Regulation of cardiac sarcoplasmic reticulum calcium transport by calcium-calmodulin-dependent phosphorylation

Cardiac sarcoplasmic reticulum contains an endogenous calcium-calmodulin-dependent protein kinase and a 22,000-Da substrate, phospholamban. This kinase is half-maximally activated (EC50) by 3.8 +/- 0.3 microM calcium and is absolutely dependent on exogenous calmodulin (EC50 = 49 nM). To determine the effect of this phosphorylation on calcium transport, sarcoplasmic reticulum vesicles (0.5 mg/ml) were preincubated under conditions for optimal phosphorylation (50 mM potassium phosphate, pH 7.0, 10 mM MgCl2, 0.5 mM EGTA, 0.478 mM CACl2, 0.1 microM calmodulin, 0.5 mM ATP). Control sarcoplasmic reticulum was preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both control and phosphorylated vesicles were centrifuged and resuspended in 0.3 M sucrose, 20 mM Tris-HCl, 100 mM KCl, pH 7.0, to remove calmodulin and subsequently assayed for calcium (45Ca) transport in the presence of 2.5 mM Tris-oxalate. Phosphorylation of sarcoplasmic reticulum vesicles by calcium-calmodulin-dependent protein kinase resulted in a significant increase (2- to 4-fold) in the rate of calcium transport at low calcium concentrations (less than 3 microM), while calcium transport was minimally affected at higher calcium. Hill coefficients (n) derived from Hill plots of transport data showed no difference between control and phosphorylated sarcoplasmic reticulum (n = 2.0), indicating that phosphorylation does not alter the cooperativity between calcium sites on the calcium pump. The EC50 for calcium activation of calcium transport by control vesicles was 0.86 +/- 0.1 microM calcium, and phosphorylation of phospholamban decreased this value to 0.61 +/- 0.07 microM calcium (n = 7, p less than 0.028), indicating an increase in the apparent affinity for calcium upon phosphorylation. These results were found to be specific for calcium-calmodulin-dependent phosphorylation of phospholamban. Control experiments on the effects of the reactants used in the phosphorylation assay and subsequent centrifugation of sarcoplasmic reticulum showed no alteration of the rate of calcium transport. Therefore, the calcium pump in cardiac sarcoplasmic reticulum appears to be regulated by an endogenous calcium-calmodulin-dependent protein kinase, and this may provide an important regulatory mechanism for the myocardium.     

Regulation of calcium transport by protein phosphatase activity associated with cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium-calmodulin-dependent protein kinase on a 22,000 proteolipid, called phospholamban. Phosphorylation by the calcium-calmodulin-dependent protein kinase is associated with stimulation of the initial rates of calcium transport (Davis, B. A., Schwartz, A., Samaha, F. J., and Kranias, E. G. (1983) J. Biol. Chem. 258, 13587-13591). The present study shows that protein phosphatase activity, associated with canine cardiac sarcoplasmic reticulum vesicles, can catalyze dephosphorylation of the calcium-calmodulin-dependent sites on phospholamban. The activity was maximally stimulated by manganese; fluoride was inhibitory, but its effect was reversible. Dephosphorylation of phospholamban, which was prephosphorylated by calcium-calmodulin-dependent protein kinase, resulted in a reduction of the stimulation on calcium transport rates, particularly at submaximal calcium concentrations. The decrease in calcium transport was associated with a statistically significant decrease in the apparent affinity (EC50) for calcium. Rephosphorylation of phospholamban by the endogenous calcium-calmodulin-dependent protein kinase caused full recovery of the stimulation on calcium transport rates and reversal of the effects mediated by the protein phosphatase. Thus, the calcium pump in cardiac sarcoplasmic reticulum appears to be under reversible regulation mediated by endogenous calcium-calmodulin-dependent protein kinase and protein phosphatase. Such regulation may represent an important control mechanism for the myocardium.     

Decrease in calcium transport associated with phosphoprotein phosphatase-catalyzed dephosphorylation of cardiac sarcoplasmic reticulum.

Phosphoprotein phosphatase activity is found in preparations of sarcoplasmic reticulum isolated from canine heart when assayed with either phosphate or phosphorylated sarcoplasmic reticulum as substrate. Phosphoprotein phosphatase-catalyzed dephosphorylation of the 22,000 dalton phosphoprotein of cardiac sarcoplasmic reticulum is stimulated markedly by MnCl2 (5 mM) and to a lesser extent by MgCl2 (5 mM); inorganic phosphate (50 mM) and NaF (25 mM) are inhibitory. Dephosphorylation of this 22,000 dalton phosphoprotein is correlated with a decreased initial rate of calcium transport. The close structural and functional relationship of phosphoprotein phosphatase to the cardiac sarcoplasmic reticulum suggests a possible role of this enzyme in reversing the relaxation-promoting effects of catecholamines on the intact heart.     

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.     

Calcium transport by cardiac sarcoplasmic reticulum and phosphorylation of phospholamban

Summary Active calcium transport by cardiac sarcoplasmic reticulum assumes a central role in the excitation-concentration coupling of the myocardium, in that Ca²⁺-dependent ATPase (mol.wt. 100 000) of cardiac sarcoplasmic reticulum serves as an energy transducer and a translocator of Ca²⁺ across the membrane. During the translocation of Ca²⁺, the ATPase undergoes a complex series of reactions during which the phosphorylated intermediate EP is formed. We documented how the elementary steps of the ATPase reaction are coupled with calcium translocation, and provided evidences to indicate that two key steps of ATPase correspond to the conformational change of the enzyme, and appear to alter the affinity of the enzyme for Ca²⁺.A line of evidence also indicated that Ca²⁺-dependent ATPase of cardiac sarcoplasmic reticulum is regulated by a specific protein named phospholamban (mol.wt. 22 000), which serves as a substrate for cyclic AMP-dependent protein kinase. Cyclic AMP-dependent phosphorylation of phospholamban resulted in a marked increase in the rate of turnover of the ATPase, by enhancing the rates of the key elementary steps, i.e. the steps at which the intermediate EP is formed and decomposed. Thus phospholamban is putatively thought to serve as a modulator of Cat²⁺-dependent ATPase of cardiac sarcoplasmic reticulum. A working model was proposed to interpret the mechanism. Also documented is a possibility that another protein kinase activatable by Ca²⁺ and calmodulin is functional in regulating the phospholamban-ATPase system, thus suggesting the existence of a dual control system, in which both cyclic AMP- and calmodulin-dependent phosphorylation are in control of the Cat²⁺-dependent ATPase.Such a control mechanism may provide the interpretation, at the cellular level, that catecholamines exert actions on myocardial contractility. Thus, catecholamine-mediated increases in intracellular cyclic AMP could enhance calcium fluxes across the membrane of sarcoplasmic reticulum, thus resulting in the increased rates of relaxation and, at the same time, the increased rate and extent of contraction. Such a mechanism could also be operational in the tissues, other than the myocardium, in which catecholamines and other hormones serve as the first messenger, producing intracellular cyclic AMP as the second messenger.     

Characterization of cardiac sarcoplasmic reticulum ATP-ADP phosphate exchange and phosphorylation of the calcium transport adenosine triphosphatase.

1. The terminal phosphate of (gamma-32P)ATP is rapidly incorporated into cardiac sarcoplasmic reticulum membranes (0.7--1.3 mumol/g protein) in the presence of calcium and magnesium. Cardiac sarcoplasmic reticulum membranes catalize an ATP-ADP phosphate exchange in the presence of calcium and magnesium. 2. Half-maximum activation of the phosphoprotein formation and ATP-ADP phosphate exchange is reached at an ionized calcium concentration of about 0.3 muM. The Hill coefficients are 1.3. 3. Transphosphorylation and ATP-ADP phosphate exchange require magnesium and are maximally activated at magnesium concentrations close to or equal to the ATP concentration. 4. The phosphoprotein level is reduced to about 45% at an ADP/ATP ratio of 0.1. The rate of calcium-dependent ATP splitting declines, whilst the rate of the calcium-dependent ATP-ADP phosphate exchange increases when the ADP/ATP ratio is varied from 0.1 to 1. The sum of both, the rate of ATP splitting and the rate of ADP-ATP phosphate exchange remains constant. 5. Phosphoprotein formation and ATP-ADP phosphate exchange are not affected by azide, dinitrophenol, dicyclohexyl carbodiimide and oubain, whilst both activities are reduced by blockade of -SH groups localized on the outside of the sarcoplasmic reticulum membrane. 6. The isolated phosphoprotein is acid stable. The trichloroacetic acid denatured 32P-labelled membrane complex is dephosphorylated by hydroxylamine, which might indicate that the phosphorylated protein is an acyl-phosphate. 7. Polyacrylamide gel elctrophoresis (performed with phenol/acetic acid/water) of phosphorylated sarcoplasmic reticulum fractions demonstrates that the 32P-incorporation occurs into a protein of about 100000 molecular weight. 8. It is suggested that the phosphoprotein represents a phosphorylated intermediate of the calcium-dependent ATPase which formation occurs as an early step in the reaction sequence of calcium translocation by cardiac sarcoplasmic reticulum similar as in skeletal muscle.     

Phospholamban involvement in the maintenance of basal calcium transport in cardiac sarcoplasmic reticulum

Phosphorylation of cardiac sarcoplasmic reticulum membrane vesicles by exogenous c-AMP and c-AMP-dependent protein kinase stimulates calcium uptake and Ca2+-dependent ATP hydrolysis by 40-50% and results in the incorporation of 32P into a 22-KDa protein, phospholamban. Treatment of the membrane with DOC (0.0002% or 5 X 10(-6) M) solubilizes phospholamban from the membrane and induces a 90% inhibition of basal calcium uptake. This inhibition cannot be attributed to an alteration in vesicle integrity or membrane permeability. The (Ca2+ + Mg2+)-ATPase remains associated with the membrane fraction and exhibits optimal levels of Ca2+-stimulated ATP hydrolysis. Phosphorylation prior to DOC treatment allows retention of the phospholamban in the membrane, concomitant with maintenance of the calcium transport activity. The results presented suggest that phospholamban is involved in the maintenance of basal calcium transport function in cardiac sarcoplasmic reticulum and that its phosphorylation stimulates Ca2+ transport.     

Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adenosinetriphosphatase

The roles of the phosphorylation (phosphorylated enzyme intermediate) and nucleotide binding domains in calcium transport were studied by comparing acetyl phosphate and ATP as substrates for the Ca2+-ATPase of sarcoplasmic reticulum vesicles. We found that the maximal level of phosphoenzyme obtained with either substrate is approximately 4 nmol/mg of protein, corresponding to the stoichiometry of catalytic sites in our preparation. The initial burst of phosphoenzyme formation observed in the transient state, following addition of either substrate, is accompanied by internalization of 2 mol of calcium per mole of phosphoenzyme. The internalized calcium is then translocated with a sequential pattern, independent of the substrate used. Following a rate-limiting step, the phosphoenzyme undergoes hydrolytic cleavage and proceeds to the steady-state activity which is soon "back inhibited" by the rise of Ca2+ concentration in the lumen of the vesicles. When the "back inhibition" is released by the addition of oxalate, substrate utilization and calcium transport occur with a ratio of 1:2, independent of the substrate and its concentration. When the nucleotide binding site is derivatized with FITP, the enzyme can still utilize acetyl phosphate (but not ATP) for calcium transport. No secondary activation of acetyl phosphate utilization by the FITC-enzyme was obtained with millimolar nucleotide. These observations demonstrate that the basic coupling mechanism of catalysis and calcium transport involves the phosphorylation and calcium binding domains, and not the nucleotide binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphatidate releases calcium from cardiac sarcoplasmic reticulum

Phosphatidate (PA) inhibits calcium accumulation by cardiac sarcoplasmic reticulum (SR) and enhances its Ca⁺⁺ ATPase activity. These effects seem to be related to a phosphatidate-induced increase in the calcium permeability of the SR membrane with resultant calcium release. The amount of calcium released by phosphatidate is dependent both on the calcium concentration outside the SR vesicles and the internal calcium concentration. The ionophoric effects of phosphatidate on the sarcoplasmic membrane provide a novel pathway for controlling Ca⁺⁺ transport in the cardiac cell.     

The role of phospholamban in the regulation of calcium transport by cardiac sarcoplasmic reticulum

The calcium transport mechanism of cardiac sarcoplasmic reticulum (SR) is regulated by a phosphoregulatory mechanism involving the phosphorylation-dephosphorylation of an integral membrane component, termed phospholamban. Phospholamban, a 27,000 Da proteolipid, contains phosphorylation sites for three independent protein kinases: 1) cAMP-dependent, 2) Ca²⁺-calmodulin-dependent, and 3) Ca²⁺-phospholipid-dependent. Phosphorylation of phospholamban by any one of these kinases is associated with stimulation of the calcium transport rates in isolated SR vesicles. Dephosphorylation of phosphorylated phospholamban results in the reversal of the stimulatory effects produced by the protein kinases. Studies conducted on perfused hearts have shown that during exposure to beta-adrenergic agents, a good correlation exists between the in situ phosphorylation of phospholamban and the relaxation of the left ventricle. Phosphorylation of phospholamban in situ is also associated with stimulation of calcium transport rates by cardiac SR, similar to in vitro findings. Removal of beta-adrenergic agents results in the reversal of the inotropic response and this is associated with dephosphorylation of phospholamban. These findings indicate that a phospho-regulatory mechanism involving phospholamban may provide at least one of the controls for regulation of the contractile properties of the myocardium.     

Lysophospholipid-mediated alterations in the calcium transport systems of skeletal and cardiac muscle sarcoplasmic reticulum

Summary The effects of various lysophospholipids on the calcium transport activity of sarcoplasmic reticulum (SR) from rabbit skeletal and canine cardiac muscles were examined. The lipids decreased calcium transport activity in both membrane types; the effectiveness being in the order lysoPC > lsyoPS, lysoPG > lysoPE. The maximum inhibition induced by lysoPC, lysoPG and lysoPS was greater than 85% of the normal Ca²⁺-transport rate. In cardiac SR lysoPE had a maximal inhibition of about 50%. Half maximal inhibition of calcium transport by lysoPC was achieved at 110 nmoles lysoPC/mg SR. At this concentration of lysoPC, the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺-uptake activities were inhibited to the same extent (about 60%) in skeletal sarcoplasmic reticulum, while in cardiac sarcoplasmic reticulum, there was less than 20% inhibition of the Ca²⁺ + Mg²⁺-ATPase activity. Studies with EGTA-induced passive calcium efflux showed that up to 200 nmoles lysoPC/mg SR did not alter calcium permeability significantly in cardiac sarcoplasmic reticulum. In skeletal muscle membranes the lysophospholipid mediated decrease in calcium uptake correlated well with the increase in passive calcium efflux due to lysophosphatidylcholine. The difference in the lysophospholipid-induced effects on the sarcoplasmic reticulum from the two muscle types probably reflects variations in protein and other membrane components related to the respective calcium transport systems.     

Characterization of calmodulin-mediated phosphorylation of cardiac muscle sarcoplasmic reticulum

Sarcoplasmic reticulum, isolated from canine cardiac muscle, was phosphorylated in the presence of exogenous cAMP-dependent protein kinase or calmodulin. This phosphorylation has been shown previously to activate sarcoplasmic reticulum calcium uptake (LePeuch et al. (1979) Biochemistry18, 5150–5157). Calmodulin appeared to activate an endogenous protein kinase present in sarcoplasmic reticulum membranes. The incorporation of phosphate increased with time. However, once all the ATP was consumed, the level of phosphorylated protein started to decrease due to the action of an endogenous protein phosphatase. Dephosphorylation occurred even when the level of phosphorylated sarcoplasmic reticulum remained constant at high ATP concentrations. The phosphorylation of sarcoplasmic reticulum in the presence of calmodulin, increased as the pH was increased from pH 5.5 to 8.5. This phosphorylation was only inhibited by KCl concentrations greater than 100 mm. The apparent K_m of cAMP-dependent protein kinase for ATP was 5.2 ± 0.2 × 10^?5m, and of the calmodulin-dependent protein kinase for ATP was 3.67 ± 0.29 × 10^?5m. Phosphorylation was maximally activated by 5–10 mm MgCl₂; higher MgCl₂ concentrations inhibited this phosphorylation. Thus the calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum could be maximally activated at sarcoplasmic concentrations of K⁺, Mg²⁺, and ATP. The calmodulindependent phosphorylation was half-maximally activated at Ca²⁺ concentrations that were significantly greater than those required to promote the formation of the sarcoplasmic reticulum Ca-activated ATPase phosphoprotein intermediate. Thus at sarcoplasmic Ca²⁺ concentrations that might be expected during systole, the sarcoplasmic reticulum calcium pump would be fully activated before any significant calmodul-independent sarcoplasmic reticulum phosphorylation occurred. However, under certain pathological conditions when the sarcoplasmic Ca²⁺ becomes elevated (e.g., in ischemia) the kinase could be activated so that the sarcoplasmic reticulum would be phosphorylated and calcium uptake augmented. Thus, the calmodulin-dependent protein kinase may only function when the heart needs to rescue itself from a possibly fatal calcium overload.     

Coupling of calcium transport with ATP hydrolysis in scallop sarcoplasmic reticulum

Previously, we showed that incubation of the scallop sarcoplasmic reticulum (SR) with EGTA at above 37 degrees C resulted in the uncoupling of ATP hydrolysis with Ca2+ transport [Nagata et al. (1996) J. Biochem. 119, 1100-1105]. We have extended this study by comparing the kinetic behavior of Ca2+ release and binding to the uncoupled SR with that of intact scallop or rabbit SR. The change in the Ca2+ concentration in the reaction medium, as determined as the absorption of APIII, was followed using a stopped flow system. Intact scallop SR was preincubated with Ca2+ in the presence of a Ca2+ ionophore, A23187, and then ATP was added to initiate the reaction. The Ca2+ level in the medium increased to the maximum level in several seconds, and then slowly decreased to the initial low level. The rising and subsequent slow decay phases could be related to the dissociation and reassociation of Ca2+ with the Ca-ATPase, respectively. When uncoupled scallop SR vesicles were preincubated with CaCl2 in the absence of A23187 and then the reaction was initiated by the addition of ATP, a remarkable amount of Ca2+ was released from the SR vesicles into the cytosolic solution, whereas, with intact scallop or rabbit SR, only a sharp decrease in the Ca2+ level was observed. Based on these findings, we concluded that the heat treatment of scallop SR in EGTA may alter the conformation of the Ca-ATPase, thereby causing Ca2+ to be released from the enzyme, during the catalytic cycle, at the cytoplasmic surface, but not at the lumenal surface of SR vesicles.     

The interaction of calcium and ryanodine with cardiac sarcoplasmic reticulum

The binding of [3H]ryanodine with cardiac sarcoplasmic reticulum vesicles depends on the calcium concentration. Binding in the absence of calcium appears to be non-specific because it shows no saturation up to 20 microM ryanodine. The apparent Km value for calcium varied between 2 and 0.8 microM when the ryanodine concentration varied between 10 and 265 nM. The Hill coefficient for the calcium dependence of [3H]ryanodine binding was near two. Scatchard analysis of ryanodine binding indicated a high-affinity site with a Bmax of 5.2 +/- 0.4 pmol/mg with a Kd of 6.8 +/- 0.1 nM. Preincubation under conditions in which the high-affinity sites were saturated did not result in stimulation of the calcium uptake rate indicative of closure of the calcium channel. Stimulation of calcium uptake rate occurred only at higher concentrations of ryanodine (apparent Km = 17 microM). This stimulation of the calcium uptake rate also required calcium in the submicromolar range. The data obtained support the hypothesis that ryanodine binding to the low-affinity site (Km about 17 microM) is responsible for closure of the calcium release channel and the subsequent increase in the calcium uptake rate of the sarcoplasmic reticulum. Because the number of ryanodine-binding sites is much less than the number of calcium transport pumps the channel is probably distinct from the pump.     

Correlation between protein kinase-mediated stimulation of calcium transport by cardiac sarcoplasmic reticulum and phosphorylation of a 22000 dalton protein.

Increases in protein kinase-catalyzed phosphorylation of a 22000 dalton protein correlated closely with increases in phosphate-facilitated calcium transport measured concurrently in canine cardiac sarcoplasmic reticulum under similar conditions in the presence of varying concentrations of bovine cardiac protein kinase. A correlation coefficient of 0.93 and a P value of less than 0.001 were obtained. Protein kinase-catalyzed phosphorylation of the 22000 dalton microsomal protein may mediate the abbreviation of systole seen in the mammalian heart in response to inotropic agents like catecholamines.     

Correlation between protein kinase-mediated stimulation of calcium transport by cardiac sarcoplasmic reticulum and phosphorylation of a 22 000 dalton protein

Increases in protein kinase-catalyzed phosphorylation of a 22 000 dalton protein correlated closely with increases in phosphate-facilitated calcium transport measured concurrently in canine cardiac sarcoplasmic reticulum under similar conditions in the presence of varying concentrations of bovine cardiac protein kinase. A correlation coefficient of 0.93 and a P value of < 0.001 were obtained. Protein kinase-catalyzed phosphorylation of the 22 000 dalton microsomal protein may mediate the abbreviation of systole seen in the mammalian heart in response to inotropic agents like catecholamines.     

Dependence of cardiac sarcoplasmic reticulum calcium pump activity on the phosphorylation status of phospholamban.

The application of electrophoretic resolution of the different phosphorylation species of pentameric phospholamban as a measure of phosphorylation stoichiometry was examined and verified. This enabled a critical evaluation of a number of issues central to current models of calcium pump regulation in cardiac sarcoplasmic reticulum. The phospholamban content of numerous preparations was calculated from 32P incorporation at a given stoichiometry, and compared with the respective calcium pump concentration (derived by comparison with a Coomassie-stained calibration curve of the fast-twitch skeletal muscle isozyme). A relationship of 2 mol of phospholamban:1 mol of ATPase resulted (phospholamban monomer:ATPase monomer), which was maintained throughout all vesicle subpopulations. The precise mechanism of coupling of phospholamban phosphorylation to calcium pump stimulation was probed, with particular emphasis on the individual contributions of each phosphorylated species (P1 to P5). This relationship could be adequately explained in three ways: (i) each phosphorylation event contributed equally to calcium pump stimulation; (ii) P1 and P2 were incapable of stimulating calcium pump activity, but full stimulation occurred upon generation of species P3; or (iii) the phosphospecies P1 was without effect on basal calcium pump activity, but successive phosphorylations contributed equally to stimulation. Finally, the functional implication of dual site phosphorylation of phospholamban (cAMP- and the endogenous calmodulin-dependent kinases) was examined. No change in calcium pump activity accompanied the second tier of phosphorylation over that achieved by the first.