Role of phospholamban in regulating cardiac sarcoplasmic reticulum calcium pump

Cardiac sarcoplasmic reticulum plays a critical role in the excitation-contraction cycle and hormonal regulation of heart cells. Catecholamines exert their ionotropic action through the regulation of calcium transport into the sarcoplasmic reticulum. Cyclic 3'-5'-adenosine monophosphate (cAMP) causes the cAMP-dependent protein kinase to phosphorylate the regulatory protein phospholamban, which results in the stimulation of calcium transport. Calmodulin also phosphorylates phospholamban by a calcium-dependent mechanism. We have reported the isolation and purification of phospholamban with low deoxycholate (DOC) concentrations (5 X 10(-6) M). We have also reported the isolation and purification of Ca2+ + Mg2+-ATPase with a similar procedure. Both phospholamban and Ca2+ + Mg2+-ATPase retained their native properties associated with sarcoplasmic reticulum vesicles. Further, we have shown that the removal of phospholamban from membranes of sarcoplasmic reticulum vesicles uncouples Ca2+-uptake from ATPase without any effect on Ca2+ + Mg2+-ATPase activity or Ca2+ efflux. Phospholamban appears to be the substrate for both the Ca2+-calmodulin system and the cAMP-dependent protein kinase system. It is found that the phosphorylation of phospholamban by the Ca2+-calmodulin system is required for the normal basal level of Ca2+ transport, and that the phosphorylation of phospholamban at another site by the cAMP-dependent protein kinase system causes the stimulation of Ca2+-transport above the basal level. The functional effects of the phosphorylation of phospholamban by cAMP-dependent protein kinase system are expressed only after the phosphorylation of phospholamban with Ca2+-calmodulin system. We propose a model for the cardiac Ca2+ + Mg2+-ATPase, whereby the enzyme is normally uncoupled from Ca2+ uptake. The enzyme becomes coupled to Ca2+ transport after the first site of phospholamban is phosphorylated with the Ca2+-calmodulin system. When the second site of phospholamban is phosphorylated with cAMP-dependent protein kinase both Ca2+ transport and ATPase are stimulated and phospholamban becomes inaccessible to DOC solubilization and trypsin.     

Regulation of cardiac sarcoplasmic reticulum function by phospholamban

Calcium fluxes across the sarcoplasmic reticulum membrane are regulated by phosphorylation of a 27,000-dalton membrane-bound protein termed phospholamban. Phospholamban is phosphorylated by three different protein kinases (cAMP-dependent, Ca2+.CAM-dependent and Ca2+.phospholipid dependent) at apparently distinct sites. Phosphorylation by each of the protein kinases increases the rates of active calcium transport by sarcoplasmic reticulum vesicles. The stimulatory effects of protein kinases on the calcium pump may be reversed by an endogenous protein phosphatase activity. The phosphoprotein phosphatase can dephosphorylate both the cAMP-dependent and the Ca2+.CAM-dependent sites of phospholamban. Phosphorylation of phospholamban also occurs in situ, in perfused beating hearts, during the peak of the inotropic response to beta-adrenergic stimulation. Reversal of the stimulatory effects is associated with dephosphorylation of phospholamban. Thus, in vivo and in vitro studies suggest that phospholamban is a regulator for the calcium pump in cardiac sarcoplasmic reticulum. The degree of phospholamban phosphorylation determined by the interaction of specific protein kinases and phosphatases may represent an important control for sarcoplasmic reticulum function and, thus, for the contraction-relaxation cycle in the myocardium. In this review, we summarize recent evidence on physical and structural properties of phospholamban, the proposed structural molecular models for this protein, and the significance of its regulatory role both in vitro and in situ.     

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.     

Mechanism of the stimulation of cardiac sarcoplasmic reticulum calcium pump by calmodulin

Calmodulin has been shown to stimulate the initial rates of Ca2+-uptake and Ca2+-ATPase in cardiac sarcoplasmic reticulum, when it is present in the reaction assay media for these activities. To determine whether the stimulatory effect of calmodulin is mediated directly through its interaction with the Ca2+-ATPase, or indirectly through phosphorylation of phospholamban by an endogenous protein kinase, two approaches were taken in the present study. In the first approach, the effects of calmodulin were studied on a Ca2+-ATPase preparation, isolated from cardiac sarcoplasmic reticulum, which was essentially free of phospholamban. The enzyme was preincubated with various concentrations of calmodulin at 0 degrees C and 37 degrees C, but there was no effect on the Ca2+-ATPase activity assayed over a wide range of [Ca2+] (0.1-10 microM). In the second approach, cardiac sarcoplasmic reticulum vesicles were prephosphorylated by an endogenous protein kinase in the presence of calmodulin. Phosphorylation occurred predominantly on phospholamban, an oligomeric proteolipid. The sarcoplasmic reticulum vesicles were washed prior to assaying for Ca2+ uptake and Ca2+-ATPase activity in order to remove the added calmodulin. Phosphorylation of phospholamban enhanced the initial rates of Ca2+-uptake and Ca2+-ATPase, and this stimulation was associated with an increase in the affinity of the Ca2+-pump for calcium. The EC50 values for calcium activation of Ca2+-uptake and Ca2+-ATPase were 0.96 +/- 0.03 microM and 0.96 +/- 0.1 microM calcium by control vesicles, respectively. Phosphorylation decreased these values to 0.64 +/- 0.12 microM calcium for Ca2+-uptake and 0.62 +/- 0.11 microM calcium for Ca2+-ATPase. The stimulatory effect was associated with increases in the apparent initial rates of formation and decomposition of the phosphorylated intermediate of the Ca2+-ATPase. These findings suggest that calmodulin regulates cardiac sarcoplasmic reticulum function by protein kinase-mediated phosphorylation of phospholamban.     

Protein-Protein Interactions in Calcium Transport Regulation Probed by Saturation Transfer Electron Paramagnetic Resonance

We have used electron paramagnetic resonance (EPR) to probe the homo- and heterooligomeric interactions of reconstituted sarcoplasmic reticulum Ca-ATPase (SERCA) and its regulator phospholamban (PLB). SERCA is responsible for restoring calcium to the sarcoplasmic reticulum to allow muscle relaxation, whereas PLB inhibits cardiac SERCA unless phosphorylated at Ser¹⁶. To determine whether changes in protein association play essential roles in regulation, we detected the microsecond rotational diffusion of both proteins using saturation transfer EPR. Peptide synthesis was used to create a fully functional and monomeric PLB mutant with a spin label rigidly coupled to the backbone of the transmembrane helix, while SERCA was reacted with a Cys-specific spin label. Saturation transfer EPR revealed that sufficiently high lipid/protein ratios minimized self-association for both proteins. Under these dilute conditions, labeled PLB was substantially immobilized after co-reconstitution with unlabeled SERCA, reflecting their association to form the regulatory complex. Ser¹⁶ phosphorylation slightly increased this immobilization. Complementary measurements with labeled SERCA showed no change in mobility after co-reconstitution with unlabeled PLB, regardless of its phosphorylation state. We conclude that phosphorylating monomeric PLB can relieve SERCA inhibition without changes in the oligomeric states of these proteins, indicating a structural rearrangement within the heterodimeric regulatory complex.     

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.     

Molecular dynamics in mouse atrial tumor sarcoplasmic reticulum.

We have determined directly the effects of the inhibitory peptide phospholamban (PLB) on the rotational dynamics of the calcium pump (Ca-ATPase) of cardiac sarcoplasmic reticulum (SR). This was accomplished by comparing mouse ventricular SR, which has PLB levels similar to those found in other mammals, with mouse atrial SR, which is effectively devoid of PLB and thus has much higher (unregulated) calcium pump activity. To obtain sufficient quantities of atrial SR, we isolated the membranes from atrial tumor cells. We used time-resolved phosphorescence anisotropy of an erythrosin isothiocyanate label attached selectively and rigidly to the Ca-ATPase, to detect the microsecond rotational motion of the Ca-ATPase in the two preparations. The time-resolved phosphorescence anisotropy decays of both preparations at 25 degrees C were multi-exponential, because of the presence of different oligomeric species. The rotational correlation times for the different oligomers were similar for the two preparations, but the total decay amplitude was substantially greater for atrial tumor SR, indicating that a smaller fraction of the Ca-ATPase molecules exists as large aggregates. Phosphorylation of PLB in ventricular SR decreased the population of large-scale Ca-ATPase aggregates to a level similar to that of atrial tumor SR. Lipid chain mobility (fluidity), detected by electron paramagnetic resonance of stearic acid spin labels, was very similar in the two preparations, indicating that the higher protein mobility in atrial tumor SR is not due to higher lipid fluidity. We conclude that PLB inhibits by inducing Ca-ATPase lateral aggregation, which can be relieved either by phosphorylating or removing PLB.     

The use of a water-soluble carbodiimide to cross-link cytochrome c to plastocyanin

Canine cardiac sarcoplasmic reticulum is phosphorylated by adenosine 3',5'-monophosphate (cAMP)-dependent and by Ca2+-calmodulin-dependent protein kinases on an Mr 22 000 protein called phospholamban. Both types of phosphorylation are associated with an increase in the initial rate of Ca2+ transport. Thus, phospholamban appears to be a regulator for the calcium pump in cardiac sarcoplasmic reticulum. However, there is conflicting evidence as to the degree of association of the Ca2+-ATPase with its regulator, phospholamban. In this study, we report that phospholamban does not copurify with a Ca2+-ATPase preparation of high specific activity. Although 32P-labeled phospholamban is solubilized in the same fraction as the Ca2+-ATPase from cardiac sarcoplasmic reticulum, it dissociates from the Ca2+ pump during subsequent purification steps. Our isolation procedure results in an increase of over 4-fold in the specific activity of the Ca2+-ATPase, but a decrease of 2.5-fold in the specific activity of 32Pi-phosphoester bonds (pmol Pi/mg). Furthermore, the purified Ca2+-ATPase enzyme preparation is not a substrate for protein kinase in vitro to any significant extent. These data indicate that phospholamban does not copurify with the Ca2+-ATPase from cardiac sarcoplasmic reticulum. Isolation of a Ca2+-ATPase preparation essentially free of phospholamban will aid in future kinetic studies designed to elucidate similarities and differences in the Ca2+-ATPase parameters from cardiac and skeletal muscle (which is known not to contain phospholamban).     

Anesthetics alter the physical and functional properties of the Ca-ATPase in cardiac sarcoplasmic reticulum.

We have studied the effects of the local anesthetic lidocaine, and the general anesthetic halothane, on the function and oligomeric state of the CA-ATPase in cardiac sarcoplasmic reticulum (SR). Oligomeric changes were detected by time-resolved phosphorescence anisotropy (TPA). Lidocaine inhibited and aggregated the Ca-ATPase in cardiac SR. Micromolar calcium or 0.5 M lithium chloride protected against lidocaine-induced inhibition, indicating that electrostatic interactions are essential to lidocaine inhibition of the Ca-ATPase. The phospholamban (PLB) antibody 2D12, which mimics PLB phosphorylation, had no effect on lidocaine inhibition of the Ca-ATPase in cardiac SR. Inhibition and aggregation of the Ca-ATPase in cardiac SR occurred at lower concentrations of lidocaine than necessary to inhibit and aggregate the Ca-ATPase in skeletal SR, suggesting that the cardiac isoform of the enzyme has a higher affinity for lidocaine. Halothane inhibited and aggregated the Ca-ATPase in cardiac SR. Both inhibition and aggregation of the Ca-ATPase by halothane were much greater in the presence of PLB antibody or when PLB was phosphorylated, indicating a protective effect of PLB on halothane-induced inhibition and aggregation. The effects of halothane on cardiac SR are opposite from the effects of halothane observed in skeletal SR, where halothane activates and dissociates the Ca-ATPase. These results underscore the crucial role of protein-protein interactions on Ca-ATPase regulation and anesthetic perturbation of cardiac SR.     

Heterogeneous distribution of calmodulin- and cAMP-dependent regulation of Ca2+ uptake in cardiac sarcoplasmic reticulum subfractions

The activity of the Ca2+-pumping ATPase of cardiac sarcoplasmic reticulum is controlled by the phosphorylation level of the intrinsic membrane protein phospholamban. Phospholamban monomers contain two distinct phosphorylation sites for either the cAMP-dependent or the calmodulin-dependent kinase. The two kinases, however, preferentially phosphorylate different populations of phospholamban molecules and double phosphorylation of the same subunit by their concerted action is a phenomenon that occurs only under particular experimental conditions. This study investigates the phosphorylation pattern of phospholamban in various subfractions derived from dog cardiac sarcoplasmic reticulum. The results show that the endogenous calmodulin-dependent kinase preferentially phosphorylates the phospholamban population found in association with the cisternal compartments of the reticulum. The differential phosphorylation occurs despite the presence of sufficient amounts of the kinase in all sarcoplasmic reticulum subfractions. On the other hand, phospholamban molecules localized on the longitudinal system are preferential substrates for the cAMP-dependent kinase. Possibly, the different lipid and/or protein microenvironment of phospholamban in the various sarcoplasmic reticulum domains is responsible for the apparent heterogeneity of phosphorylation. The present findings are compatible with the concept of additive and independent action of the cAMP-dependent and calmodulin-dependent kinases on cardiac sarcoplasmic reticulum. The imply, however, that different regions of the sarcoplasmic reticulum network are controlled by the two regulatory mechanisms.     

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.     

An autoinhibitory peptide from the erythrocyte Ca-ATPase aggregates and inhibits both muscle Ca-ATPase isoforms.

We have studied the effects of C28R2, a basic peptide derived from the autoinhibitory domain of the plasma membrane Ca-ATPase, on enzyme activity, oligomeric state, and E1-E2 conformational equilibrium of the Ca-ATPase from skeletal and cardiac sarcoplasmic reticulum (SR). Time-resolved phosphorescence anisotropy (TPA) was used to determine changes in the distribution of Ca-ATPase among its different oligomeric species in SR. C28R2, at a concentration of 1-10 microM, inhibits the Ca-ATPase activity of both skeletal and cardiac SR (CSR). In skeletal SR, this inhibition by C28R2 is much greater at low (0.15 microM) than at high (10 microM) Ca2+, whereas in CSR the inhibition is the same at low and high Ca2+. The effects of the peptide on the rotational mobility of the Ca-ATPase correlated well with function, indicating that C28R2-induced protein aggregation and Ca-ATPase inhibition are much more Ca-dependent in skeletal than in CSR. In CSR at low Ca2+, phospholamban (PLB) antibody (functionally equivalent to PLB phosphorylation) increased the inhibitory effect of C28R2 slightly. Fluorescence of fluorescein 5-isothiocyanate-labeled SR suggests that C28R2 stabilizes the E1 conformation of the Ca-ATPase in skeletal SR, whereas in CSR it stabilizes E2. After the addition of PLB antibody, C28R2 still stabilizes the E2 conformational state of CSR. Therefore, we conclude that C28R2 affects Ca-ATPase activity, conformation, and self-association differently in cardiac and skeletal SR and that PLB is probably not responsible for the differences.     

Comparable levels of Ca-ATPase inhibition by phospholamban in slow-twitch skeletal and cardiac sarcoplasmic reticulum

Alterations in expression levels of phospholamban (PLB) relative to the sarcoplasmic reticulum (SR) Ca-ATPase have been suggested to underlie defects of calcium regulation in the failing heart and other cardiac pathologies. To understand how variation in PLB expression relative to that of the Ca-ATPase can modulate calcium transport, we have investigated the inhibition of the Ca-ATPase by PLB in native SR membranes from slow-twitch skeletal and cardiac muscle and in reconstituted proteoliposomes. Quantitative immunoblotting in combination with affinity-purified protein standards was used to measure protein concentrations of PLB and of the Ca-ATPase. Functional inhibition of the Ca-ATPase was determined from both the calcium concentrations for half-maximal activation (Ca(1/2)) and the shift in the calcium concentrations following release of PLB inhibition (i.e., (Delta)Ca(1/2)) by incubation with monoclonal antibodies against PLB, which are equivalent to phosphorylation of PLB by cAMP-dependent protein kinase. We report that equivalent levels of PLB inhibition and antibody-induced activation ((Delta)Ca(1/2) = 0.25 +/- 0.02 microM) are observed in SR membranes from slow-twitch skeletal and cardiac muscle, where molar stoichiometries of PLB expressed per Ca-ATPase vary, respectively, from 0.9 +/- 0.1 to 4.1 +/- 0.8. Similar levels of inhibition to those observed in isolated SR vesicles were observed using reconstituted proteoliposomes following co-reconstitution of affinity-purified Ca-ATPase with PLB. These results indicate that total expression levels of one PLB per Ca-ATPase result in full inhibition of the Ca-ATPase and, based on the measured K(D) (140 +/- 30 microM), suggests one PLB complexed with two Ca-ATPase molecules is sufficient for full inhibition of activity. Therefore, the excess PLB expressed in the heart over that required for inhibition suggests a capability for graded responses of the Ca-ATPase activity to endogenous kinases and phosphatases that modulate the level of phosphorylation necessary to relieve inhibition of the Ca-ATPase by PLB.     

Calcium/calmodulin-dependent protein kinase II activity is increased in sarcoplasmic reticulum from coronary artery ligated rabbit hearts.

A protein kinase activity intrinsic to the sarcoplasmic reticulum was studied in normal and hypertrophied rabbit hearts. The relationship between this kinase activity and phospholamban phosphorylation was examined. Calmodulin-dependent kinase II activity was found to be increased in sarcoplasmic reticulum preparations from hypertrophied hearts compared with normal. This was evident by measuring the phosphotransferase activity of the kinase and also by examining phospholamban phosphorylation by electrophoretic band shift analysis. Increased phospholamban phosphorylation by Calmodulin-dependent protein kinase II was dependent on prior phosphorylation by cAMP-dependent protein kinase, indicating potential crosstalk. Specific immunoblot analysis of the rabbit sarcoplasmic reticulum identified the presence of the delta form of calmodulin dependent protein kinase II and showed it to be up-regulated in hypertrophied hearts.     

Phosphorylation of phospholamban by calcium-activated, phospholipid-dependent protein kinase. Stimulation of cardiac sarcoplasmic reticulum calcium uptake

Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) is able to catalyze the phosphorylation of phospholamban in a canine cardiac sarcoplasmic reticulum preparation. This phosphorylation is associated with a 2-fold stimulation of Ca2+ uptake by cardiac sarcoplasmic reticulum similar to that seen following phosphorylation of phospholamban by an endogenous calmodulin-dependent protein kinase or by the catalytic subunit of cAMP-dependent protein kinase. Two-dimensional peptide maps of the tryptic fragments of phospholamban indicate that the three protein kinases differ in their selectivity for sites of phosphorylation. However, one common peptide appears to be phosphorylated by all three protein kinases. These findings suggest that protein kinase C may play a role similar to those played by cAMP- and calmodulin-dependent protein kinases in the regulation of Ca2+ uptake by cardiac sarcoplasmic reticulum, and raise the possibility that the effects of all three protein kinases are mediated through phosphorylation of a common peptide in phospholamban.     

Microsecond rotational dynamics of spin-labeled Ca-ATPase during enzymatic cycling initiated by photolysis of caged ATP.

We have measured the microsecond rotational motions of the sarcoplasmic reticulum (SR) Ca-ATPase as a function of enzyme-specific ligands, including those that induce active calcium transport. We labeled the Ca-ATPase with a maleimide spin probe and detected rotational dynamics using saturation-transfer electron paramagnetic resonance (ST-EPR). This probe's ST-EPR spectra have been shown to be sensitive to microsecond protein rotational motion, corresponding to large-scale protein rotations that should be affected by changes in the enzyme's shape, flexibility, protein-protein interactions (oligomeric state), and protein-lipid interactions. We found that the motions of the enzyme-nucleotide and the enzyme-nucleotide/Ca states are indistinguishable from the motions in the absence of ligands. Rotational mobility does decrease in response to the addition of DMSO, a solvent that inhibits Ca-ATPase activity and stabilizes the phosphoenzyme. However, the addition of phosphate to form phosphoenzyme, in the presence or absence of DMSO, does not change the motions significantly. During the steady state of active calcium transport, the microsecond rotational mobility is indistinguishable from that of the resting enzyme. In order to detect any transient changes in mobility that might not be detectable in the steady state and to improve the precision of steady-state measurements, we photolyzed caged ATP with a laser pulse in the presence of calcium and detected the ST-EPR response from the spin-labeled enzyme, with a time resolution of 1 s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Ca2+ transport by cyclic 3′,5′-AMP-dependent and calcium-calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by cyclic AMP-dependent and by Ca²⁺-calmodulin-dependent protein kinases on a 22 kDa protein, called phospholamban. Both types of phosphorylation have been shown to stimulate the initial rates of Ca²⁺ transport. To establish the interrelationship of the cAMP-dependent and Ca²⁺-calmodulin-dependent phosphorylation on Ca²⁺ transport, cardiac sarcoplasmic reticulum vesicles were preincubated under optimum conditions for: (a) cAMP-dependent phosphorylation, (b) Ca²⁺-calmodulin-dependent phosphorylation, and (c) combined cAMP-dependent and Ca²⁺-calmodulin-dependent phosphorylation. Control vesicles were treated under identical conditions, but in the absence of ATP, to avoid phosphorylation. Control and phosphorylated sarcoplasmic reticulum vesicles were subsequently centrifuged and assayed for Ca²⁺ transport in the presence of 2.5 mM Tris-oxalate. Our results indicate that cAMP-dependent and Ca²⁺-calmodulin-dependent phosphorylation can each stimulate calcium transport in an independent manner and when both are operating, they appear to have an additive effect. Stimulation of Ca²⁺ transport was associated with a statistically significant increase in the apparent affinity for calcium by each type of phosphorylation. The degree of stimulation of the calcium affinity was relatively proportional to the degree of phospholamban phosphorylation. These findings suggest the presence of a dual control system which may operate in independent and combined manners for regulating cardiac sarcoplasmic reticulum function.     

Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum

Phospholamban, the putative protein regulator of the Ca2+ pump of cardiac sarcoplasmic reticulum, was purified to apparent homogeneity from canine cardiac sarcoplasmic reticulum vesicles by selective extraction with sodium cholate, followed by adsorption to calcium oxalate, solubilization in Zwittergent 3-14, and specific elution from p-hydroxymercuribenzoate-agarose. Phospholamban, isolated in the dephosphorylated state, was purified 80-fold in 15% yield (approximately 2 mg of phospholamban/g of sarcoplasmic reticulum protein). Nondissociated phospholamban exhibited an apparent Mr = 25,000 in sodium dodecyl sulfate-polyacrylamide gels. Partially dissociated phospholamban, induced by boiling in sodium dodecyl sulfate, exhibited five distinct mobility forms in sodium dodecyl sulfate-polyacrylamide gels, of apparent molecular weights between 5,000-6,000 and 25,000. Phospholamban was phosphorylated to a level of 190 nmol of Pi/mg of protein by cAMP-dependent protein kinase, consistent by minimum stoichiometry with a subunit molecular weight of approximately 5,000. Phospholamban prepared by the present method was different in several respects from the proteins that have been isolated in other laboratories. Pure phospholamban was cysteine rich, containing 6 residues/100 amino acid residues. Dephosphorylated phospholamban was strongly basic with a pI = 10; phosphorylation decreased the pI to approximately 6.7. Pure phospholamban (and phospholamban present in sarcoplasmic reticulum vesicles) was not readily extracted into acidified chloroform/methanol, suggesting that the protein does not behave as an acidic proteolipid. The purified protein was highly antigenic. Phospholamban was localized by immunochemical methods to cardiac membranes enriched in sarcoplasmic reticulum, but was absent from sarcoplasmic reticulum membranes prepared from fast skeletal muscle. The method described for isolation of cardiac phospholamban is highly reproducible and relatively simple, and should be useful for further detailed studies designed to probe the molecular structure of the protein.     

Structural and functional modifications of phospholamban and regulation of calcium transport in the sarcoplasmic reticulum under conditions of metabolic cardiac insufficiency

The phosphorylation of phospholamban from dog myocardium is shown to correlate with the protein sensitivity to tryptic attack both in undamaged myocardium and under conditions of circulatory hypoxia and experimental infarction. In the absence of "exogenous" protein kinase a decreased phosphorylation of phospholamban is observed in the incubation mixture containing 10(-6) M cAMP both for circulatory hypoxia and myocardial infarction. In the latter case the phosphorylation remains diminished in the presence of "exogenous" protein kinase as well. The sarcoplasmic reticulum from damaged myocardium exhibited changes in the velocity of the oxalate-dependent transport of calcium which correlated with the corresponding degree of the phospholamban phosphorylation.     

A phospholamban protein phosphatase activity associated with cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic phospholamban protein phosphatase activity, which is also effective in dephosphorylating phosphorylase a. The phosphatase associated with sarcoplasmic reticulum membranes was solubilized with Triton X-100 and subjected to chromatography on Mono Q HR 5/5 and polylysine-agarose. A single peak of phosphatase activity was eluted from each column and it was coincident for both phospholamban and phosphorylase a, used as substrates. Thermal denaturation of the enzyme resulted in progressive and coincident loss of both phospholamban and phosphorylase a phosphatase activities. Enzymic activity was partially inhibited by protein phosphatase inhibitor 1. Migration of the enzyme during sucrose density gradient ultracentrifugation corresponded to a globular protein with an apparent Mr of 46,000. This enzyme preparation could dephosphorylate both the calcium-calmodulin-dependent as well as the cAMP-dependent sites on phospholamban. Thus, dephosphorylation of phospholamban by this sarcoplasmic reticulum-associated phosphatase may participate in modulating sarcoplasmic reticulum function in cardiac muscle.