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.     

Functional characterization of junctional terminal cisternae from mammalian fast skeletal muscle sarcoplasmic reticulum

Junctional terminal cisternae are a recently isolated sarcoplasmic reticulum fraction containing two types of membranes, the junctional face membrane with morphologically intact "feet" structures and the calcium pump membrane [Saito, A., Seiler, S., Chu, A., & Fleischer, S. (1984) J. Cell Biol. 99, 875-885]. In this study, the Ca2+ fluxes of junctional terminal cisternae are characterized and compared with three other well-defined fractions derived from the sarcotubular system of fast-twitch skeletal muscle, including light and heavy sarcoplasmic reticulum, corresponding to longitudinal and terminal cisternae regions of the sarcoplasmic reticulum, and isolated triads. Functionally, junctional terminal cisternae have low net energized Ca2+ transport measured in the presence or absence of a Ca2+-trapping anion, as compared to light and heavy sarcoplasmic reticulum and triads. Ca2+ transport and Ca2+ pumping efficiency can be restored to values similar to those of light sarcoplasmic reticulum with ruthenium red or high [Mg2+]. In contrast to junctional terminal cisternae, heavy sarcoplasmic reticulum and triads have higher Ca2+ transport and are stimulated less by ruthenium red. Heavy sarcoplasmic reticulum appears to be derived from the nonjunctional portion of the terminal cisternae. Our studies indicate that the decreased Ca2+ transport is referable to the enhanced permeability to Ca2+, reflecting the predominant localization of Ca2+ release channels in junctional terminal cisternae. This conclusion is based on the following observations: The Ca2+, -Mg2+ -dependent ATPase activity of junctional terminal cisternae in the presence of a Ca2+ ionophore is comparable to that of light sarcoplasmic reticulum when normalized for the calcium pump protein content; i.e., the enhanced Ca2+ transport cannot be explained by a faster turnover of the pump. Ruthenium red or elevated [Mg2+] enhances energized Ca2+ transport and Ca2+ pumping efficiency in junctional terminal cisternae so that values approaching those of light sarcoplasmic reticulum are obtained. Rapid Ca2+ efflux in junctional terminal cisternae can be directly measured and is blocked by ruthenium red or high [Mg2+]. Ryanodine at pharmacologically significant concentrations blocks the ruthenium red stimulation of Ca2+ loading. Ryanodine binding in junctional terminal cisternae, which appears to titrate Ca2+ release channels, is 2 orders of magnitude lower than the concentration of the calcium pump protein. By contrast, light sarcoplasmic reticulum has a high Ca2+ loading rate and slow Ca2+ efflux that are not modulated by ruthenium red, ryanodine, or Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+ dependence of stimulated 45Ca efflux in skinned muscle fibers

Stimulation of sarcoplasmic reticulum Ca release by Mg reduction of caffeine was studied in situ, to characterize further the Ca2+ dependence observed previously with stimulation by Cl ion. 45Ca efflux and isometric force were measured simultaneously at 19 degrees C in frog skeletal muscle fibers skinned by microdissection; EGTA was added to chelate myofilament space Ca either before or after the stimulus. Both Mg2+ reduction (20 or 110 microM to 4 microM) and caffeine (5 mM) induced large force responses and 45Ca release, which were inhibited by pretreatment with 5 mM EGTA. In the case of Mg reduction, residual efflux stimulation was undetectable, and 45Ca efflux in EGTA at 4 microM Mg2+ was not significantly increased. Residual caffeine stimulation at 20 microM Mg2+ was substantial and was reduced further in increased EGTA (10 mM); at 600 microM Mg2+, residual stimulation in 5 mM EGTA was undetectable. Caffeine appears to initiate a small Ca2+-insensitive efflux that produces a large Ca2+-dependent efflux. Additional experiments suggested that caffeine also inhibited influx. The results suggest that stimulated efflux is mediated mainly or entirely by a channel controlled by an intrinsic Ca2+ receptor, which responds to local [Ca2+] in or near the channel. Receptor affinity for Ca2+ probably is influenced by Mg2+, but inhibition is weak unless local [Ca2+] is very low.     

Formation of magnesium-phosphoenzyme and magnesium-calcium-phosphoenzyme in the phosphorylation of adenosine triphosphatase by orthophosphate in sarcoplasmic reticulum. Models of a reaction sequence

The aim of the present study was to test simple reaction sequences which describe calcium-independent plus calcium-dependent phosphorylation of sarcoplasmic reticulum transport. ATPase by orthophosphate including the function of magnesium in phosphoenzyme formation. The reaction schemes considered were based on the reaction sequence for calcium-independent phosphorylation proposed previously; namely that the transport enzyme (E) forms a ternary complex (Mg . E . Pi), by random binding of free magnesium and free orthophosphate, which is in equilibrium with the magnesium-phosphoenzyme (Mg . E-P). Phosphorylation, performed at pH 7.0 20 degrees C and a constant free orthophosphate concentration using sarcoplasmic reticulum vesicles either unloaded or loaded passively with calcium in the presence of 5 mM or 40 mM CaCl2, resulted in a gradual decrease in the apparent magnesium half-saturation constant and an increase in maximum phosphoprotein formation with increasing calcium loads. When phosphorylation of sarcoplasmic reticulum vesicles preloaded in the presence of 5 mM CaCl2 was performed at a constant free magnesium concentration, a decrease in the apparent orthophosphate half-saturation constant and an increase in maximum phosphoprotein formation was observed as compared with vesicles from which calcium inside has been removed by ionophore X-537A plus EGTA treatment; however, both parameters remained unchanged by increasing free magnesium from 20 mM to 30 mM. When phosphorylation of sarcoplasmic reticulum vesicles passively loaded with calcium in the presence of 40 mM CaCl2, at which the saturation of the low-affinity calcium binding sites of the ATPase is presumably near maximum, was performed at increasing concentrations of free orthophosphate, there was a parallel shift of phosphoprotein formation as a function of free magnesium and vice versa, with no change in the maximum phosphoenzyme formation. Comparison of the experimental data with the pattern of phosphoprotein formation predicted from model equations for various theoretical possible reaction sequences suggests that phosphoenzyme formation from orthophosphate possesses the following features. Firstly, calcium present at the inside of the sarcoplasmic reticulum membrane binds to the free enzyme and in sequential order to E . Mg . Pi or Mg . E-P or to both, but neither to E. Mg nor to E . Pi. Secondly, calcium-independent and calcium-dependent phosphoproteins are magnesium-phosphoenzymes. Calcium-dependent phosphoenzyme is a magnesium-calcium-enzyme phosphate complex with 1 magnesium, 2 calciums and 1 orthophosphate (the last covalently) bound to the enzyme [Mg . E-P . (Cai)2], and not a 'calcium-phosphoprotein' without bound magnesium.     

Single channel and 45Ca2+ flux measurements of the cardiac sarcoplasmic reticulum calcium channel.

Purified canine cardiac sarcoplasmic reticulum vesicles were passively loaded with 45CaCl2 and assayed for Ca2+ releasing activity according to a rapid quench protocol. Ca2+ release from a subpopulation of vesicles was found to be activated by micromolar Ca2+ and millimolar adenine nucleotides, and inhibited by millimolar Mg2+ and micromolar ruthenium red. 45Ca2+ release in the presence of 10 microM free Ca2+ gave a half-time for efflux of 20 ms. Addition of 5 mM ATP to 10 microM free Ca2+ increased efflux twofold (t1/2 = 10 ms). A high-conductance calcium-conducting channel was incorporated into planar lipid bilayers from the purified cardiac sarcoplasmic reticulum fractions. The channel displayed a unitary conductance of 75 +/- 3 pS in 53 mM trans Ca2+ and was selective for Ca2+ vs. Tris+ by a ratio of 8.74. The channel was dependent on cis Ca2+ for activity and was also stimulated by millimolar ATP. Micromolar ruthenium red and millimolar Mg2+ were inhibitory, and reduced open probability in single-channel recordings. These studies suggest that cardiac sarcoplasmic reticulum contains a high-conductance Ca2+ channel that releases Ca2+ with rates significant to excitation-contraction coupling.     

Activation of an islet cell plasma membrane (Ca2+ + Mg2+)-ATPase by calmodulin and Ca-EGTA

Islet cell plasma membranes contain a calcium-stimulated and magnesium-dependent ATPase (Ca2+ + Mg2+)-ATPase) which requires calmodulin for maximum enzyme activity (Kotagal, N., Patke, C., Landt, M., McDonald, J., Colca, J., Lacy, P., and McDaniel, M. (1982) FEBS Lett. 137, 249-252). Investigations indicated that exogenously added calmodulin increases the velocity and decreases the Km for Ca2+ of the high affinity (Ca2+ + Mg2+)-ATPase. These studies routinely employed the chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to maintain Ca2+ concentrations in the submicromolar range. During the course of these investigations, it was found unexpectedly that increasing the concentrations of EGTA (0.1-4 mM) and total calcium in the media, while maintaining constant free Ca2+ levels, increased the velocity of the high affinity (Ca2+ + Mg2+)-ATPase. The free calcium concentrations under these conditions were verified by a calcium-sensitive electrode. The (Ca2+ + Mg2+)-ATPase maximally activated by 2-4 mM EGTA was not further stimulated by calmodulin, whereas camodulin stimulation increased as the concentration of EGTA in the media was decreased. A similar enhancement by Ca-EGTA was observed on active calcium transport by the plasma membrane-enriched fraction. Moreover, Ca-EGTA had a negligible effect on both active calcium transport as well as Ca2+-stimulated ATPase activity by the islet cell endoplasmic reticulum, processes which are not stimulated by calmodulin. The results indicate that stimulation by Ca-EGTA may be used to differentiate calcium transport systems by these subcellular organelles. Furthermore, the concentration of EGTA routinely employed to maintain free Ca2+ levels may itself obscure effects of calmodulin and other physiological agents on calcium-dependent activities.     

Effects of Mg2+ on calcium accumulation by two fractions of sarcoplasmic reticulum from rabbit skeletal muscle

Calcium accumulation by two fractions of sarcoplasmic reticulum presumably derived from longitudinal tubules (light vesicles) and terminal cisternae (heavy vesicles) was examined radiochemically in the presence of various free Mg2+ concentrations. Both fractions of sarcoplasmic reticulum exhibited a Mg2+-dependent increase in phosphate-supported calcium uptake velocity, though half-maximal velocity in heavy vesicles occurred at a much higher free Mg2+ concentration than that in light vesicles (i.e., approx. 0.90 mM vs. approx. 0.02 mM Mg2+). Calcium uptake velocity in light vesicles correlated with Ca2+-dependent ATPase activity, suggesting that Mg2+ stimulated the calcium pump. Calcium uptake velocity in heavy vesicles did not correlate with Ca2+-dependent ATPase activity, although a Mg2+-dependent increase in calcium influx was observed. Thus, Mg2+ may increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles. Analyses of calcium sequestration (in the absence of phosphate) showed a similar trend in that elevation of Mg2+ from 0.07 to 5 mM stimulated calcium sequestration in heavy vesicles much more than in light vesicles. This difference between the two fractions of sarcoplasmic reticulum was not explained by phosphoenzyme (EP) level or distribution. Analyses of calcium uptake, Ca2+-dependent ATPase activity, and unidirectional calcium flux in the presence of approx. 0.4 mM Mg2+ suggested that ruthenium red (0.5 microM) can also increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles, with no effect in light vesicles. These functional differences between light and heavy vesicles suggest that calcium transport in terminal cisternae is regulated differently from that in longitudinal tubules.     

Lanthanum inhibits steady-state turnover of the sarcoplasmic reticulum calcium ATPase by replacing magnesium as the catalytic ion

LaATP is shown to be an effective inhibitor of the calcium ATPase of sarcoplasmic reticulum because the binding of LaATP to cE.Ca2 results in the formation of lanthanum phosphoenzyme, which decays slowly. Steady-state activity of the calcium ATPase in leaky sarcoplasmic reticulum vesicles is inhibited 50% by 0.16 microM LaCl3 (15 nM free La3+, 21 nM LaATP) in the presence of 25 microM Ca2+ and 49 microM MgATP (5 mM MgSO4, 100 mM KCl, 40 mM 4-morpholinepropanesulfonic acid, pH 7.0, 25 degrees C). However, 50% inhibition of the uptake of 45Ca and phosphorylation by [gamma-32P]ATP in a single turnover experiment requires 100 microM LaCl3 (28 microM free La3+) in the presence of 25 microM Ca2+; this inhibition is reversed by calcium but inhibition of steady-state turnover is not. Therefore, binding of La3+ to the cytoplasmic calcium transport site is not responsible for the inhibition of steady-state ATPase activity. The addition of 6.7 microM LaCl3 (1.1 microM free La3+) has no effect on the rate of dephosphorylation of phosphoenzyme formed from MgATP and enzyme in leaky vesicles, while 6.7 mM CaCl2 slows the rate of phosphoenzyme hydrolysis as expected; 6.7 microM LaCl3 and 6.7 mM CaCl2 cause 95 and 98% inhibition of steady-state ATPase activity, respectively. This shows that inhibition of ATPase activity in the steady state is not caused by binding of La3+ to the intravesicular calcium transport site of the phosphoenzyme. Inhibition of ATPase activity by 2 microM LaCl3 (0.16 microM free La3+, 0.31 microM LaATP) requires greater than 5 s, which corresponds to approximately 50 turnovers, to reach a steady-state level of greater than or equal to 80% inhibition. Inhibition by La3+ is fully reversed by the addition of 0.55 mM CaCl2 and 0.50 mM EGTA; this reactivation is slow with t1/2 approximately 9 s. Two forms of phosphoenzyme are present in reactions that are partially inhibited by La3+: phosphoenzyme with Mg2+ at the catalytic site and phosphoenzyme with La3+ at the catalytic site, which undergo hydrolysis with observed rate constants of greater than 4 and 0.05 s-1, respectively. We conclude, therefore, that La3+ inhibits steady-state ATPase activity under these conditions by replacing Mg2+ as the catalytic ion for phosphoryl transfer. The slow development of inhibition corresponds to the accumulation of lanthanum phosphoenzyme. Initially, most of the enzyme catalyzes MgATP hydrolysis, but the fraction of enzyme with La3+ bound to the catalytic site gradually increases because lanthanum phosphoenzyme undergoes hydrolysis much more slowly than does magnesium phosphoenzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

The vectorial specificity for calcium binding to the CaATPase of sarcoplasmic reticulum is controlled by phosphorylation, not by an E-E* conformational change.

The E-E* model for calcium pumping by the CaATPase of sarcoplasmic reticulum includes two distinct conformational states of the enzyme, E and E*. Exterior Ca2+ binds only to E and interior Ca2+ binds only to E*. Therefore, it is expected that there will be competition between the binding of calcium to the unphosphorylated enzyme from the two sides of the membrane. The equilibrium concentration of cECa2, the enzyme with Ca2+ bound at the exterior site, was measured at different Ca2+ concentrations with empty sarcoplasmic reticulum vesicles (SRV) and with SRV loaded with 40 mM Ca2+ by reaction with 0.5 mM [gamma-32P]ATP plus 20 mM EGTA for 13 ms (100 mM KCl, 5 mM MgSO4, 40 mM Mops/KOH, pH 7.0, 25 degrees C). The sigmoidal dependence on free exterior calcium concentration of the concentration of cECa2, measured as [32P]phosphoenzyme, is identical with empty and loaded SRV, within experimental error. The value of K0.5 is 2.8 microM, and the Hill coefficient is 2. This result shows that there is no competition between binding of Ca2+ to the outside and the inside of the membrane. This is consistent with a model in which the vectorial specificity for calcium binding is controlled by the chemical state of the enzyme, rather than a simple conformational change. It is concluded that there are not two interconverting forms of the free enzyme, E and E*, instead the vectorial specificity for binding and dissociation of Ca2+ is determined by the state of phosphorylation of the CaATPase.     

Quercetin interaction with the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

In sarcoplasmic reticulum vesicles or in the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum, quercetin inhibited ATP hydrolysis, Ca2+ uptake, ATP-Pi exchange, ATP synthesis coupled to Ca2+ efflux, ATP-ADP exchange, and steady state phosphorylation of the ATPase by inorganic phosphate. Steady state phosphorylation of the ATPase by ATP was not inhibited. Quercetin also inhibited ATP and ADP binding but not the binding of Ca2+. The inhibition of ATP-dependent Ca2+ transport by quercetin was reversible, and ATP, Ca2+, and dithiothreitol did not affect the inhibitory action of quercetin.     

Ryanodine activation and inhibition of the Ca2+ release channel of sarcoplasmic reticulum

The effect of the plant alkaloid ryanodine on the skeletal muscle sarcoplasmic reticulum Ca2+ release channel was studied by determining the Ca2+ permeability of "heavy" vesicles passively loaded with 45Ca2+ in the presence or absence of ryanodine. Depending on the experimental conditions, ryanodine either stimulated or inhibited Ca2+ efflux. Vesicles were rendered permeable to 45Ca2+ at a ryanodine concentration of 0.01 microM when diluted into a medium containing the two Ca2+ release channel inhibitors Mg2+ and ruthenium red. At ryanodine concentrations greater than 10 microM, 45Ca2+ efflux was inhibited in channel-activating (5 microM Ca2+) or -inhibiting (10 mM Mg2+ plus 10 microM ruthenium red) media. An optimal stimulatory effect was observed when vesicles were incubated with ryanodine at 37 degrees C and in media that caused partial opening of the channel. Similar results to those described above were obtained using cardiac sarcoplasmic reticulum vesicles that were capable of rapid 45Ca2+ efflux. Use of the slowly permeating molecule L-[3H]glucose allowed measurement of channel-mediated efflux rates from vesicles in the presence and absence of ryanodine. At low activating concentrations, ryanodine did not appreciably change the regulation of L-glucose efflux rates by external Ca2+, Mg2+, and adenine nucleotide. These results suggested two possible modes of action of ryanodine: 1) a change in the gating mechanism of the channel which is not readily detected using the slowly permeating molecule L-glucose or 2) a change in channel structure which prevents its complete closing.     

Enzymatic activity of dystrophic chicken sarcoplasmic reticulum

We have isolated sarcoplasmic reticulum from normal and dystrophic chicken muscle, using an improved isolation procedure. Dystrophic sarcoplasmic reticulum has a reduced level of calcium-sensitive ATPase activity, phosphoenzyme formation, and steady-state calcium transport. Anion-stimulated calcium transport by dystrophic sarcoplasmic reticulum is also reduced when measured under the proper conditions, and dystrophic sarcoplasmic reticulum shows no alteration in calcium efflux rate. Active calcium phosphate loading of the normal and dystrophic sarcoplasmic reticulum preparations indicates that a reduced percentage jof the dystrophic vesicles are capable of active calcium transport. The loaded dystrophic sarcoplasmic reticulum vesicles exhibit the same relative reductions in enzymatic activity as the starting sarcoplasmic reticulum preparations. However, the enzyme activities of normal and dystrophic sarcoplasmic reticulum are similar in the presence of detergent and exogenous phospholipid. On the basis of these results, we suggest that the lipid microenvironment of the dystrophic enzyme is altered.     

Oxidation induced by phthalocyanine dyes causes rapid calcium release from sarcoplasmic reticulum vesicles

The copper containing phthalocyanine dyes, alcian blue, copper phthalocyanine tetrasulfonic acid, and Luxol fast blue MBSN are found to induce rapid calcium efflux from actively loaded sarcoplasmic reticulum (SR) vesicles. Alcian blue (5 microM), with 1 mM free Mg2+ triggered Ca2+ efflux at rates greater than 20 nmol/mg of SR/s. As in the case of Ca2+ efflux induced by calcium, heavy metals, or SH oxidation with Cu2+/cysteine, efflux induced by phthalocyanines is also stimulated by adenine containing nucleotides and inhibited by millimolar Mg2+ and submicromolar ruthenium red (RR). In addition, analogs of RR, such as hexamminecobalt(III) chloride or hexammineruthenium(III) chloride also inhibit Ca2+ efflux but are effective at somewhat higher concentrations (approximately 50 microM). Calcium release stimulated by phthalocyanines is specific for SR derived from the terminal cisternae region rather than longitudinal SR. Preincubation of alcian blue with the reducing agents, sodium dithionite, dithiothreitol, or cysteine causes complete loss of Ca2+ release activity from SR vesicles. Reoxidation of the alcian blue leads to return of the Ca2+ release activity of the phthalocyanine dye. The copper containing phthalocyanine dyes appear to cause rapid Ca2+ release from SR vesicles by oxidizing sulfhydryl groups associated with the calcium release channel. Moreover, phthalocyanines appear to act by oxidizing a pair of neighboring sulfhydryls to a disulfide because subsequent additions of the reducing agent dithiothreitol promote the closure of the Ca2+ channel and calcium re-uptake.     

Passive Ca2+ fluxes across the membrane of sarcoplasmatic reticulum of skeletal muscles. The effect of calcium channel blockers

It was found that the initial rate of passive KC1-stimulated Ca2+ influx into sarcoplasmic reticulum (SR) vesicles follows the saturation kinetics at Ca2+ concentrations of 8-10 mM. The inhibitory effect of Ca2+ channel blockers (La3+, Mn2+, Co2+, Cd2+, Mg2+) on passive Ca2+ influx into SR vesicles is competitive with respect to Ca2+. These blockers also inhibit the initial fast phase of Ca2+ efflux from Ca2+-loaded SR vesicles. Verapamil (0.1-0.5 mM) added to the incubation mixture has no effect on passive Ca2+ fluxes across the SR vesicle membrane or on Ca2+ binding and ATP-dependent Ca2+ accumulation. However, preincubation of SR vesicles with verapamil (18 hours, 4 degrees C) or its introduction into the medium for SR vesicle isolation leads to the inhibition of passive Ca2+ fluxes.     

Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds

The caffeine-sensitive Ca2+ release pathway in skeletal muscle was identified and characterized by studying the release of 45Ca2+ from heavy sarcoplasmic reticulum (SR) vesicles and by incorporating the vesicles or the purified Ca2+ release channel protein complex into planar lipid bilayers. First-order rate constants for 45Ca2+ efflux of 1 s-1 were obtained in the presence of 1-10 microM free Ca2+ or 2 X 10(-9) M free Ca2+ plus 20 mM caffeine. Caffeine- and Ca2+-induced 45Ca2+ release were potentiated by ATP and Mg.ATP, and were both inhibited by Mg2+. Dimethylxanthines were similarly (3,9-dimethylxanthine) or more (1,7-, 1,3-, and 3,7-dimethylxanthine) effective than caffeine in increasing the 45Ca2+ efflux rate. 1,9-Dimethylxanthine and 1,3-dimethyluracil (which lacks the imidazole ring) did not appreciably stimulate 45Ca2+ efflux. Recordings of calcium ion currents through single channels showed that the Ca2+- and ATP-gated SR Ca2+ release channel is activated by addition of caffeine to the cis (cytoplasmic) and not the trans (lumenal) side of the channel in the bilayer. The single channel measurements further revealed that caffeine activated Ca2+ release by increasing the number and duration of open channel events without a change of unit conductance (107 pS in 50 mM Ca2+ trans). These results suggest that caffeine exerts its Ca2+ releasing effects in muscle by activating the high-conductance, ligand-gated Ca2+ release channel of sarcoplasmic reticulum.     

Lack of effects of calcium X calmodulin-dependent phosphorylation on Ca2+ release from cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium X calmodulin-dependent protein kinase and phosphorylation occurs mainly on a 27 kDa proteolipid, called phospholamban. To determine whether this phosphorylation has any effect on Ca2+ release, sarcoplasmic reticulum vesicles were phosphorylated by the calcium X calmodulin-dependent protein kinase, while non-phosphorylated vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both non-phosphorylated and phosphorylated vesicles were centrifuged to remove calmodulin, and subsequently used for Ca2+ release studies. Calcium loading was carried out either by the active calcium pump or by incubation with high (5 mM) calcium for longer periods. Phosphorylation of sarcoplasmic reticulum by calcium X calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca2+ released from cardiac sarcoplasmic reticulum vesicles loaded under passive conditions and on the apparent 45Ca2+-40Ca2+ exchange from cardiac sarcoplasmic reticulum vesicles loaded under active conditions. Thus, it appears that calcium X calmodulin-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum is not involved in the regulation of Ca2+ release and 45Ca2+-40Ca2+ exchange.     

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).     

Characterization of Ca2+ release from heterogeneous Ca2+ stores in sarcoplasmic reticulum isolated from arterial and gastric smooth muscle

Ca2+ transport was investigated in vesicles of sarcoplasmic reticulum subfractionated from bovine main pulmonary artery and porcine gastric antrum using digitonin binding and zonal density gradient centrifugation. Gradient fractions recovered at 15-33% sucrose were studied as the sarcoplasmic reticulum component using Fluo-3 fluorescence or 45Ca2+ Millipore filtration. Thapsigargin blocked active Ca2+ uptake and induced a slow Ca2+ release from actively loaded vesicles. Unidirectional 45Ca2+ efflux from passively loaded vesicles showed multicompartmental kinetics. The time course of an initial fast component could not be quantitatively measured with the sampling method. The slow release had a half-time of several minutes. Both components were inhibited by 20 microM ruthenium red and 10 mM Mg2+. Caffeine, inositol 1,4,5-trisphosphate, ATP, and diltiazem accelerated the slow component. A Ca2+ release component activated by ryanodine or cyclic adenosine diphosphate ribose was resolved with Fluo-3. Comparison of tissue responses showed that the fast Ca2+ release was significantly smaller and more sensitive to inhibition by Mg2+ and ruthenium red in arterial vesicles. They released more Ca2+ in response to inositol 1,4,5-trisphosphate and were more sensitive to activation by cyclic adenosine diphosphate ribose. Ryanodine and caffeine, in contrast, were more effective in gastric antrum. In each tissue, the fraction of the Ca2+ store released by sequential application of caffeine and inositol 1,4,5-trisphosphate depended on the order applied and was additive. The results indicate that sarcoplasmic reticulum purified from arterial and gastric smooth muscle represents vesicle subpopulations that retain functional Ca2+ channels that reflect tissue-specific pharmacological modulation. The relationship of these differences to physiological responses has not been determined.     

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.     

Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent on Ca2+ and is modulated by Mg2+, adenine nucleotide, and calmodulin

A subpopulation of canine cardiac sarcoplasmic reticulum vesicles has been found to contain a "Ca2+ release channel" which mediates the release of intravesicular Ca2+ stores with rates sufficiently rapid to contribute to excitation-contraction coupling in cardiac muscle. 45Ca2+ release behavior of passively and actively loaded vesicles was determined by Millipore filtration and with the use of a rapid quench apparatus using the two Ca2+ channel inhibitors, Mg2+ and ruthenium red. At pH 7.0 and 5-20 microM external Ca2+, cardiac vesicles released half of their 45Ca2+ stores within 20 ms. Ca2+-induced Ca2+ release was inhibited by raising and lowering external Ca2+ concentration, by the addition of Mg2+, and by decreasing the pH. Calmodulin reduced the Ca2+-induced Ca2+ release rate 3-6-fold in a reaction that did not appear to involve a calmodulin-dependent protein kinase. Under various experimental conditions, ATP or the nonhydrolyzable ATP analog, adenosine 5'-(beta, gamma-methylene)triphosphate (AMP-PCP), and caffeine stimulated 45Ca2+ release 2-500-fold. Maximal release rates (t1/2 = 10 ms) were observed in media containing 10 microM Ca2+ and 5 mM AMP-PCP or 10 mM caffeine. An increased external Ca2+ concentration (greater than or equal to 1 mM) was required to optimize the 45Ca2+ efflux rate in the presence of 8 mM Mg2+ and 5 mM AMP-PCP. These results suggest that cardiac sarcoplasmic reticulum contains a ligand-gated Ca2+ channel which is activated by Ca2+, adenine nucleotide, and caffeine, and inhibited by Mg2+, H+, and calmodulin.