

Depletion of intracellular calcium stores activates a calcium current in mast cells.

In many cell types, receptor-mediated Ca2+ release from internal stores is followed by Ca2+ influx across the plasma membrane. The sustained entry of Ca2+ is thought to result partly from the depletion of intracellular Ca2+ pools. Most investigations have characterized Ca2+ influx indirectly by measuring Ca(2+)-activated currents or using Fura-2 quenching by Mn2+, which in some cells enters the cells by the same influx pathway. But only a few studies have investigated this Ca2+ entry pathway more directly. We have combined patch-clamp and Fura-2 measurements to monitor membrane currents in mast cells under conditions where intracellular Ca2+ stores were emptied by either inositol 1,4,5-trisphosphate, ionomycin, or excess of the Ca2+ chelator EGTA. The depletion of Ca2+ pools by these independent mechanisms commonly induced activation of a sustained calcium inward current that was highly selective for Ca2+ ions over Ba2+, Sr2+ and Mn2+. This Ca2+ current, which we term ICRAC (calcium release-activated calcium), is not voltage-activated and shows a characteristic inward rectification. It may be the mechanism by which electrically nonexcitable cells maintain raised intracellular Ca2+ concentrations and replenish their empty Ca2+ stores after receptor stimulation.     

Calcium transients in aequorin-injected frog cardiac muscle.

The Ca2+ -sensitive bioluminescent protein aequorin was microinjected into cells of frog atrial trabeculae to study intracellular calcium transients associated with excitation-contraction coupling. The amplitude of the aequorin signal increased with extracellular Ca2+ concentration and stimulus frequency, but decreased with stretch. Isoprenaline and acetylstrophanthidin both increased the amplitude, but had strikingly different effects on the time course of the signal.