Ca2+-dependent stimulation of 3-O-methylglucose transport in mouse fibroblast Swiss 3T3 cells induced by phorbol-12,13-dibutyrate

Binding of phorbol-12,13-dibutyrate (PDBu), a tumor promoter, to quiescent Swiss 3T3 cells increased the number of hexose carriers, resulting in stimulation of membrane transport of 3-O-methylglucose (3MeGlu) in a Ca2+-dependent fashion. Extracellular Ca2+ was necessary to initiate the binding of PDBu to its receptor, and intracellular Ca2+ was required to maintain it. The loss of PDBu-binding, caused by elimination of Ca2+, was accompanied by a loss of stimulation of hexose transport. These results indicated that Ca2+-dependent, continuous binding of PDBu to its receptor was essential to induce the stimulation of hexose transport.     

Alcohol stimulates Na+/Ca2+ exchange in brain mitochondria

Ethanol, at low concentrations, specifically stimulates the Na(+)-dependent Ca2(+)-efflux in brain mitochondria. In addition, at higher concentrations, ethanol inhibits the Na(+)-independent Ca2(+)-efflux. The electrogenic Ca(+)-uptake system is not affected by ethanol. The specific stimulation of Na+/Ca2+ exchange reaches a maximum of 60% stimulation, with half-maximal stimulation at 130 mM ethanol. The inhibition of the Na(+)-independent efflux is proportional to the ethanol concentration, becoming significant only above 200 mM, with 50% inhibition at 0.5 M. The inhibition of the Na(+)-independent efflux is, in large part, due to an inhibition of the activation of the Cyclosporin-sensitive pore. Long-term ethanol-feeding had no effect on the Ca2+ transport systems and their sensitivity to acute ethanol treatment. It is suggested that the stimulation of the Na(+)-dependent Ca2(+)-efflux, which is the dominant Ca2+ efflux pathway in brain mitochondria, contributes to the intoxicating effects of ethanol.     

Ca2+ ions and the stimulation of 3-O-methylglucose transport by uncouplers in rat thymocytes.

The uptake of 3-O-methylglucose by rat thymocytes follows a biphasic time course. 2,4-Dinitrophenol (10-3 M), carbonyl cyanide m-chlorophenylhydrazone (10-5 M) and oligomycin (5 microgram/ml) each reduce ATP levels in rat thymocytes by 85% and bring about 3- to 4-fold stimulation of the slow phase of 3-O-methylglucose uptake. No consistent effect is observed on either the half-time of the rapid phase of uptake or the relative proportions of the two phases of uptake in the presence of these agents. Ca2+ ions do not appear to play a necessary role in the stimulation of transport activity since cells depleted of exchangeable Ca2+ by treatment with the Ca2+-Mg2+ ionophore, A23187, plus [ethylenebis(oxyethylenenitrilo)]tetraacetic acid respond to uncouplers in exactly the same manner as untreated cells. The effect of dinitrophenol on the slow phase of 3-O-methylglucose uptake is reversible after 10 min of incubation. After 60 min however, cells washed free of dinitrophenol and incubated at 37 degrees exhibited an additional acceleration in transport activity. This stimulation of transport is characterized by an increase in the proportion of the rapid phase of uptake with little or no change in its half-time. The results suggest that rat thymocytes regulate their 3-O-methylglucose transport activity in two distinct fashions.     

A mechanism of passive transport of Ca2+ in muscle sarcoplasmic reticulum

Basing on the data available in literature and authors' investigations the mechanism of local alkalization of the myoplasm by proton efflux attended by Ca2+ influx is mic reticulum and may be the main link in the process of electrochemical coupling in the skeletal and cardiac muscle cells. Experimental evidence for participation of Ca2(+)-ATPase in the passive transport of calcium through sarcoplasmic membrane is given.     

Stimulation of ATP-driven Ca2+ pump in the basal-lateral plasma membranes of kidney cortex during compensatory renal growth

During compensatory renal growth 45Ca2+ transport in basal-lateral plasma membrane vesicles isolated from the rat renal cortex have been investigated. Stimulation of Ca2(+)-ATPase activity was observed, without an effect of compensatory renal growth on Na+/Ca2+ exchanger activity and on passive Ca2+ permeability of the vesicles. Twelve hours following unilateral nephrectomy about 40% increase of Ca2(+)-ATPase activity above control value was observed and this effect was present until the end of the experimental period (7 days). When kinetic parameters for Ca2(+)-ATPase were studied in native membranes, an increase of Vmax was observed, whereas the Km for Ca2+ was similar in control vesicles and vesicles isolated from the remnant kidney. Depletion of endogenous calmodulin resulted in a decrease of Vmax and an increase of Km (Ca2+), while its addition reversed these parameters and increased the Hill coefficient from about 1 to about 2. Once again, only a significant increase of Vmax in vesicles isolated from the remnant kidney above the control value was observed. Finally, increase of Ca2(+)-ATPase activity during compensatory renal growth could be abolished by actinomycin D, indicating that its stimulation is due to protein synthesis.     

Thyroid hormone markedly increases the mRNA coding for sarcoplasmic reticulum Ca2+-ATPase in the rat heart

Previous findings have shown that thyroid hormone markedly increases the speed of diastolic relaxation in the heart. This thyroid hormone-dependent change is also accompanied by an increased Ca2+ pumping ability in the sarcoplasmic reticulum. In an effort to determine the underlying cause of improved Ca2+ transport, mRNA levels of the slow Ca2+-ATPase of the sarcoplasmic reticulum were quantified on Northern blots. In hypothyroid rat hearts, the steady state level of Ca2+-ATPase mRNA was only 36% of control levels, whereas hyperthyroid rat heart mRNA levels were 136% of control. Ca2+-ATPase mRNA responded rapidly to T3, as the mRNA level was significantly increased by 2 h and normalized by 5 h after T3 injection into hypothyroid rats. The well established effect of thyroid hormone on improved myocardial contractility and increased speed of diastolic relaxation may in part relate to specific alterations in the level of the mRNA coding for Ca2+-ATPase, resulting in increased pump units.     

Fast efflux of Ca2+ mediated by the sarcoplasmic reticulum Ca2(+)-ATPase.

The Ca2(+)-ATPase found in the light fraction of sarcoplasmic reticulum vesicles can be phosphorylated by Pi, forming an acylphosphate residue at the catalytic site of the enzyme. This reaction was inhibited by the phenothiazines trifluoperazine, chlorpromazine, imipramine, and fluphenazine and by the beta-adrenergic blocking agents propranolol and alprenolol. The inhibition was reversed by raising either the Pi or the Mg2+ concentration in the medium and was not affected by the presence of K+. Phosphorylation of the Ca2(+)-ATPase by Pi was also inhibited by ruthenium red and spermidine. These compounds compete with Mg2+, but, unlike the phenothiazines, they did not compete with Pi at the catalytic site, and the inhibition was abolished when K+ was included in the assay medium. The efflux of Ca2+ from loaded vesicles was greatly increased by the phenothiazines and by propranolol and alprenolol. In the presence of 200 microM trifluoperazine, the rate of Ca2+ efflux was higher than 3 mumol of Ca2+/mg of protein/10 s. The activation of efflux by these drugs was antagonized by Pi, Mg2+, K+, Ca2+, ADP, dimethyl sulfoxide, ruthenium red, and spermidine. The increase of Ca2+ efflux caused by trifluoperazine was not correlated with binding of the drug to the membrane lipids. It is concluded that the Ca2+ pump can be uncoupled by different drugs, thereby greatly increasing the efflux of Ca2+ through the ATPase. Displacement of these drugs by the natural ligands of the ATPase blocks the efflux through the uncoupled pathway and limits it to a much smaller rate. Thus, the Ca2(+)-ATPase can operate either as a pump (coupled) or as a Ca2+ channel (uncoupled).     

Ca2+-mediated activation of human erythrocyte membrane Ca2+-ATPase

Ca2+-ATPase of human erythrocyte membranes, after being washed to remove Ca2+ after incubation with the ion, was found to be activated. Stimulation of the ATPase was related neither to fluidity change nor to cytoskeletal degradation of the membranes mediated by Ca2+. Activation of the transport enzyme was also unaffected by detergent treatment of the membrane, but was suppressed when leupeptin was included during incubation of the membranes with Ca2+. Stimulation of the ATPase by a membrane-associated Ca2+-dependent proteinase was thus suggested. Much less 138 kDa Ca2+-ATPase protein could be harvested from a Triton extract of membranes incubated with Ca2+ than without Ca2+. Activity of the activated enzyme could not be further elevated by exogenous calpain, even after treatment of the membranes with glycodeoxycholate. There was also an overlap in the effect of calmodulin and the Ca2+-mediated stimulation of membrane Ca2+-ATPase. While Km(ATP) of the stimulated ATPase remained unchanged, a significant drop in the free-Ca2+ concentration for half-maximal activation of the enzyme was observed.     

The Na(+)-independent Ca2+ efflux mechanism of liver mitochondria is not a passive Ca2+/2H+ exchanger

Whether the Na(2+)-independent Ca2+ efflux mechanism of liver mitochondria is a Ca2+/2H+ exchanger and whether this exchanger is a passive mechanism have been controversial since shortly after the discovery of this mechanism. Here, a new approach to determining if the mechanism is passive is developed based on the energy available to a passive Ca2+/2H+ exchanger. Conditions are identified in which the Na(+)-independent Ca2+ efflux mechanism transports Ca2+ out of mitochondria against a Ca2+ gradient many times greater than that possible for a passive Ca2+/2H+ exchanger, thus ruling this out as a possible mechanism.     

Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line

Addition of either vasoactive intestinal peptide (VIP) or the Ca2+ ionophore, A23187, to confluent monolayers of the T84 epithelial cell line derived from a human colon carcinoma increased the rate of 86Rb+ or 42K+ efflux from preloaded cells. Stimulation of the rate of efflux by VIP and A23187 still occurred in the presence of ouabain and bumetanide, inhibitors of the Na+,K+-ATPase and Na+,K+,Cl- cotransport, respectively. The effect of A23187 required extracellular Ca2+, while that of VIP correlated with its known effect on cyclic AMP production. Other agents which increased cyclic AMP production or mimicked its effect also increased 86Rb+ efflux. VIP- or A23187-stimulated efflux was inhibited by 5 mM Ba2+ or 1 mM quinidine, but not by 20 mM tetraethylammonium, 4 mM 4-aminopyridine, or 1 microM apamin. Under appropriate conditions, VIP and A23187 also increased the rate of 86Rb+ or 42K+ uptake. Stimulation of the initial rate of uptake by either agent required high intracellular K+ and was not markedly affected by the imposition of transcellular pH gradients. The effect of A23187, but not VIP or dibutyryl cyclic AMP, was refractory to depletion of cellular energy stores. A23187-stimulated uptake was not significantly affected by anion substitution, however, stimulation of uptake by VIP required the presence of a permeant anion. This result may be due to the simultaneous activation of a cyclic AMP-dependent Cl- transport system. The kinetics of both VIP- and A23187-stimulated uptake and efflux were consistent with a channel-rather than a carrier-mediated K+ transport mechanism. The results also suggest that cyclic AMP and Ca2+ may activate two different kinds of K+ transport systems. Finally, both transport systems have been localized to the basolateral membrane of T84 monolayers, a result compatible with their possible regulatory role in hormone-activated electrogenic Cl- secretion.     

Anomalous regulation of the Drosophila Na(+)-Ca2+ exchanger by Ca2+

The Na(+)-Ca2+ exchanger from Drosophila was expressed in Xenopus and characterized electrophysiologically using the giant excised patch technique. This protein, termed Calx, shares 49% amino acid identity to the canine cardiac Na(+)-Ca2+ exchanger, NCX1. Calx exhibits properties similar to previously characterized Na(+)-Ca2+ exchangers including intracellular Na+ affinities, current-voltage relationships, and sensitivity to the peptide inhibitor, XIP. However, the Drosophila Na(+)-Ca2+ exchanger shows a completely opposite response to cytoplasmic Ca2+. Previously cloned Na(+)-Ca2+ exchangers (NCX1 and NCX2) are stimulated by cytoplasmic Ca2+ in the micromolar range (0.1- 10 microM). This stimulation of exchange current is mediated by occupancy of a regulatory Ca2+ binding site separate from the Ca2+ transport site. In contrast, Calx is inhibited by cytoplasmic Ca2+ over this same concentration range. The inhibition of exchange current is evident for both forward and reverse modes of transport. The characteristics of the inhibition are consistent with the binding of Ca2+ at a regulatory site distinct from the transport site. These data provide a rational basis for subsequent structure-function studies targeting the intracellular Ca2+ regulatory mechanism.     

Effects of platelet-derived growth factor on Ca2+ in 3T3 cells

The effect of platelet-derived growth factor (PDGF) on cellular Ca2+ was examined in BALB/c-3T3 cells. PDGF induced: A decrease in cell 45Ca2+ content. An apparent increased rate of efflux of preloaded 45Ca2+. A decrease in residual intracellular 45Ca2+ remaining after rapid efflux. When added after the rapid phase of efflux of 45Ca2+ had occurred, an immediate decrease in post-efflux residual intracellular 45Ca2+. All of the observed changes in 45Ca2+ induced by PDGF are consistent with a rapid release of Ca2+ from an intracellular Ca2+ pool that has the slowest efflux and is relatively inaccessible to extracellular EDTA. When incubated with chlortetracycline (CTC), a fluorescent Ca2+ probe, 3T3 cell mitochondria became intensely fluorescent. Addition of PDGF resulted in a rapid decrease in CTC fluorescence intensity in both adherent and suspended 3T3 cells. The effects of PDGF on 3T3 cell Ca2+ stores and CTC fluorescence intensity were identical with the effects of the Ca2+ ionophore A23187 and of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone. Serum, which contains PDGF, also altered intracellular Ca2+ stores, but platelet-poor plasma, which does not contain PDGF, had no effect. EGF, insulin, and tetradecanoyl phorbol acetate (TPA), other factors which stimulate 3T3 cell growth, did not alter 3T3 cell Ca2+ stores. Release of Ca2+ from intracellular sequestration sites may be a mechanism by which PDGF stimulates cell growth.     

Effects of heavy metal on rat liver microsomal Ca2(+)-ATPase and Ca2+ sequestering. Relation to SH groups.

In isolated hepatic microsomal vesicles the heavy metals Cd2+, Cu2+, and Zn2+ inhibit Ca2+ uptake and evoke a prompt efflux of Ca2+ from preloaded vesicles in a dose-dependent manner. N-Ethylmaleimide also inhibits Ca2+ uptake and causes Ca2+ release, but it is less effective in these respects than the heavy metals. Measurement of mannose-6-phosphatase activity indicate that the heavy metal-induced Ca2+ efflux is not caused by a general increase in membrane permeability. Heavy metals also inhibit the Ca2(+)-ATPase activity and the formation of the phosphorylated intermediate of the enzyme. In contrast, the sulfhydryl modifying reagent, N-ethylmaleimide inhibits the Ca2(+)-ATPase activity while it has a relatively small effect on Ca2+ release. Thus, the effects of these agents on Ca2+ sequestering and Ca2(+)-ATPase activity are not strictly proportional. The sulfhydryl group reducing agent dithiothreitol protects the microsomes from the effects of heavy metals, while glutathione is less protective. Addition of vanadate to vesicles, at a concentration which completely blocked the activity of the Ca2(+)-ATPase, resulted in a small and slow release of the accumulated Ca2+. Subsequent additions of heavy metals evoked a massive Ca2+ release. Thus, the effects of heavy metals on Ca2+ efflux cannot be due entirely to their inhibition of the Ca2+ pump. The heavy metal-induced Ca2+ efflux is not inhibited either by ruthenium red or tetracaine.     

Tributyltin increases cytosolic free Ca2+ concentration in thymocytes by mobilizing intracellular Ca2+, activating a Ca2+ entry pathway, and inhibiting Ca2+ efflux.

The immunotoxic environmental pollutant tri-n-butyltin (TBT) kills thymocytes by apoptosis through a mechanism that requires an increase in intracellular Ca2+ concentration. The addition of TBT (EC50 = 2 microM) to fura-2-loaded rat thymocytes resulted in a rapid and sustained increase in the cytosolic free Ca2+ concentration ([Ca2+]i) to greater than 1 microM. In nominally Ca(2+)-free medium, TBT slightly but consistently increased thymocyte [Ca2+]i by about 0.11 microM. The subsequent restoration of CaCl2 to the medium resulted in a sustained overshoot in [Ca2+]i; similarly, the addition of MnCl2 produced a rapid decrease in the intracellular fura-2 fluorescence in thymocytes exposed to TBT. The rates of Ca2+ and Mn2+ entry stimulated by TBT were essentially identical to the rates stimulated by 2,5-di-(tert.-butyl)-1,4-benzohydroquinone (tBuBHQ), which has previously been shown to empty the agonist-sensitive endoplasmic reticular Ca2+ store and to stimulate subsequent Ca2+ influx by a capacitative mechanism. The addition of excess [ethylenebis(oxyethylenenitrilo)]tetraacetic acid to thymocytes produced a rapid return to basal [Ca2+]i after tBuBHQ treatment but a similar rapid return to basal [Ca2+]i was not observed after TBT treatment. In addition, TBT produced a marked inhibition of both Ca2+ efflux from the cells and the plasma membrane Ca(2+)-ATPase activity. Also, TBT treatment resulted in a rapid decrease in thymocyte ATP level. Taken together, our results show that TBT increases [Ca2+]i in thymocytes by the combination of intracellular Ca2+ mobilization, stimulation of Ca2+ entry, and inhibition of the Ca2+ efflux process. Furthermore, the ability of TBT to apparently mobilize the tBuBHQ-sensitive intracellular Ca2+ store followed by Ca2+ and Mn2+ entry suggests that the TBT-induced [Ca2+]i increase involves a capacitative type of Ca2+ entry.     

Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium.

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.     

Calcium homeostasis and transport are affected by disruption of cta3, a novel gene encoding Ca2(+)-ATPase in Schizosaccharomyces pombe

A new P-type ATPase gene, cta3, has been identified in Schizosaccharomyces pombe. The deduced amino acid sequence presents a 45% identity with the Saccharomyces cerevisiae putative Ca2(+)-ATPase encoded by the PMR2 gene. The cta3 protein contains 7 out of the 8 amino acid residues involved in high affinity Ca2+ binding in the sarcoplasmic reticulum Ca2(+)-ATPase from muscles. It also contains a region similar to the phospholamban-binding domain that characterizes this Ca2+ pump. A null mutation of cta3 leads to higher levels of cytosolic free Ca2+ and to lower amounts of sequestered and bound Ca2+. Cellular Ca2+ efflux and rates of uptake into intracellular compartments are reduced by the loss of cta3 function. The sequence analysis and the physiological results strongly support the conclusion that the cta3 gene encodes a Ca2(+)-ATPase, probably located in intracellular membranes.     

Functional evidence of a transmembrane channel within the Ca2+ transport ATPase of sarcoplasmic reticulum.

Ca2+ efflux can be studied conveniently following dilution of sarcoplasmic reticulum (SR) vesicles preloaded with 45Ca2+ by active transport. The rates of efflux are highly dependent on ATPase substrates and cofactors (Pi, Mg2+, Ca2+ and ADP) in the efflux medium. On the other hand, phenothiazines stimulate efflux through a passive permeability channel with no coupled catalytic events. Efflux activation by manipulation of catalytically active ATPase ligands, as well as by the catalytically inactive phenothiazines, can be prevented by thapsigargin, which is a highly specific inhibitor of the Ca(2+)-ATPase. This demonstrates that the passive channel activated by phenothiazines is an integral part of the ATPase, and can operate either uncoupled or coupled to catalytic events.     

Limited tryptic modification stimulates activation of Ca2+ release from isolated sarcoplasmic reticulum vesicles

Tryptic modification appears to potentiate activation of the Ca2+ channels of isolated sarcoplasmic reticulum vesicles. In the presence of 1 mM free Mg2+ we observe that: 1) cAMP and doxorubicin activation of passive efflux from tryptically modified vesicles is approximately 20-fold greater than from native SR. 2) Ruthenium red inhibits Ca2+ efflux from modified vesicles. 3) The binding affinities and Hill coefficients of activation of efflux by cAMP and doxorubicin are the same in modified vesicles as in native vesicles. 4) Proteolysis stimulates passive efflux from heavy SR much more than from light SR. 5) Stimulation of cAMP- and doxorubicin-activated Ca2+ release is biphasic, whereas Hg2+-activated Ca2+ efflux is monophasic. 6) In the absence of Mg2+, the Ca2+ dependence of cAMP-activated efflux from tryptically modified vesicles is similar to that of native vesicles, with peak efflux rates occurring between approximately 1 and 10 microM Ca2+. 7) The Mg2+ dependence of efflux from modified vesicles is similar to that of native vesicles. 8) SDS-polyacrylamide gels indicate that the Ca2+, Mg2+-ATPase and the high molecular weight ryanodine receptor are both cleaved faster than the stimulation of efflux.     

Ca2(+)-induced conformational changes and location of Ca2+ transport sites in sarcoplasmic reticulum Ca2(+)-ATPase as detected by the use of proteolytic enzyme (V8)

Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureus produced appreciable amounts of a Ca2(+)-ATPase fragment (p85) in the presence of Ca2+ (E1 conformation of the enzyme), along with many other peptide fragments that were also formed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (E2 conformation). p85 was formed as a carboxyl-terminal cleavage product of Ca2(+)-ATPase by a split of the peptide bond between Glu-231 and Ile-232. Other conformation-dependent V8 splits were localized to the "hinge" region, involved in ATP binding, between the middle and COOH-terminal one-third of the Ca2(+)-ATPase polypeptide chain. Representative split products in this region (p48,p31) were identified as NH2-terminal and COOH-terminal cleavage products of p85. In the membrane p85 probably remains associated with its complementary NH2-terminal fragment(s) and retains the capacity to bind Ca2+ as evidenced by resistance to V8 degradation in Ca2+ and ability to become phosphorylated by ATP. However, the hydrolysis rate of the phosphorylated enzyme is reduced, indicating that peptide cleavage at Glu-231 interferes with Ca2+ transport steps after phosphorylation. Binding of Ca2+ to V8 and tryptic fragments of Ca2(+)-ATPase was studied on the basis of Ca2(+)-induced changes in electrophoretic mobility and 45Ca2+ autoradiography after transfer of peptides to Immobilon membranes. These data indicate binding by the NH2-terminal 1-198 amino acid residues (corresponding to the tryptic A2 fragment) and the COOH-terminal 715-1001 amino acid residues (corresponding to p31). By contrast the central portion of Ca2(+)-ATPase, including the NH2-terminal portion of p85, is devoid of Ca2+ binding. These results question an earlier proposition that Ca2(+)-binding is located to the "stalk" region of Ca2(+)-ATPase (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H.) (1986) Cell 44, 597-607) but are in agreement with recent data obtained by oligonucleotide-directed mutagenesis of Ca2(+)-ATPase (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). These different studies suggest that Ca2+ translocation sites may have an intramembranous location and are formed predominantly by the carboxyl-terminal part of the Ca2(+)-ATPase polypeptide chain.     

Ca2+-induced Ca2+ release from fragmented sarcoplasmic reticulum: Ca2+-dependent passive Ca2+ efflux

Characterization of the putative Ca2+-gated Ca2+ channel of sarcoplasmic reticulum, which is thought to mediate Ca2+-induced Ca2+ release, was carried out in order to elucidate the mechanism of Ca2+-induced Ca2+ release. Heavy and light fractions of fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle were loaded passively with Ca2+, and then passive Ca2+ efflux was measured under various conditions. The fast phase of the Ca2+ efflux depended on the extravesicular free Ca2+ concentration and was assigned to the Ca2+ efflux through the Ca2+-gated Ca2+ channel. Vesicles with the Ca2+-gated Ca2+ channels comprised about 85% of the heavy fraction and about 40% of the light fraction. The amount of Ca2+ loaded in FSR was found to be much larger than that estimated on the basis of vesicle inner volume and the equilibration of intravesicular with extravesicular Ca2+, indicating Ca2+ binding inside FSR. Taking this fact into account, the Ca2+ efflux curve was quantitatively analyzed and the dependence of the Ca2+ efflux rate constant on the extravesicular free Ca2+ concentration was determined. The Ca2+ efflux was maximal, with the rate constant of 0.75 s-1, when the extravesicular free Ca2+ was at 3 microM. Caffeine increased the affinity for Ca2+ of Ca2+-binding sites for opening the channel with only a slight change in the maximum rate of Ca2+ efflux. Mg2+ inhibited the Ca2+ binding to the sites for opening the channel while procaine seemed to inhibit the Ca2+ efflux by blocking the ionophore moiety of the channel.