ATPase Of Cardiac Sarcoplasmic Reticulum Research Articles

Super-resolution Imaging of Cardiac Sarcoplasmic Reticulum ATPase in Relation to Myofibril Distribution in Rat Tissue Sections

Super-resolution Imaging of Cardiac Sarcoplasmic Reticulum ATPase in Relation to Myofibril Distribution in Rat Tissue Sections

Plasma Antioxidant Enzyme Levels in Acute Coronary Syndrome

had increased by more than 100% and neuronal excitation was observed within the paraventricular nucleus of the hypothalamus (PVN), as determined by Fra-protein expression. In contrast, similar levels of nNOS positive cellswere observed in PVNof the shamandMI rats. These experiments suggest that sympathetic activation occurs early in the progression of heart failure, independent of changes in nNOS regulation in the PVN. http://dx.doi.org/10.1016/j.hlc.2012.08.028 Distribution of G s and Co-localisation with Calcium Handling Proteins in Rat Cardiomyocytes M.L. Munro, D. Baddelely, C. Soeller Department of Physiology, School ofMedical Sciences, University of Auckland Background: The β-adrenergic pathway modulates cardiac function via phosphorylation of proteins involved in excitation-contraction-coupling, including RyR and SERCA.G s is one of the components of this pathway and it has been suggested that proteins in this pathway form signalosomes, which may localise to caveolae. Methods: Isolated rat cardiomyocytes were immunolabelled for G s with caveolin-3, RyR or SERCA and imaged using either confocal laser scanning microscopy or superresolution localisation microscopy. taneous coronary intervention. Impedance aggregometry in whole blood was used to measure platelet reactivity in response to both arachidonic acid and ADP stimulation. Plasma GPx and SOD activity levels were measured using commercially available colourimetric assay kits. In 76 ACS patients we report that there was a significant correlation betweenplatelet reactivity andGPx activity levels (p< 0.05) in response to arachidonic acid stimulation. Further investigation into themechanismof this relationship is the focus of this ongoing study. http://dx.doi.org/10.1016/j.hlc.2012.08.030 Super-resolution Imaging of Cardiac Sarcoplasmic ReticulumATPase inRelation toMyofibrilDistribution inRat

Highly Specific, Conformationally-Dependent Cross-Linking of Lys27 of PLB to SERCA2a in Cardiac SR Vesicles from Humans

Phospholamban (PLB) inhibits SERCA2a, the Ca2+-ATPase of cardiac sarcoplasmic reticulum (SR), by decreasing the apparent Ca2+ affinity of the enzyme. The mechanism of Ca2+ pump inhibition by PLB has been studied by our group using chemical cross-linking agents, which enable PLB-binding interactions with SERCA2a to be measured simultaneously with enzyme inhibition. Previously, cross-linking of canine PLB to SERCA2a was only attainable with Cys-scanning point mutants of PLB. For example, N27C of canine PLB cross-links exclusively to Lys328 of canine SERCA2a with heterobifunctional thiol-to-amine cross-linking agents after co-expression of the two proteins in Sf21 insect cells. Here, we show with SR vesicles prepared from human hearts, that PLB and SERCA2a are cross-linkable using DSG (disuccinimidyl glutarate), a 7.7 Å long, homobifunctional, amine-specific cross-linking agent. Cross-linking of human PLB to SERCA2a takes advantage of the unique Lys residue at position 27 of human PLB, making it susceptible to amine-specific cross-linkers without the need for mutagenesis. This was confirmed by testing SR vesicles prepared from both human and canine ventricles; DSG cross-linked human, but not canine, PLB to SERCA2a. Cross-linking of human PLB to SERCA2a was completely inhibited by either Ca2+ (Ki = 0.50 µM), or the Ca2+ pump inhibitor thapsigargin, but substantially augmented by ATP. This is the first demonstration that PLB binds exclusively to the E2 conformation of SERCA2a in SR vesicles, preferentially the state with bound nucleotide (E2·ATP), and not the state stabilized by thapsigargin (protonated E2). Importantly, similar results were obtained with SR vesicles prepared from both normal and failed human hearts, indicating that PLB-binding interactions with SERCA2a are unchanged in failing myocardium. Studies are in progress to demonstrate with human SR vesicles that Lys27 of PLB cross-links exclusively to Lys328 of SERCA2a.

Ca2+ Binding to Site I of the Cardiac Ca2+ Pump Is Sufficient to Dissociate Phospholamban

Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca(2+). Recent cross-linking studies have suggested that PLB binding and Ca(2+) binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca(2+)-ATPase, preventing formation of E1, the conformation that binds two Ca(2+) (at sites I and II) with high affinity and is required for ATP hydrolysis. Here we determined whether Ca(2+) binding to site I, site II, or both sites is sufficient to dissociate PLB from the Ca(2+) pump. Seven SERCA2a mutants with amino acid substitutions at Ca(2+)-binding site I (E770Q, T798A, and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q) were made, and the effects of Ca(2+) on N30C-PLB cross-linking to Lys(328) of SERCA2a were measured. In agreement with earlier reports with the skeletal muscle Ca(2+)-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP in the presence of Ca(2+); however, all were phosphorylatable by P(i) to form E2P. Ca(2+) inhibition of E2P formation was observed only in SERCA2a mutants retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB and each Ca(2+)-ATPase mutant was observed in the absence of Ca(2+). Importantly, however, micromolar Ca(2+) inhibited PLB cross-linking only to mutants retaining a functional Ca(2+)-binding site I. The dynamic equilibrium between Ca(2+) pumps and N30C-PLB was retained by all mutants, demonstrating normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody. From these results we conclude that site I is the key Ca(2+)-binding site regulating the physical association between PLB and SERCA2a.

The Ca2+ ATPase of cardiac sarcoplasmic reticulum: Physiological role and relevance to diseases

The Ca2+ ATPase of sarcoplasmic reticulum has a prominent role in excitation/contraction coupling of cardiac muscle, as it induces relaxation by sequestering Ca2+ from the cytoplasm. The stored Ca2+ is in turn released to trigger contraction. We review here experiments demonstrating that in cardiac myocytes Ca2+ signaling and contractile activation are strongly altered by pharmacological inhibition or transcriptional down-regulation of SERCA. On the other hand, kinetics, and intensity of Ca2+ signaling are improved by SERCA overexpression following delivery of exogenous cDNA by adenovirus vectors. Experiments on adrenergic hypertrophy demonstrate SERCA down-regulation, consistent with its pathogenetic involvement in cardiac hypertrophy and failure, as also shown in other experimental models and clinical studies. Compensation by alternate Ca2+ signaling proteins, including functional activation and increased expression of Na+/Ca2+ exchanger and TRPC proteins has been observed. These compensatory mechanisms, including calcineurin activation, remain to be clarified and are a most important subject of current studies.

Central Role for the Cardiotonic Steroid Marinobufagenin in the Pathogenesis of Experimental Uremic Cardiomyopathy

Patients with chronic renal failure develop a "uremic" cardiomyopathy characterized by diastolic dysfunction, cardiac hypertrophy, and systemic oxidant stress. Patients with chronic renal failure are also known to have increases in the circulating concentrations of the cardiotonic steroid marinobufagenin (MBG). On this background, we hypothesized that elevations in circulating MBG may be involved in the cardiomyopathy. First, we observed that administration of MBG (10 microg/kg per day) for 4 weeks caused comparable increases in plasma MBG as partial nephrectomy at 4 weeks. MBG infusion caused increases in conscious blood pressure, cardiac weight, and the time constant for left ventricular relaxation similar to partial nephrectomy. Decreases in the expression of the cardiac sarcoplasmic reticulum ATPase, cardiac fibrosis, and systemic oxidant stress were observed with both MBG infusion and partial nephrectomy. Next, rats were actively immunized against a MBG-BSA conjugate or BSA control, and partial nephrectomy was subsequently performed. Immunization against MBG attenuated the cardiac hypertrophy, impairment of diastolic function, cardiac fibrosis, and systemic oxidant stress seen with partial nephrectomy without a significant effect on conscious blood pressure. These data suggest that the increased concentrations of MBG are important in the cardiac disease and oxidant stress state seen with renal failure.

Free radical-induced protein modification and inhibition of Ca2+-ATPase of cardiac sarcoplasmic reticulum.

The effect of oxidative stress on the Ca2+-ATPase activity, lipid peroxidation and protein modification of cardiac sarcoplasmic reticulum (SR) membranes was investigated. Isolated SR vesicles were exposed to FeSO4/EDTA (0.2 micromol Fe2+ per mg of protein) at 37 degrees C for 1 h in the presence or absence of antioxidants. FeSO4/EDTA decreased the maximum velocity of Ca2+-ATPase reaction without a change of affinity for Ca2+ or Hill coefficient. Treatment with radical-generating system led also to conjugated diene formation, loss of sulfhydryl groups, changes in tryptophan and bityrosine fluorescences and to production of lysine conjugates with lipid peroxidation end-products. Lipid antioxidants butylated hydroxytoluene (BHT) and stobadine partially prevented inhibition of Ca2+-ATPase and decrease in tryptophan fluorescence, while the loss of -SH groups and formation of bityrosines or lysine conjugates were completely prevented. Glutathione also partially protected Ca2+-ATPase activity and decreased formation of bityrosine, but it was not able to prevent oxidative modification of tryptophan and lysine. These findings suggest that combination of amino acid modifications, rather than oxidation of amino acids of one kind, is responsible for inhibition of SR Ca2+-ATPase activity.

S2-4 Intermolecular interactions between phospholamban and the calcium ATPase of cardiac sarcoplasmic reticulum

S2-4 Intermolecular interactions between phospholamban and the calcium ATPase of cardiac sarcoplasmic reticulum

Close Proximity between Residue 30 of Phospholamban and Cysteine 318 of the Cardiac Ca2+ Pump Revealed by Intermolecular Thiol Cross-linking

Phospholamban (PLB) is a 52-amino acid inhibitor of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SERCA2a), which acts by decreasing the apparent affinity of the enzyme for Ca(2+). To localize binding sites of SERCA2a for PLB, we performed Cys-scanning mutagenesis of PLB, co-expressed the PLB mutants with SERCA2a in insect cell microsomes, and tested for cross-linking of the mutated PLB molecules to SERCA2a using 1,6-bismaleimidohexane, a 10-A-long, homobifunctional thiol cross-linking agent. Of several mutants tested, only PLB with a Cys replacement at position 30 (N30C-PLB) cross-linked to SERCA2a. Cross-linking occurred specifically and with high efficiency. The process was abolished by micromolar Ca(2+) or by an anti-PLB monoclonal antibody and was inhibited 50% by phosphorylation of PLB by cAMP-dependent protein kinase. The SERCA2a inhibitors thapsigargin and cyclopiazonic acid also completely prevented cross-linking. The two essential requirements for cross-linking of N30C-PLB to SERCA2a were a Ca(2+)-free enzyme and, unexpectedly, a micromolar concentration of ATP or ADP, demonstrating that N30C-PLB cross-links preferentially to the nucleotide-bound, E2 state of SERCA2a. Sequencing of a purified proteolytic fragment in combination with SERCA2a mutagenesis identified Cys(318) of SERCA2a as the sole amino acid cross-linked to N30C-PLB. The proximity of residue 30 of PLB to Cys(318) of SERCA2a suggests that PLB may interfere with Ca(2+) activation of SERCA2a by a protein interaction occurring near transmembrane helix M4.

Reconstitution of the cytoplasmic interaction between phospholamban and Ca(2+)-ATPase of cardiac sarcoplasmic reticulum.

Phospholamban (PLN) reversibly inhibits the Ca(2+)-ATPase of cardiac sarcoplasmic reticulum (SERCA2a) through a direct protein-protein interaction, playing a pivotal role in the regulation of intracellular Ca(2+) in heart muscle cells. The interaction between PLN and SERCA2a occurs at multiple sites within the cytoplasmic and membrane domains. Here, we have reconstituted the cytoplasmic protein-protein interaction using bacterially expressed fusion proteins of the cytoplasmic domain of PLN and the long cytoplasmic loop of SERCA2a. We have developed two methods to evaluate the binding of the fusion proteins, one with glutathione-Sepharose beads and the other with a 96-well plate. Essentially the same results were obtained by the two methods. The affinity of the binding (K(D)) was 0.70 microM. The association was inhibited by cAMP-dependent phosphorylation of the PLN fusion protein and by usage of anti-PLN monoclonal antibody. It was also diminished by substitution at the phosphorylation site of PLN of Ser(16) to Asp. These results suggest that PLN can bind SERCA2a in the absence of the membrane domains and that the modifications of the cytoplasmic domain of PLN that activate SERCA2a parallel the disruption of the association between the two fusion proteins. It has been shown that the removal of PLN inhibition of SERCA2a rescues cardiac function and morphology in the mouse dilated cardiomyopathy model. Our assay system can be applied to the screening of novel inotropic agents that remove the inhibition of SERCA2a by PLN, improving the relaxation as well as the contractility of the failing heart.

The effect of Mg2+ on cardiac muscle function: is CaATP the substrate for priming myofibril cross-bridge formation and Ca2+ reuptake by the sarcoplasmic reticulum?

Kinetics are established for the activation of the myofibril from the relaxed state [Smith, Dixon, Kirschenlohr, Grace, Metcalfe and Vandenberg (2000) Biochem. J. 346, 393–402]. These require two troponin Ca2+-binding sites, one for each myosin head, to act as a single unit in initial cross-bridge formation. This defines the first, or activating, ATPase reaction, as distinct from the further activity of the enzyme that continues when a cross-bridge to actin is already established. The pairing of myosin heads to act as one unit suggests a possible alternating mechanism for muscle action. A large positive inotropic (contraction-intensifying) effect of loading the Mg2+ chelator citrate, via its acetoxymethyl ester, into the heart has confirmed the competitive inhibition of the Ca2+ activation by Mg2+, previously seen in vitro. In the absence of a recognized second Ca2+-binding site on the myofibril, with appropriate binding properties, the bound ATP is proposed as the second activating Ca2+-binding site. As ATP, free or bound to protein, can bind either Mg2+ or Ca2+, this leads to competitive inhibition by Mg2+. Published physico-chemical studies on skeletal muscle have shown that CaATP is potentially a more effective substrate than MgATP for cross-bridge formation. The above considerations allow calculation of the observed variation of fractional activation by Ca2+ as a function of [Mg2+] and in turn reveal simple Michaelis–Menten kinetics for the activation of the ATPase by sub-millimolar [Mg2+]. Furthermore the ability of bound ATP to bind either cation, and the much better promotion of cross-bridge formation by CaATP binding, give rise to the observed variation of the Hill coefficient for Ca2+ activation with altered [Mg2+]. The inclusion of CaADP within the initiating cross-bridge and replacement by MgADP during the second cycle is consistent with the observed fall in the rate of the myofibril ATPase that occurs after two phosphates are released. The similarity of the kinetics of the cardiac sarcoplasmic reticulum ATPase to those of the myofibril, in particular the positive co-operativity of both Mg2+ inhibition and Ca2+ activation, leads to the conclusion that this ATPase also has an initiation step that utilizes CaATP. The first-order activation by sub-millimolar [Mg2+], similar to that of the myofibril, may be explained by Mg2+ involvement in the phosphate-release step of the ATPase. The inhibition of both the myofibril and sarcoplasmic reticulum Ca2+-transporting ATPases by Mg2+ offers an explanation for the specific requirement for phosphocreatine (PCr) for full activity of both enzymes in situ and its effect on their apparent affinities for ATP. This explanation is based on the slow diffusion of Mg2+ within the myofibril and on the contrast of PCr with both ATP and phosphoenolpyruvate, in that PCr does not bind Mg2+ under physiological conditions, whereas both the other two bind it more tightly than the products of their hydrolysis do. The switch to supply of energy by diffusion of MgATP into the myofibril when depletion of PCr raises [ATP]/[PCr] greatly, e.g. during anoxia, results in a local [Mg2+] increase, which inhibits the ATPase. It is possible that mechanisms similar to those described above occur in skeletal muscle but the Ca2+ co-operativity involved would be masked by the presence of two Ca2+-binding sites on each troponin.

The effect of Mg2+ on cardiac muscle function: Is CaATP the substrate for priming myofibril cross-bridge formation and Ca2+ reuptake by the sarcoplasmic reticulum?

Kinetics are established for the activation of the myofibril from the relaxed state [Smith, Dixon, Kirschenlohr, Grace, Metcalfe and Vandenberg (2000) Biochem. J. 346, 393-402]. These require two troponin Ca2+-binding sites, one for each myosin head, to act as a single unit in initial cross-bridge formation. This defines the first, or activating, ATPase reaction, as distinct from the further activity of the enzyme that continues when a cross-bridge to actin is already established. The pairing of myosin heads to act as one unit suggests a possible alternating mechanism for muscle action. A large positive inotropic (contraction-intensifying) effect of loading the Mg2+ chelator citrate, via its acetoxymethyl ester, into the heart has confirmed the competitive inhibition of the Ca2+ activation by Mg2+, previously seen in vitro. In the absence of a recognized second Ca2+ binding site on the myofibril, with appropriate binding properties, the bound ATP is proposed as the second activating Ca2+-binding site. As ATP, free or bound to protein, can bind either Mg2+ or Ca2+, this leads to competitive inhibition by Mg2+. Published physico-chemical studies on skeletal muscle have shown that CaATP is potentially a more effective substrate than MgATP for cross-bridge formation. The above considerations allow calculation of the observed variation of fractional activation by Ca2+ as a function of [Mg2+] and in turn reveal simple Michaelis-Menten kinetics for the activation of the ATPase by sub-millimolar [Mg2+]. Furthermore the ability of bound ATP to bind either cation, and the much better promotion of cross-bridge formation by CaATP binding, give rise to the observed variation of the Hill coefficient for Ca2+ activation with altered [Mg2+]. The inclusion of CaADP within the initiating cross-bridge and replacement by MgADP during the second cycle is consistent with the observed fall in the rate of the myofibril ATPase that occurs after two phosphates are released. The similarity of the kinetics of the cardiac sarcoplasmic reticulum ATPase to those of the myofibril, in particular the positive co-operativity of both Mg2+ inhibition and Ca2+ activation, leads to the conclusion that this ATPase also has an initiation step that utilizes CaATP. The first-order activation by sub-millimolar [Mg2+], similar to that of the myofibril, may be explained by Mg2+ involvement in the phosphate-release step of the ATPase. The inhibition of both the myofibril and sarcoplasmic reticulum Ca2+ transporting ATPases by Mg2+ offers an explanation for the specific requirement for phosphocreatine (PCr) for full activity of both enzymes in situ and its effect on their apparent affinities for ATP. This explanation is based on the slow diffusion of Mg2+ within the myofibril and on the contrast of PCr with both ATP and phosphoenolpyruvate, in that PCr does not bind Mg2+ under physiological conditions, whereas both the other two bind it more tightly than the products of their hydrolysis do. The switch to supply of energy by diffusion of MgATP into the myofibril when depletion of PCr raises [ATP]/[PCr] greatly, e.g. during anoxia, results in a local [Mg2+] increase, which inhibits the ATPase. It is possible that mechanisms similar to those described above occur in skeletal muscle but the Ca2+ co-operativity involved would be masked by the presence of two Ca2+ binding sites on each troponin.

THE DECOMPENSATED DETRUSOR III: IMPACT OF BLADDER OUTLET OBSTRUCTION ON SARCOPLASMIC ENDOPLASMIC RETICULUM PROTEIN AND GENE EXPRESSION

THE DECOMPENSATED DETRUSOR III: IMPACT OF BLADDER OUTLET OBSTRUCTION ON SARCOPLASMIC ENDOPLASMIC RETICULUM PROTEIN AND GENE EXPRESSION

Physical association between the cytoplasmic domains of the Ca2+-ATPase of cardiac sarcoplasmic reticulum and phospholamban

Physical association between the cytoplasmic domains of the Ca2+-ATPase of cardiac sarcoplasmic reticulum and phospholamban

Serine Phosphorylation of the Sarcoplasmic Reticulum Ca2+-ATPase in the Intact Beating Rabbit Heart

Recent studies have demonstrated that Ca2+/calmodulin-dependent protein kinase phosphorylates the Ca2+-pumping ATPase of cardiac sarcoplasmic reticulum (SR) in vitro. Also, evidence from in vitro studies suggested that this phosphorylation, occurring at Ser38, results in stimulation of Ca2+ transport. In the present study, we investigated whether serine phosphorylation of the SR Ca2+-ATPase occurs in the intact functioning heart. Hearts removed from anesthetized rabbits were subjected to retrograde aortic perfusion of the coronary arteries with oxygenated mammalian Ringer solution containing 32Pi and contractions were monitored by recording systolic left ventricular pressure development. Following 45–50 min of 32P perfusion, the hearts were freeze-clamped, SR isolated, and analyzed for protein phosphorylation. SDS–polyacrylamide gel electrophoresis and autoradiography showed phosphorylation of several peptides including the Ca2+-ATPase and Ca2+ release channel (ryanodine receptor). The identity of Ca2+-ATPase as a phosphorylated substrate was confirmed by Western immunoblotting as well as immunoprecipitation using a cardiac SR Ca2+-ATPase-specific monoclonal antibody. The Ca2+-ATPase showed immunoreactivity with a phosphoserine monoclonal antibody indicating that the in situ phosphorylation occurred at the serine residue. Quantification of Ca2+-ATPase phosphorylation in situ yielded a value of 208 ± 12 pmol 32P/mg SR protein which corresponded to the phosphorylation of ∼20% of the Ca2+ pump units in the SR membrane. Since this phosphorylation occurred under basal conditions (i.e., in the absence of any inotropic intervention), a considerable steady-state pool of serine-phosphorylated Ca2+-ATPase likely exists in the normally beating heart. These findings demonstrate that serine phosphorylation of the Ca2+-ATPase is a physiological event which may be important in the regulation of SR function.

Phospholamban: protein structure, mechanism of action, and role in cardiac function.

A comprehensive discussion is presented of advances in understanding the structure and function of phospholamban (PLB), the principal regulator of the Ca2+-ATPase of cardiac sarcoplasmic reticulum. Extensive historical studies are reviewed to provide perspective on recent developments. Phospholamban gene structure, expression, and regulation are presented in addition to in vitro and in vivo studies of PLB protein structure and activity. Applications of breakthrough experimental technologies in identifying PLB structure-function relationships and in defining its interaction with the Ca2+-ATPase are also highlighted. The current leading viewpoint of PLB's mechanism of action emerges from a critical examination of alternative hypotheses and the most recent experimental evidence. The potential physiological relevance of PLB function in human heart failure is also covered. The interest in PLB across diverse biochemical disciplines portends its continued intense scrutiny and its potential exploitation as a therapeutic target.

Phencyclidine block of Ca 2+ ATPase in rat heart sarcoplasmic reticulum

Phencyclidine hydrochloride (PCP) also known as Angel Dust is a very potent psychotomimetic drug of abuse. Besides its central nervous system (CNS) effects PCP produces a number of adverse effects in a variety of tissues including the cardiovascular system. Since PCP is known to alter the cellular calcium homeostasis the present studies were initiated to determine the changes in cardiac Ca 2+ ATPase activity in rats treated with PCP. For in vitro studies the cardiac sarcoplasmic reticulum (SR) fractions prepared from normal rats were incubated with 25, 50 and 100 μM PCP and the enzyme activities were estimated. Whereas, for in vivo studies the cardiac SR fractions prepared from rats treated with PCP (10 mg/kg body wt. single dose, intra-peritoneally (i.p.)) and sacrificed at different time intervals were used. PCP reduced the Ca 2+ ATPase activity significantly both in vitro and in vivo. A 50% inhibition of the enzyme activity was obtained with 100 μM PCP in vitro. A significant reduction of SR Ca 2+ ATPase was also evident as early as 1 h after treatment of rats with PCP. The reduction of Ca 2+ ATPase activity in SR was irreversible even at 12 h after treatment. The in vitro kinetic studies revealed that PCP was found to be a competitive inhibitor of Ca 2+ ATPase with respect to the substrate, ATP, and non-competitive with respect to Ca 2+ activation. These results indicate that PCP alters the myocardial Ca 2+ homeostasis by inhibiting the Ca 2+ ATPase in cardiac SR in rats. Inhibition of SR Ca 2+ ATPase may result in the impairment of contraction and relaxation coupling processes in the myocardium.

Phospholamban domain I/cytochrome b5 transmembrane sequence chimeras do not inhibit SERCA2a

A series of chimeras between the transmembrane domains of phospholamban (PLN) and cytochrome b 5 were coexpressed with the Ca 2+-ATPase of cardiac sarcoplasmic reticulum (SERCA2a). The chimeric molecules were not inhibitory, in line with our view that inhibitory PLN/SERCA2a interactions occur in transmembrane sequences, while cytoplasmic interactions regulate the inhibitory interactions in a four-base circuit.

Phosphorylation of Na,K-ATPase by protein kinase C at Ser18 occurs in intact cells but does not result in direct inhibition of ATP hydrolysis.

Na,K-ATPase activity has been demonstrated to be regulated by a variety of hormones in different tissues. It is known to be directly phosphorylated on its alpha-subunit, but the functional effects of protein kinases remain controversial. We have developed a sensitive, antibody-based assay for detection of the level of phosphorylation of the alpha1-isoform of rat Na,K-ATPase at the serine residue that is most readily phosphorylated by protein kinase C (PKC) in vitro, Ser18. By stimulation of endogenous PKC and inhibition of phosphatase activity, it was possible to consistently obtain a very high stoichiometry of phosphorylation (close to 0.9) in several types of intact cells. This demonstrates the accessibility and competency of the site for endogenous phosphorylation. The cells used were derived from rat (NRK 52E, C6, L6, and primary cultures of cerebellar granule cells, representing epithelial cells, glia, muscle cells, and neurons). In the presence of the phosphatase inhibitor calyculin A, full phosphorylation was preserved during subsequent assays of enzyme activity in vitro. Assay of the hydrolysis of ATP in NRK and C6 cells, however, indicated that there was no significant effect of phosphorylation on the Vmax of the Na, K-ATPase or on the apparent affinity for Na+. Any regulatory effect of PKC on sodium pump activity thus must be lost upon disruption or permeabilization of the cells and is not a direct consequence of enzyme alteration by covalent phosphorylation of Ser18.

Phosphorylation and regulation of the Ca(2+)-pumping ATPase in cardiac sarcoplasmic reticulum by calcium/calmodulin-dependent protein kinase.

In cardiac muscle, a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase) phosphorylates the Ca(2+)-pumping ATPase in addition to its previously characterized substrates, phospholamban and Ca(2+)-release channel (ryanodine receptor). The phosphorylated amino acid in the Ca(2+)-ATPase has been identified as serine. Posphorylation of the Ca(2+)-ATPase is rapid and is reversible by a membrane-associated protein phosphatase, Ca(2+)-ATPase purified from cardiac SR underwent phosphorylation by exogenous CaM kinase, and the phosphorylated enzyme displayed twofold greater catalytic activity without alteration in its Ca(2+)-sensitivity. The phosphorylation of the Ca(2+)-ATPase was found to be isoform-specific in that the cardiac and slow-twitch skeletal muscle isoform (SERCA 2), but not the fast-twitch skeletal muscle isoform (SERCA 1), underwent phosphorylation by CaM kinase. Studies using SERCA 1 and SERCA 2 isoforms and their mutants expressed in a heterelogous cell system have resulted in i) confirmation of the isoform specificity of Ca(2+)-ATPase phosphorylation by CaM kinase, ii) identification of Ser38 as the site in SERCA 2 phosphorylated by CaM kinase, and iii) demonstration of phosphorylation-induced increase in Vmax of Ca2+ transport by the SERCA 2 enzyme. These observations suggest that in cardiac and slow-twitch skeletal muscle direct phosphorylation of the SR Ca(2+)-ATPase by the membrane-bound CaM kinase may serve to stimulate Ca2+ sequestration and therefore, the speed of muscle relaxation.