Native Sarcoplasmic Reticulum Membranes Research Articles

Calcium-dependent shifts between phospholamban (PLB) monomers and homo-pentamers reveal the interactions between PLB and SERCA in native cardiac membranes.

Phospholamban monomer (PLB1) binds to the cardiac calcium pump (SERCA2a) and inhibits the enzyme. PLB homopentamers (PLB5) are inactive species, but stable in SDS that correlate well to that in the native membranes. In contrast, SDS separates PLB1 from SERCA2a. Using this feature, we developed assays to probe the constant exchanging PLB1 with SERCA2a, and with PLB5, in native cardiac sarcoplasmic reticulum (SR) membrane vesicles. Dog cardiac SR was pre-incubated at 37°C in 1mM EGTA, allowing PLB1 binding to SERCA2a. Then, 8 µg samples were incubated for 1 min with control buffer, 1mM Ca (free [Ca] = 16.4 µM), 1 mM Ca first and then 5 mM EGTA (free [Ca] = 0.06 µM), or 10µM SERCA2a inhibitor thapsigargin. These reactions were stopped by 1% SDS before SDS-PAGE and immunoblotting to measure PLB5/PLB1 ratios. Addition of Ca significantly increased PLB5/PLB1 to 2.91 ± 0.38 from 1.22 ± 0.08 in the control, Ca-free condition, suggesting that Ca activation of the pump dissociates the whole PLB monomer from inhibitory site on SERCA2a in cardiac membranes. Furthermore, subsequent addition of EGTA reversed Ca-induced increased in PLB5/PLB1 back to 1.38 ± 0.15. Meanwhile, thapsigargin virtually eliminated PLB1 binding to SERCA2a, increasing PLB5/PLB1 to 3.13 ± 0.40. In control experiments, none of these factors affected PLB5/PLB1 of WT-PLB expressed alone in insect cells. Therefore, the detected changes in PLB5/PLB1 must occur in the native SR membranes, reflecting fully reversible processes of Ca-dependent PLB1 association and dissociation from the inhibitory site on SERCA2a, followed by disassembling and assemblage into PLB5, respectively. Such assays will allow further studies to determine the effect of allosteric factors and proteins phosphorylation on the interactions between PLB and SERCA2a directly in native cardiac SR membranes.

Structure of RyR1 in native membranes.

Ryanodine receptor 1 (RyR1) mediates excitation–contraction coupling by releasing Ca2+ from sarcoplasmic reticulum (SR) to the cytoplasm of skeletal muscle cells. RyR1 activation is regulated by several proteins from both the cytoplasm and lumen of the SR. Here, we report the structure of RyR1 from native SR membranes in closed and open states. Compared to the previously reported structures of purified RyR1, our structure reveals helix‐like densities traversing the bilayer approximately 5 nm from the RyR1 transmembrane domain and sarcoplasmic extensions linking RyR1 to a putative calsequestrin network. We document the primary conformation of RyR1 in situ and its structural variations. The activation of RyR1 is associated with changes in membrane curvature and movement in the sarcoplasmic extensions. Our results provide structural insight into the mechanism of RyR1 in its native environment.

Screening for SERCA Activators using a High-throughput Time-Resolved FRET Assay

We used a prototype time-resolved fluorescence lifetime microplate reader to carry out a high-throughput screen designed to identify compounds that interact with the sarcoplasmic reticulum calcium ATPase (SERCA). SERCA is essential for the Ca homeostasis in many cell types. Insufficient SERCA activity leads to cardiovascular disease, muscular dystrophy, skin disease, and diabetes. Our goal is to discover activators of SERCA that can be developed into drugs to treat diseases in which Ca transport is deficient. The fluorescence lifetime plate reader was made possible by our recent development of fast time-resolved fluorescence by direct waveform recording, which achieves 105 higher throughput than the conventional single-photon counting technology. using this plate reader, we detected fluorescence resonance energy transfer (FRET) between IAEDANS-labeled SERCA and nucleotide analog TNPADP in native sarcoplasmic reticulum membranes. This assay was designed to detect compounds that interact with SERCA and modify either the enzyme's structure or the binding affinity of TNP-ADP. Initial hit compounds were further analyzed in functional assays. Upon screening a small (1300 compound) library, we determined that the time-resolved microplate reader has at least 10x higher precision than a conventional intensity-based microplate reader, raising the quality index (z') of our assay from marginal, in the intensity reader, to excellent. A 384-well plate is read with high precision in 2 min, which allows screening of thousands of compounds/day. An important advantage of the time-resolved fluorescence measurement is that it provides detailed structural information, thus enabling discovery of multiple classes of compounds during the primary screen.

Lethal, Hereditary Mutants of Phospholamban Elude Phosphorylation by Protein Kinase A

The sarcoplasmic reticulum calcium pump (SERCA) and its regulator, phospholamban, are essential components of cardiac contractility. Phospholamban modulates contractility by inhibiting SERCA, and this process is dynamically regulated by β-adrenergic stimulation and phosphorylation of phospholamban. Herein we reveal mechanistic insight into how four hereditary mutants of phospholamban, Arg(9) to Cys, Arg(9) to Leu, Arg(9) to His, and Arg(14) deletion, alter regulation of SERCA. Deletion of Arg(14) disrupts the protein kinase A recognition motif, which abrogates phospholamban phosphorylation and results in constitutive SERCA inhibition. Mutation of Arg(9) causes more complex changes in function, where hydrophobic substitutions such as cysteine and leucine eliminate both SERCA inhibition and phospholamban phosphorylation, whereas an aromatic substitution such as histidine selectively disrupts phosphorylation. We demonstrate that the role of Arg(9) in phospholamban function is multifaceted: it is important for inhibition of SERCA, it increases the efficiency of phosphorylation, and it is critical for protein kinase A recognition in the context of the phospholamban pentamer. Given the synergistic consequences on contractility, it is not surprising that the mutants cause lethal, hereditary dilated cardiomyopathy.

Use of Glycerol-Containing Media To Study the Intrinsic Fluorescence Properties of Detergent-Solubilized Native or Expressed SERCA1a

Rapid irreversible inactivation of Ca (2+)-free states of detergent-solubilized SERCA1a (sarco-endoplasmic reticulum calcium ATPase 1a) has so far prevented the use of Trp fluorescence for functional characterization of this ATPase after its solubilization in various detergents. Here we show that using 20-40% glycerol for protection makes this fluorescence characterization possible. Most of the ligand-induced Trp fluorescence changes previously demonstrated to occur for SERCA1a embedded in native sarcoplasmic reticulum membranes were observed in the combined presence of glycerol and detergent, although the results greatly depended on the detergent used, namely, octaethylene glycol mono- n-dodecyl ether (C 12E 8) or dodecyl maltoside (DDM). In particular, at pH 6, we found a C 12E 8-dependent unexpectedly huge reduction in SERCA1a affinity for Ca (2+). We suggest that a major reason for the different effects of the two detergents is that high concentrations of C 12E 8, but not of DDM, slow down the E2 to E1 transition in solubilized and delipidated SERCA1a. Independently of the characterization of the specific effects of various detergents on SR vesicles, our results open the way to functional characterization by Trp fluorescence of heterologously expressed and purified mutants of SERCA1a in the presence of detergent, without their preliminary reconstitution into liposomes. As an example, we used the E309Q mutant to demonstrate our previous suspicion that Ca (2+) binding to Site I of SERCA1a in fact slightly reduces Trp fluorescence, and consequently that the rise in this fluorescence generally observed when two Ca (2+) ions bind to WT SERCA1a mainly reflects Ca (2+) binding at Site II of SERCA1a.

The Presence of Sarcolipin Results in Increased Heat Production by Ca2+-ATPase

Skeletal muscle sarcoplasmic reticulum of large mammals such as rabbit contains sarcolipin (SLN), a small peptide with a single transmembrane alpha-helix. When reconstituted with the Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum into sealed vesicles, the presence of SLN leads to a reduced level of accumulation of Ca(2+). Heats of reaction of the reconstituted Ca(2+)-ATPase with ATP were measured using isothermal calorimetry. The heat released increased linearly with time over 30 min and increased with increasing SLN content. Rates ATP hydrolysis by the reconstituted Ca(2+)-ATPase were constant over a 30-min time period and were the same when measured in the presence or absence of an ATP-regenerating system. The calculated values of heat released per mol of ATP hydrolyzed increased with increasing SLN content and fitted to a simple binding equation with a dissociation constant for the SLN.ATPase complex of 6.9 x 10(-4) +/- 2.9 x 10(-4) in units of mol fraction per monolayer. It is suggested that the interaction between Ca(2+)-ATPase and SLN in the sarcoplasmic reticulum could be important in thermogenesis by the sarcoplasmic reticulum.

Glycogen- and PP1c-targeting Subunit GM Is Phosphorylated at Ser48 by Sarcoplasmic Reticulum-bound Ca2+-Calmodulin Protein Kinase in Rabbit Fast Twitch Skeletal Muscle

Multifunctional Ca(2+)-calmodulin-dependent protein kinase (CaMKII) is a Ser/Thr protein kinase uniformly distributed within the sarcoplasmic reticulum (SR) of skeletal muscle. In fast twitch muscle, no specific substrates of CaMKII have yet been identified in nonjunctional SR. Previous electron microscopy data showed that glycogen particles containing glycogen synthase (GS) associate with SR at the I band level. Furthermore, recent evidence implicates CaMKII in regulation of glucose and glycogen metabolism. Here, we demonstrate that the glycogen- and protein phosphatase 1-targeting subunit, also known as G(M), selectively localizes to the SR membranes of rabbit skeletal muscle and that G(M) and GS co-localize at the level of the I band. We further show that G(M), GS, and PP1c assemble in a structural complex that selectively localizes to nonjunctional SR and that G(M) is phosphorylated by SR-bound CaMKII and dephosphorylated by PP1c. On the other hand, no evidence for a structural interaction between G(M) and CaMKII was obtained. Using His-tagged G(M) recombinant fragments and site-directed mutagenesis, we demonstrate that the target of CaMKII is Ser(48). Taken together, these data suggest that SR-bound CaMKII participates in the regulation of GS activity through changes in the phosphorylation state of G(M). Based on these findings, we propose that SR-bound CaMKII participates in the regulation of glycogen metabolism, under physiological conditions involving repetitive raises elevations of [Ca(2+)](i).

The random-coil 'C' fragment of the dihydropyridine receptor II-III loop can activate or inhibit native skeletal ryanodine receptors.

The actions of peptide C, corresponding to (724)Glu-Pro(760) of the II-III loop of the skeletal dihydropyridine receptor, on ryanodine receptor (RyR) channels incorporated into lipid bilayers with the native sarcoplasmic reticulum membrane show that the peptide is a high-affinity activator of native skeletal RyRs at cytoplasmic concentrations of 100 nM-10 microM. In addition, we found that peptide C inhibits RyRs in a voltage-independent manner when added for longer times or at higher concentrations (up to 150 microM). Peptide C had a random-coil structure indicating that it briefly assumes a variety of structures, some of which might activate and others which might inhibit RyRs. The results suggest that RyR activation and inhibition by peptide C arise from independent stochastic processes. A rate constant of 7.5 x 10(5) s(-1).M(-1) was obtained for activation and a lower estimate for the rate constant for inhibition of 5.9 x 10(3) s(-1).M(-1). The combined actions of peptide C and peptide A (II-III loop sequence (671)Thr-Leu(690)) showed that peptide C prevented activation but not blockage of RyRs by peptide A. We suggest that the effects of peptide C indicate functional interactions between a part of the dihydropyridine receptor and the RyR. These interactions could reflect either dynamic changes that occur during excitation-contraction coupling or interactions between the proteins at rest.

Calsequestrin binds to monomeric and complexed forms of key calcium-handling proteins in native sarcoplasmic reticulum membranes from rabbit skeletal muscle

Ca 2+-handling proteins are important regulators of the excitation–contraction–relaxation cycle in skeletal muscle fibres. Although domain binding studies suggest protein coupling between various Ca 2+-regulatory elements of triad junctions, no direct biochemical evidence exists demonstrating high-molecular-mass complex formation in native microsomal membranes. Calsequestrin represents the protein backbone of the luminal Ca 2+ reservoir and thereby occupies a central position in Ca 2+ homeostasis; we therefore used calsequestrin blot overlay assays in order to determine complex formation between sarcoplasmic reticulum components. Peroxidase-conjugated calsequestrin clearly labelled four major protein bands in one-dimensional (1D) and 2D electrophoretically separated membrane preparations from adult skeletal muscle. Immunoblotting identified the calsequestrin-binding proteins of approximately 26, 63, 94 and 560 kDa as junctin, calsequestrin itself, triadin and the ryanodine receptor, respectively. Protein–protein coupling could be modified by ionic detergents, non-ionic detergents, changes in Ca 2+ concentration, as well as antibody and purified calsequestrin binding. Importantly, complex formation as determined by blot overlay assays was confirmed by differential co-immunoprecipitation experiments and chemical crosslinking analysis. Hence, the key Ca 2+-regulatory membrane components of skeletal muscle form a supramolecular membrane assembly. The formation of this tightly associated junctional sarcoplasmic reticulum complex seems to underlie the physiological regulation of skeletal muscle contraction and relaxation, which supports the biochemical concept that Ca 2+ homeostasis is regulated by direct protein–protein interactions.

Identification of the cardiac ryanodine receptor channel in membrane blebs of sarcoplasmic reticulum

Blebs of the sarcoplasmic reticulum (SR) membrane of heart muscle cells were generated after saponin perforation of the plasma membrane followed by complete hypercontraction of the cell. Although characteristic proteins of the plasma membrane, namely the β1-adrenoreceptor and Gαi, were stained by monoclonal antibodies in the hypercontracted cells, these proteins could not be detected in the adjacent blebs. Monoclonal antibodies to the cardiac ryanodine receptor (RyR2), calsequestrin and SERCA2 bound at different amounts to surface components of the blebs and to components of the hypercontracted cells. From the immunofluorescence signals we conclude that the blebs are mainly constituted of corbular and junctional SR membrane, and only to a lesser extent of network SR membrane. Deconvolution microscopy revealed that the membrane location of RyR2, calsequestrin and SERCA2 in the bleb is comparable to native SR membrane. At the bleb membrane giga-ohm seals could be obtained and patches could be excised in a way that single-channel currents could be measured, although these are not completely identified.

Oligomeric interactions between phospholamban molecules regulate Ca-ATPase activity in functionally reconstituted membranes.

Phospholamban (PLB) is a major target of the beta-adrenergic cascade in the heart, and functions as an endogenous inhibitor of Ca-ATPase transport activity. To identify whether oligomeric interactions between PLB molecules are involved in regulating Ca-ATPase transport activity, we have investigated functional interactions between PLB and the Ca-ATPase in proteoliposomes of purified PLB functionally co-reconstituted with the SERCA2a isoform of the Ca-ATPase isolated from cardiac sarcoplasmic reticulum (SR). The calcium sensitivity of this reconstituted preparation and functional stimulation by cAMP-dependent protein kinase (PKA) are virtually identical to those of the Ca-ATPase in cardiac SR microsomes, ensuring the functional relevance of this reconstituted preparation. Interactions between PLB molecules were measured following covalent modification of the single lysine (i.e., Lys(3)) in PLB isolated from cardiac SR membranes with fluorescein isothiocyanate (FITC) prior to co-reconstitution with the Ca-ATPase. FITC modification of PLB does not interfere with the ability of PLB to inhibit the Ca-ATPase, since FITC-PLB co-reconstituted with the Ca-ATPase exhibits a similar calcium dependence of Ca-ATPase activation to that observed in native SR membranes. Thus, the functional arrangement of PLB with the Ca-ATPase is not modified by FITC modification. Using changes in the anisotropy of FITC-PLB resulting from fluorescence resonance energy transfer (FRET) between proximal PLB molecules to measure the average size and spatial arrangement of FITC chromophores, we find that PLB self-associates to form oligomers whose spatial arrangement with respect to one another is in agreement with earlier suggestions that PLB exists predominantly as a homopentamer. The inability of PKA to activate PLB following covalent modification with FITC permits functional interactions between PLB molecules associated with the Ca-ATPase activation to be identified. A second-order loss of Ca-ATPase activation by PKA is observed as a function of the fractional contribution of FITC-PLB, indicating that PKA-dependent activation of two PLB molecules within a quaternary complex containing the Ca-ATPase is necessary for activation of the Ca-ATPase. We suggest that the requirement for activation of two PLB molecules by PKA represents a physiological mechanism to ensure that activation of the Ca-ATPase following beta-adrenergic stimulation in the heart only occurs above a threshold level of PKA activation.

Ca2+/Calmodulin-Dependent Phosphorylation of the Ca2+-ATPase, Uncoupled from Phospholamban, Stimulates Ca2+-Pumping in Native Cardiac Sarcoplasmic Reticulum

Recent studies have demonstrated phosphorylation of the cardiac and slow-twitch muscle isoform (SERCA2a) of the sarcoplasmic reticulum (SR) Ca2+-ATPase (at Ser38) by a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase). Analysis of the functional consequence of Ca2+-ATPase phosphorylation in the native SR membranes, however, is complicated by the concurrent phosphorylation of the SR proteins phospholamban (PLN) which stimulates Ca2+sequestration by the Ca2+-ATPase, and the ryanodine receptor-Ca2+release channel (RYR-CRC) which likely augments Ca2+release from the SR. In the present study, we achieved selective phosphorylation of the Ca2+-ATPase by endogenous CaM kinase in isolated rabbit cardiac SR vesicles utilizing a PLN monoclonal antibody (PLN AB) which inhibits PLN phosphorylation, and the RYR-CRC blocking drug, ruthenium red, which inhibits phosphorylation of RYR-CRC. Analysis of the Ca2+concentration-dependence of ATP-energized Ca2+uptake by SR showed that endogenous CaM kinase mediated phosphorylation of the Ca2+-ATPase, in the absence of PLN and/or RYR-CRC phosphorylation, results in a significant increase (∼50-70%) in the Vmaxof Ca2+sequestration without any change in the k0.5for Ca2+activation of the Ca2+transport rate. On the other hand, treatment of SR with PLN AB (which mimics the effect of PLN phosphorylation by uncoupling Ca2+-ATPase from PLN) resulted in ∼2-fold decrease in k0.5for Ca2+without any change in Vmaxof Ca2+sequestration. These findings suggest that, besides PLN phosphorylation, direct phosphorylation of the Ca2+-ATPase by SR-associated CaM kinase serves to enhance the speed of cardiac muscle relaxation.

Lysophosphatidylcholine modulates catalytically important motions of the Ca-ATPase phosphorylation domain.

Catalytically important motions of the Ca-ATPase, modulated by the physical properties of surrounding membrane phospholipids, have been suggested to be rate-limiting under physiological conditions. To identify the nature of the structural coupling between the Ca-ATPase and membrane phospholipids, we have investigated the functional and structural effects resulting from the incorporation of the lysophospholipid 1-myristoyl-2-hydroxy-sn-glycerol-3-phosphocholine (LPC) into native sarcoplasmic reticulum (SR) membranes. Nonsolubilizing concentrations of LPC abolish changes in fluorescence signals associated with either intrinsic or extrinsic chromophores that monitor normal conformational transitions accompanying calcium activation of the Ca-ATPase. There are corresponding decreases in the rates of calcium transport coupled to ATP hydrolysis, suggesting that LPC may increase conformational barriers associated with catalytic function. Fluorescence anisotropy measurements of the lipid analogue 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) partitioned into SR membranes indicate that LPC does not significantly modify lipid acyl chain rotational dynamics, suggesting differences in headgroup conformation between LPC and diacylglycerol phosphatidylcholines. Complementary measurements using phosphorescence anisotropy of erythrosin isothiocyanate at Lys464 on the Ca-ATPase provide a measure of the dynamic structure of the phosphorylation domain, and indicate that LPC restricts the amplitude of rotational motion. These results suggest a structural linkage between the cytosolic phosphorylation domain and the conformation of membrane phospholipid headgroups. Thus, changes in membrane phospholipid composition can modulate membrane surface properties and affect catalytically important motions of the Ca-ATPase in a manner that suggests a role for LPC generated during signal transduction.

Phosphatidylethanolamine modulates Ca-ATPase function and dynamics.

Phospholipids containing phosphoethanolamine (PE) headgroups within biological membranes have been suggested to be important with respect to the functional regulation of membrane proteins, including the Ca-ATPase in sarcoplasmic reticulum (SR). To investigate the role of PE headgroups in modulating the catalytic activity of the Ca-ATPase, we have reconstituted the Ca-ATPase into unilamellar liposomes containing defined amounts of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC). The enzymatic activity of the Ca-ATPase progressively increases upon incorporation of increasing amounts of PE into reconstituted vesicles, and approaches that characteristic of native SR membranes. To identify structural changes that correlate with enzyme activation, we have used frequency-domain phosphorescence spectroscopy to measure the rotational dynamics of erythrosin isothiocyanate covalently bound to Lys464 in the phosphorylation domain of the Ca-ATPase. Progressive increases in the rotational dynamics of the phosphorylation domain result from the incorporation of increasing amounts of DOPE, and correlate with enhanced enzymatic function. These results suggest that PE headgroups induce dynamic structural rearrangements involving the phosphorylation domain that modify the rates of nucleotide utilization. In contrast, no changes in the rotational dynamics of the lipid acyl chains are observed irrespective of the PE content. Therefore, the enhanced ATP hydrolytic activity associated with the incorporation of DOPE into these proteoliposomes is the result of specific noncovalent interactions involving PE phospholipid headgroups and the Ca-ATPase.

Pentameric Assembly of Phospholamban Facilitates Inhibition of Cardiac Function in Vivo

Phospholamban has been proposed to coexist as pentamers and monomers in native sarcoplasmic reticulum membranes. To determine its functional unit in vivo, we reintroduced wild-type (pentameric) or monomeric mutant (C41F) phospholamban in the hearts of phospholamban knockout mice. Transgenic lines, expressing similar levels of mutant or wild-type phospholamban, were identified, and their cardiac phenotypes were characterized in parallel. Sarcoplasmic reticulum Ca2+ transport assays indicated similar decreases in SERCA2 Ca2+ affinity by mutant or wild-type phospholamban. However, the time constants of relaxation and Ca2+ transient decline in isolated cardiomyocytes were diminished to a greater extent by wild-type than mutant phospholamban, even without significant differences in the amplitudes of myocyte contraction and Ca2+ transients between the two groups. Langendorff perfusion also indicated that mutant phospholamban was not capable of depressing the enhanced relaxation parameters of the phospholamban knockout hearts to the same extent as wild-type phospholamban. Moreover, in vivo assessment of mouse hemodynamics revealed a greater depression of cardiac function in wild-type than mutant phospholamban hearts. Thus, the mutant or monomeric form of phospholamban was not as effective in slowing Ca2+ decline or relaxation in cardiomyocytes, hearts, or intact animals as wild-type or pentameric phospholamban. These findings suggest that pentameric assembly of phospholamban is necessary for optimal regulation of myocardial contractility in vivo.

Monomeric phospholamban overexpression in transgenic mouse hearts.

Phospholamban, a prominent modulator of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity and basal contractility in the mammalian heart, has been proposed to form pentamers in native SR membranes. However, the monomeric form of phospholamban, which is associated with mutating Cys41 to Phe41, was shown to be as effective as pentameric phospholamban in inhibiting Ca2+ transport in expression systems. To determine whether this monomeric form of phospholamban is also functional in vivo, we generated transgenic mice with cardiac-specific overexpression of the mutant (Cys41-->Phe41) phospholamban. Quantitative immunoblotting indicated a 2-fold increase in the cardiac phospholamban protein levels compared with wild-type controls, with approximately equal to 50% of phospholamban migrating as monomers and approximately 50% as pentamers upon SDS-PAGE. The mutant-phospholamban transgenic hearts were analyzed in parallel with transgenic hearts overexpressing (2-fold) wild-type phospholamban, which migrated as pentamers upon SDS-PAGE. SR Ca(2+)-uptake assays revealed that the EC50 values for Ca2+ were as follows: 0.32 +/- 0.01 mumol/L in hearts overexpressing monomeric phospholamban, 0.49 +/- 0.05 mumol/L in hearts overexpressing wild-type phospholamban, and 0.26 +/- 0.01 mumol/L in wild-type control mouse hearts. Analysis of cardiomyocyte mechanics and Ca2+ kinetics indicated that the inhibitory effects of mutant-phospholamban overexpression (mt) were less pronounced than those of wild-type phospholamban overexpression (ov) as assessed by depression of the following: (1) shortening fraction (25% mt versus 45% ov), (2) rates of shortening (27% mt versus 48% ov), (3) rates of relengthening (25% mt versus 50% ov) (4) amplitude of the Ca2+ signal (21% mt versus 40% ov), and (5) time for decay of the Ca2+ signal (25% mt versus 106% ov) compared with control (100%) myocytes. The differences in basal cardiac, myocyte mechanics and Ca2+ transients among the animal groups overexpressing monomeric or wild-type phospholamban and wild-type control mice were abolished upon isoproterenol stimulation. These findings suggest that pentameric assembly of phospholamban is important for mediating its optimal regulatory effects on myocardial contractility in vivo.

Rotational mobility of Ca 2+-ATPase of sarcoplasmic reticulum in viscous media

The rotational diffusion of Ca 2+-ATPase [Ca 2+,Mg 2+-activated ATP phosphohydrolase E.C. 3.6.1.38] was studied in native sarcoplasmic reticulum membrane by saturation transfer ESR spectroscopy after covalent labelling of intramembranous sulfhydryl groups with nitroxyl derivative of maleimide (5-MSL) as a function of sucrose and glycerol in the suspending medium. The relative enzymatic activity of sarcoplasmic reticulum was followed by increasing the viscosity of the aqueous phase. The ATP hydrolysing activity of the enzyme decreased differently on adding sucrose and glycerol. In the case of sucrose the reciprocal of power dependence of viscosity was observed, whereas for glycerol an exponential decay law was obtained, indicating solvent-protein interaction. On increasing the viscosity of the aqueous phase by either sucrose or glycerol, no changes were observed in the intramembranous viscosity as measured using intercalated spin-labelled stearic acid (16-SASL). The effective rotational correlation time of the protein was measured, as a mobility parameter, using saturation transfer ESR spectroscopy and found to be increased linearly with the viscosity of the sucrose containing medium and for the extramembranous size a height of 6.8 nm was obtained, indicating that approx. 82% of the volume of Ca 2+-ATPase protein is external to the sarcoplasmic reticulum. The addition of glycerol probably promoted protein–protein interaction, as indicated by the larger changes in rotational diffusion and non-linear viscosity dependence.

Phosphorylation of phospholamban by cAMP-dependent protein kinase enhances interactions between Ca-ATPase polypeptide chains in cardiac sarcoplasmic reticulum membranes.

We have used spin-label EPR spectroscopy to examine possible alterations in protein-protein interactions that accompany the activation of the cardiac sarcoplasmic reticulum (SR) Ca-ATPase following the phosphorylation of phospholamban (PLB). Using a radioactive derivative of a maleimide spin label (MSL), we have developed conditions for the selective spin-labeling of the Ca-ATPase in both native cardiac and skeletal sarcoplasmic reticulum membranes. The rotational dynamics of the cardiac and skeletal Ca-ATPase isoforms in native SR membranes were measured using saturation transfer EPR. We report that the phosphorylation of PLB in cardiac SR results in a (1.8 +/- 0.2)-fold reduction in the overall rotational mobility of the Ca-ATPase. The alteration in the rotational dynamics of the Ca-ATPase is the direct result of the phosphorylation of PLB, and is not related to the phosphorylation of the Ca-ATPase or any other SR proteins since no alteration in the ST-EPR spectrum is observed as a result of conditions that phosphorylate the cardiac Ca-ATPase with ATP. Neither do the use of conditions that activate the Ca-ATPase in cardiac SR result in the alteration of the rotational dynamics or catalytic properties of the Ca-ATPase in skeletal SR where PLB is not expressed. Measurements of the rotational dynamics of stearic acid spin labels (SASL) incorporated into cardiac SR membranes with a nitroxide at the 5- and 12-positions using conventional EPR indicate that there is virtually no difference in the lipid acyl chain dynamics in cardiac SR membranes upon the phosphorylation of PLB. These results indicate that the decrease in the rotational dynamics of the Ca-ATPase in cardiac SR membranes associated with the phosphorylation of PLB is related to enhanced interactions between individual Ca-ATPase polypeptide chains due to (i) an alteration in the spatial arrangement of cardiac Ca-ATPase polypeptide chains within a defined oligomeric state or (ii) increased protein-protein associations. We suggest that altered interactions between Ca-ATPase polypeptide chains and PLB serves to modulate the activation barrier associated with calcium activation of the Ca-ATPase in cardiac SR membranes.

Skeletal muscle sarcoplasmic reticulum phenotype in myotonic dystrophy

In this study we investigated the sarcoplasmic reticulum (SR), alongside myofibrillar phenotype, in muscle samples from five Myotonic Dystrophy (DM) patients and five control individuals. DM muscles exhibited as a common feature, a decrease in the slow isoform of myosin heavy chain (MHC) and of troponin C in myofibrils. We observed a match between myofibrillar changes and changes in SR membrane markers specific to fiber type, i.e. the fast (SERCAI) Ca 2+-ATPase isoform increased concomitantly with a decrease of protein phospholamban (PLB), which in native SR membranes colocalizes with the slow (SERCA2a) SR Ca 2+-ATPase, and regulates its activity depending on phosphorylation by protein kinases. Our results outline a cellular process selectively affecting slow-twitch fibers, and non-degenerative in nature, since neither the total number of Ca 2+-pumps or of ryanodine receptor/Ca 2+- release channels, or their ratio to the dihydropyridine receptor/voltage sensor in junctional transverse tubules, were found to be significantly changed in DM muscle. The only documented, apparently specific molecular changes associated with this process in the SR of DM muscle, are the defective expression of the slow/cardiac isoform of Ca 2+-binding protein calsequestrin, together with an increased phosphorylation activity of membrane-bound 60 kDa Ca 2+-calmodulin (CaM) dependent protein kinase. Enhanced phosphorylation of PLB by membrane-bound Ca 2+-CaM protein kinase also appeared to be most pronounced in biopsy from a patient with a very high CTG expansion, as was the overall ‘slow-to-fast’ transformation of the same muscle biopsy. Animal studies showed that endogenous Ca 2+-CaM protein kinase exerts a dual activatory role on SERCA2a SR Ca 2+-ATPase, i.e. either by direct phosphorylation of the Ca 2+-ATPase protein, or mediated by phosphorylation of PLB. Our results seem to be consistent with a maturational-related abnormality and/or with altered modulatory mechanisms of SR Ca 2+-transport in DM slow-twitch muscle fibers.

Conformation of Ca(2+)-ATPase in two crystal forms. Effects of Ca2+, thapsigargin, adenosine 5'-(beta, gamma-methylene)triphosphate), and chromium(III)-ATP on crystallization

The structure of Ca(2+)-ATPase has been studied by electron microscopy of two different crystal forms: one tubular form induced by vanadate in native sarcoplasmic reticulum (SR) membranes and another multilamellar form grown from detergent-solubilized SR. To determine the conformation of Ca(2+)-ATPase within each crystal form, the respective effects of Ca2+, thapsigargin, adenosine 5'-(beta, gamma-methylene)triphosphate) (AMP-PCP), and chromium(III) (Cr-ATP) on crystallization have been studied. Vanadate-induced tubes were prevented from forming by micromolar Ca2+, but if preformed in the absence of Ca2_, millimolar Ca2+ was required to disrupt these crystals. Thapsigargin promoted tube formation even in the presence of 10 mM Ca2+. Neither AMP-PCP nor Cr-ATP prevented tube formation, and the Ca2+ sensitivity of tube formation from Cr-ATP-inhibited SR was identical to controls. Multilamellar crystals required at least 0.2 mM Ca2+ and were prevented from forming by thapsigargin, AMP-PCP, or Cr-ATP. It is concluded that helical tubes are composed of the Ca(2+)-free, dephosphorylated conformation (E), and the nucleotide-bound conformation (E-ATP) is also tolerated. In contrast, multilamellar crystals are composed of the Ca(2+)-bound conformation (E.Ca2) and do not tolerate nucleotide binding. Thus, comparison of structures obtained from the two crystal forms should reveal physiologically relevant conformational differences.