Sarcoplasmic Reticulum Vesicles Research Articles

Stabilization of the Skeletal Muscle Ryanodine Receptor Ion Channel-FKBP12 Complex by the 1,4-Benzothiazepine Derivative S107

Activation of the skeletal muscle ryanodine receptor (RyR1) complex results in the rapid release of Ca2+ from the sarcoplasmic reticulum and muscle contraction. Dissociation of the small FK506-binding protein 12 subunit (FKBP12) increases RyR1 activity and leads to the impairment of muscle function. The 1,4-benzothiazepine derivative JTV519, and more specific derivative S107 (2,3,4,5,-tetrahydro-7-methoxy-4-methyl-1,4-benzothiazepine), are thought to improve skeletal muscle function by stabilizing the RyR1-FKBP12 complex. Here, we report that SR vesicles enriched in RyR1 bound a large number of S107 molecules with micromolar affinity. The effects of S107 and FKBP12 on RyR1 were examined under conditions that altered the redox state of RyR1. In SR vesicles, S107 increased FKBP12 binding to RyR1 in presence of reduced glutathione and the NO-donor NOC12. S107 was without effect in the presence of oxidized glutathione. Addition of 0.15 μM FKBP12 to SR vesicles prevented FKBP12 dissociation, whereas in the presence of oxidized glutathione and NOC12 FKBP12 dissociation was observed in skeletal muscle homogenates that contained 0.43 μM myoplasmic FKBP12, and this dissociation was attenuated by S107. In single channel measurements, pretreatment of FKBP12-depleted RyR1s with S107 in the absence and presence of NOC12 augmented the FKBP12-mediated decrease in channel activity. The data suggest that S107 can reverse the harmful effects of redox active species on SR Ca2+ release in skeletal muscle.

FRET Detection of CaM-RyR2 Binding Modulation by S100A1

Using fluorescence resonance energy transfer (FRET), we are testing the hypothesis that S100A1 competes with calmodulin (CaM) for binding to cardiac ryanodine receptors (RyR2). In isolated pig cardiac sarcoplasmic reticulum (SR) vesicles, we targeted a donor-labeled FKBP (D-FKBP) to the RyR2 cytosolic headpiece. We then detected FRET as a decrease of donor fluorescence in the presence of CaM labeled with acceptor within the N- or C-lobe (denoted AN-CaM and AC-CaM, respectively), thus directly and specifically indexing CaM binding in the proximity of D-FKBP. FRET between D-FKBP and A-CaMs (100 nM) was completely inhibited by unlabeled WT-CaM, with KI ≈ 100 nM, indicating that WT-CaM and A-CaMs compete with similar affinities for the same RyR2 binding site. However, S100A1 (ranging from 0.1 to 30 μM) had no significant effect on FRET when cardiac SR was concurrently incubated with S100A1 and AN-CaM. Upon sequentially incubating the SR with S100A1 first, then with AN-CaM, we found partial inhibition of FRET, but with KI ≈ 30 μM S100A1. This effect is more pronounced in nM Ca2+ (versus μM Ca2+). Intriguingly, FRET between D-FKBP and AC-CaM was not significantly affected by S100A1. S100A1 lowered maximum FRET for AN-CaM but did not significantly change its binding affinity. This suggests that S100A1 allosterically interacts with RyR-CaM binding, rather than directly competing for the same binding site as CaM. We are currently developing a complementary FRET approach, using acceptor-labeled S100A1, to specifically resolve the S100A1 binding to RyR. Ultimately, we aim to elucidate the interplay between S100A1 and CaM binding to RyR.

TRIC-B Channels Exhibit Labile Gating Properties; Evidence from TRIC-A Knockout Mice

Trimeric intracellular cation channels (TRIC-A and TRIC-B) are located in the sarcoplasmic/endoplasmic reticulum (SR/ER) of most cells. Identifying the distinct biophysical properties of TRIC-A and TRIC-B is difficult because both channels are present in most tissues, yet this is crucial for delineating their individual physiological roles. Skeletal muscle SR vesicles (LSR) from TRIC-A knockout mice were incorporated into artificial membranes under voltage-clamp conditions as previously described [Pitt et al., 2010, Biophys. J. 99, 417-426] and single-channel recordings of native TRIC-B were obtained in symmetrical solutions of 210 mM K-PIPES, pH 7.2. The maximum single-channel conductance of TRIC-B was 197 ± 2 pS (n=32; SEM). TRIC-B channels always exhibited sub-conductance gating states and while these were of a variable nature, the predominant sub-conductance levels were found at 156 ± 3 pS (n=17; SEM), 125 ± 2 pS (n=19; SEM), 96 ± 2 pS (n=19; SEM) and 62 ± 2 pS (n=27; SEM). TRIC-B channel gating was voltage-dependent and channels were inhibited at negative holding potentials. For example, the probability of dwelling in the full open channel level was 0.0478±0.0194 at +30 mV but only 0.0010±0.0008 at −30 mV (n=6; SEM; ∗p<0.05). Application of 300 mM KCl to the cytosolic channel side produced a parallel shift in the current-voltage relationship and a shift in the reversal potential to approximately −20 mV indicating that TRIC-B is not permeable to anions. Our study demonstrates that the single-channel properties of TRIC-B channels are exceptionally labile. This intrinsic variability may be important for enabling flexible physiological regulation of monovalent cation fluxes across the SR membrane.Supported by the British Heart Foundation and the Japan Society for the Promotion of Science.

Age-Associated Changes in Ca2+-ATPase and Oxidative Damage in Sarcoplasmic Reticulum of Rat Heart

Altered Ca(2+) handling may be responsible for the development of cardiac contractile dysfunctions with advanced age. In the present study, we investigated the roles of oxidative damage to sarcoplasmic reticulum (SR) and expression of Ca(2+)-ATPase (SERCA 2a) and phospholamban in age-associated dysfunction of cardiac SR. SR vesicles were prepared from hearts of 2-, 6-, 15-, and 26-month-old Wistar rats. Although activity of Ca(2+)-ATPase decreased with advancing age, no differences in relative amounts of SERCA 2a and phospholamban protein were observed. On the other hand, significant accumulation of protein oxidative damage occurred with aging. The results of this study suggest that age-related alteration in Ca(2+)-ATPase activity in the rat heart is not a consequence of decreased protein levels of SERCA 2a and phospholamban, but could arise from oxidative modifications of SR proteins. Cellular oxidative damage caused by reactive oxygen species could contribute to age-related alternations in myocardial relaxation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptosis by disruption of intracellular calcium homeostasis in human neuronal cell line SHSY5Y

The persistent xenobiotic agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces neurotoxic effects that alters neurodevelopment and behavior both during development and adulthood. There are many ongoing efforts to determine the molecular mechanisms of TCDD-mediated neurotoxicity, the signaling pathways involved and its molecular targets in neurons. In this work, we have used SHSY5Y human neuroblastoma cells to characterize the TCDD-induced toxicity. TCDD produces a loss of viability linked to an increased caspase-3 activity, PARP-1 fragmentation, DNA laddering, nuclear fragmentation and hypodiploid (apoptotic) DNA content, in a similar way than staurosporine, a prototypical molecule of apoptosis induction. In addition, TCDD produces a decrease of mitochondrial membrane potential and an increase of intracellular calcium concentration (P < 0.05). Finally, based on the high lipophilic properties of the dioxin, we test the TCDD effect on the membrane integrity using sarcoplasmic reticulum vesicles as a model. TCDD produces calcium efflux through the membrane and an anisotropy decrease (P < 0.05) that reflects an increase in membrane fluidity. Altogether these results support the hypothesis that TCDD toxicity in SHSY5Y neuroblastoma cells provokes the disruption of calcium homeostasis, probably affecting membrane structural integrity, leading to an apoptotic process.

Nucleotide Activation of the Ca-ATPase

We have used fluorescence spectroscopy, molecular modeling, and limited proteolysis to examine structural dynamics of the sarcoplasmic reticulum Ca-ATPase (SERCA). The Ca-ATPase in sarcoplasmic reticulum vesicles from fast twitch muscle (SERCA1a isoform) was selectively labeled with fluorescein isothiocyanate (FITC), a probe that specifically reacts with Lys-515 in the nucleotide-binding site. Conformation-specific proteolysis demonstrated that FITC labeling does not induce closure of the cytoplasmic headpiece, thereby assigning FITC-SERCA as a nucleotide-free enzyme. We used enzyme reverse mode to synthesize FITC monophosphate (FMP) on SERCA, producing a phosphorylated pseudosubstrate tethered to the nucleotide-binding site of a Ca(2+)-free enzyme (E2 state to prevent FMP hydrolysis). Conformation-specific proteolysis demonstrated that FMP formation induces SERCA headpiece closure similar to ATP binding, presumably due to the high energy phosphoryl group on the fluorescent probe (ATP·E2 analog). Subnanosecond-resolved detection of fluorescence lifetime, anisotropy, and quenching was used to characterize FMP-SERCA (ATP·E2 state) versus FITC-SERCA in Ca(2+)-free, Ca(2+)-bound, and actively cycling phosphoenzyme states (E2, E1, and EP). Time-resolved spectroscopy revealed that FMP-SERCA exhibits increased probe dynamics but decreased probe accessibility compared with FITC-SERCA, indicating that ATP exhibits enhanced dynamics within a closed cytoplasmic headpiece. Molecular modeling was used to calculate the solvent-accessible surface area of FITC and FMP bound to SERCA crystal structures, revealing a positive correlation of solvent-accessible surface area with quenching but not anisotropy. Thus, headpiece closure is coupled to substrate binding but not active site dynamics. We propose that dynamics in the nucleotide-binding site of SERCA is important for Ca(2+) binding (distal allostery) and phosphoenzyme formation (direct activation).

T.P.10 The acute effects of curcumin exposure on skeletal muscle contractile function

Curcumin is a component in the Asian spice turmeric which has demonstrated anti-inflammatory, anti-oxidant and anti-cancer effects in animal models of human disease. It has also been shown to alleviate the dystropathology in mdx mice. However, in vitro studies on isolated sarcoplasmic reticulum (SR) vesicles indicate that curcumin has inhibitory effects on SR function which may adversely affect muscle contraction. The aim of this study was to investigate the specific effects of curcumin on skeletal muscle contractile function. These experiments, conducted on isolated extensor digitorum longus (EDL) muscles from 8week old ARC mice, were approved by the animal ethics committee of the University of Western Australia. Mice were anaesthetized (pentobarbitone, 40mg/kg, IP) and the muscles surgically removed and mounted in an in vitro muscle test system containing mammalian Ringer solution bubbled with carbogen. Measures of contractile function were performed before and after 60min exposure to either 100μM curcumin (dissolved in DMSO) or control solution containing the equivalent DMSO concentration (0.05%). The effect of curcumin on the susceptibility to and recovery from fatigue was also assessed. Curcumin exposure significantly decreased maximum specific force in EDL muscles by 14% compared to control muscles exposed to DMSO alone (curcumin: 19.7±0.7N/cm2, n=6; control: 23.0±1.0N/cm2, n=6, P<0.05). Curcumin had no significant effect on peak twitch force, the time to peak or the 1/2 relaxation time of twitch contractions. The rate of muscle fatigue was significantly reduced after curcumin exposure compared to controls (P<0.01, n=6). This study shows for the first time that (100μM) curcumin exposure significantly affects skeletal muscle contractile function. As the twitch contraction and relaxation times are sensitive to changes in SR function, these results are not consistent with previous findings that curcumin inhibits SR Ca2+ handling in SR vesicles.

Uncoupling of sarcoplasmic reticulum Ca2+-ATPase by N-arachidonoyl dopamine. Members of the endocannabinoid family as thermogenic drugs

The sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) plays a role in thermogenesis. The exogenous compound capsaicin increased SERCA-mediated ATP hydrolysis not coupled to Ca²⁺ transport. Here, we have sought to identify endogenous compounds that may function as SERCA uncoupling agents. Using isolated SR vesicles from rabbits, we have screened for endogenous compounds that uncouple SERCA. We have also studied their ability to deplete cytoplasmic ATP from human skeletal muscle cells in culture. Studies on SR vesicles showed that the endogenous lipid metabolite N-arachidonoyl dopamine (NADA) was a potent stimulator of SERCA uncoupling. NADA stabilized an E₁-like pump conformation that had a lower dephosphorylation rate, low affinity for Ca²⁺ at the luminal sites and a specific proteinase K cleavage pattern involving protection of the C-terminal p83C fragment from further cleavage. Moreover, we found a significantly decreased cytoplasmic ATP levels following treatment of skeletal muscle cells with 100 nM NADA. This effect was dependent on the presence of glucose and abolished by pretreatment with the specific SERCA inhibitor thapsigargin, regardless of the presence of glucose. NADA is an endogenous molecule that may function as SERCA uncoupling agent in vivo. Members of the endocannabinoid family exert concerted actions on several Ca²⁺-handling proteins. Uncoupling of SERCA by exogenous compounds could be a novel post-mitochondrial strategy for reduction of cellular ATP levels. In addition, signalling networks leading to SERCA uncoupling can be explored to study the importance of this ion pump in pathophysiological conditions related to metabolism.

Phospholamban phosphorylation increases the passive calcium leak from cardiac sarcoplasmic reticulum

Phospholamban (PLN) is a 52 amino acid integral membrane protein of the sarcoplasmic reticulum (SR) that exists in both monomeric and pentameric forms. In its unphosphorylated state, PLN inhibits the SR Ca(2+) ATPase (SERCA). This inhibition is relieved when PLN is phosphorylated as a result of β-adrenergic stimulation of the heart. Consistent with some predictions from molecular models and from functional studies of PLN incorporated into planar lipid bilayers, it has also been postulated that pentameric PLN can also form ion-selective channels. Other molecular models contradict this hypothesis, however. In the work reported here, we used the Ca(2+)-sensitive fluorescent dye Fura-2, to examine the passive Ca(2+) permeability of the SR membrane in vesicles derived from cardiac ventricle. We have found that phosphorylation of PLN by protein kinase A (PKA) leads to an increase in the rate of Ca(2+) leak from Ca(2+)-loaded SR vesicles. This enhanced rate of Ca(2+) leak from the SR is also observed when SR vesicles are incubated with a PLN specific antibody (A1) that mimics phosphorylation of PLN. The ryanodine receptor blocker ruthenium red does not affect the increased rate of Ca(2+) leak from the SR after PLN phosphorylation with PKA or after exposure to A1 antibody, arguing against a possible role of ryanodine receptors in mediating the enhanced leak. Our results are consistent with the hypothesis that phosphorylated PLN forms or regulates a Ca(2+) leak pathway in cardiac SR membranes in situ.

The potassium channel opener CGS7184 activates Ca2+ release from the endoplasmic reticulum

CGS7184 (ethyl 1-[[(4-chlorophenyl)amino]oxo]-2-hydroxy-6-trifluoromethyl-1H-indole-3-carboxylate) is a synthetic large-conductance Ca2+-activated potassium (BKCa) channel opener. The existing literature suggests that potassium channels are involved in cardioprotection, particularly during ischemia-reperfusion events. However, the cellular mechanisms mediating the effects of CGS7184 remain unclear. In the present study, we investigated the effect of the BKCa channel opener CGS7184 on Ca2+ homeostasis in H9C2 and C2C12 cell lines, Ca2+ uptake by isolated sarcoplasmic reticulum (SR) vesicles, SR Ca2+-ATPase (SERCA) activity, and single-channel properties of the ryanodine receptor calcium release channel (RYR2) when incorporated into a planar lipid bilayer. The effects of CGS7184 on calcium homeostasis in C2C12 and H9C2 cell lines were measured with a Fura-2 fluorescent indicator. The BKCa channel opener CGS7184, when added to the H9C2 and C2C12 cells, caused a concentration-dependent release of calcium from internal stores. Calcium accumulation by the SR vesicles isolated from cardiac and skeletal muscle was inhibited by CGS7184 with a half-maximal inhibition value of 0.45±0.04μM and 0.37±0.03μM, respectively. The results of the present study indicate that the BKCa channel opener CGS7184 modulates cytosolic Ca2+ concentration in H9C2 and C1C12 cells due to its interaction with the endoplasmic reticulum (ER). CGS7184 approximately doubled the opening probability of RYR2 channels; however, the compound seemed to most strongly affect channels with a higher control activity. These results strongly suggest that the BKCa channel opener CGS7184 affects intracellular calcium homeostasis by interacting with the sarcoplasmic reticulum RYR2 channels.

3,5-Di-t-butyl catechol is a potent human ryanodine receptor 1 activator, not suitable for the diagnosis of malignant hyperthermia susceptibility

3,5-Di-t-butyl catechol (DTCAT) releases Ca2+ from rat skeletal muscle sarcoplasmic reticulum (SR) vesicles. Hence, it is a candidate for use as a substitute for halothane or caffeine in the in vitro contracture test for the diagnosis of susceptibility to malignant hyperthermia (MH).To characterize the effect of DTCAT at cell level, Ca2+ release experiments were performed on cultured, human skeletal muscle myotubes using the fluorescent Ca2+ indicator fura2-AM. DTCAT was also assayed in the in vitro contracture test on human skeletal muscle bundles obtained from individuals diagnosed susceptible (MHS), normal (MHN) or equivocal for halothane (MHEH) and compared to the standard test substances caffeine and halothane.DTCAT increased, in a concentration-dependent manner and with a higher efficacy as compared to caffeine, the free, intracellular Ca2+ levels of cultured MHN and MHS skeletal muscle myotubes. This effect was similar in both types of myotubes and involved the release of Ca2+ from SR stores as well as Ca2+-influx from the extracellular space. Inhibition of ryanodine receptors either with ryanodine or with ruthenium red markedly reduced DTCAT-induced increase in intracellular Ca2+ concentration while abolishing that induced by caffeine. In MHN skeletal muscle bundles, DTCAT induced contractures with an EC50 value of 160±91μM. However, the sensitivity of MHS or MHEH muscles to DTCAT was similar to that of MHN muscles.In conclusion, DTCAT is not suitable for the diagnosis of MH susceptibility due to its failure to discriminate between MHN and MHS muscles.

Characterizing Phospholamban to Sarco(endo)plasmic Reticulum Ca2+-ATPase 2a (SERCA2a) Protein Binding Interactions in Human Cardiac Sarcoplasmic Reticulum Vesicles Using Chemical Cross-linking

Chemical cross-linking was used to study protein binding interactions between native phospholamban (PLB) and SERCA2a in sarcoplasmic reticulum (SR) vesicles prepared from normal and failed human hearts. Lys(27) of PLB was cross-linked to the Ca(2+) pump at the cytoplasmic extension of M4 (at or near Lys(328)) with the homobifunctional cross-linker, disuccinimidyl glutarate (7.7 Å). Cross-linking was augmented by ATP but abolished by Ca(2+) or thapsigargin, confirming in native SR vesicles that PLB binds preferentially to E2 (low Ca(2+) affinity conformation of the Ca(2+)-ATPase) stabilized by ATP. To assess the functional effects of PLB binding on SERCA2a activity, the anti-PLB antibody, 2D12, was used to disrupt the physical interactions between PLB and SERCA2a in SR vesicles. We observed a tight correlation between 2D12-induced inhibition of PLB cross-linking to SERCA2a and 2D12 stimulation of Ca(2+)-ATPase activity and Ca(2+) transport. The results suggest that the inhibitory effect of PLB on Ca(2+)-ATPase activity in SR vesicles results from mutually exclusive binding of PLB and Ca(2+) to the Ca(2+) pump, requiring PLB dissociation for catalytic activation. Importantly, the same result was obtained with SR vesicles prepared from normal and failed human hearts; therefore, we conclude that PLB binding interactions with the Ca(2+) pump are largely unchanged in failing myocardium.

3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity

3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca2+) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca2+-uptake activity was significantly inhibited by 80% with 150μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA.

Time-Resolved Fluorescence and Molecular Modeling of FITC-Labeled Ca-ATPase

We have detected structural dynamics in the nucleotide-binding domain of the Ca-ATPase (SERCA) using fluorescence spectroscopy and molecular modeling. SERCA in sarcoplasmic reticulum (SR) vesicles was selectively labeled with fluorescein isothiocyanate (FITC), an affinity probe that specifically reacts with Lys-515 in the nucleotide-binding site. FITC-labeled SERCA enters the phosphorylated “E1P-like” conformation with low probe fluorescence (∼50% intensity) when SR vesicles are loaded with calcium using acetyl phosphate as the energy source, followed by chelation of extravesicular calcium to halt enzyme cycling. Here we compared FITC-SERCA in the unique low fluorescence state (E1P-like) with conformations that show high probe fluorescence: calcium-free (E2), calcium-bound (E1), and actively-cycling phosphoenzyme (EP). Direct waveform recording (DWR) and time-correlated single photon counting (TCSPC) were used to measure fluorescence emission decay and time-resolved anisotropy. Iodide quenching and detergent solubilization were used to examine FITC accessibility and SERCA oligomerization. Spectroscopy results indicate that FITC-SERCA in the E1P-like state shows increased probe dynamics, decreased probe accessibility, and decreased SERCA oligomerization in comparison to high fluorescence states (E2, E1, EP). Molecular modeling of protein and probe conformation identified Phe-487 in the nucleotide-binding domain as a critical residue involved in FITC binding, contributing to the high specificity of labeling for SERCA. Phe-487 also serves as the key residue that ‘stacks’ with the adenine moiety of ATP bound to unlabeled SERCA. We combine fluorescence data and modeling results to propose a structural mechanism for ADP release following ATP hydrolysis and E1P formation by SERCA. This work was funded by grant from NIH to DDT (R01 GM27906).

From myofibril to membrane; the transitional junction at the intercalated disc

Cardiomyocytes are coordinated by linking together at their ends through the intercalated disc. The intercalated disc with its complex folded membrane, encompasses many structural and signalling functions and is thought to play a role in cell growth and sarcomere addition. Its relationship to the contractile myofibrils is central to myocyte function. The myofibrils continue their ordered sarcomeric structure up to the edge of the intercalated disc where there is no terminal Z-disc but, instead a transitional junction. Thin actin-containing filaments from the final half sarcomere extend beyond their normal length through the transitional junction to the folded intercalated disc membrane where tension is transmitted. The peaks of the membrane folds also occur at the transitional level. They are spectrin rich and associated with sarcoplasmic reticulum vesicles. A subset of Z-disc proteins including titin, alpha-actinin and ZASP/cypher/oracle are found in the transitional region while others such as telethonin and FATZ/calsarcin/myozenin are absent. The presence of titin enables ordered sarcomeres to be maintained independently of changes in the amplitude of the membrane folds. The transitional junction is therefore poised to act as a site for a new Z-disc/SR/T-tubule complex and sarcomere addition. The evidence for this is reviewed.

Myeloperoxidase-derived oxidants inhibit sarco/endoplasmic reticulum Ca2 +-ATPase activity and perturb Ca2 + homeostasis in human coronary artery endothelial cells

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) plays a critical role in Ca2+ homeostasis via sequestration of this ion in the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in aging tissues and cardiovascular disease. We have shown previously that the myeloperoxidase (MPO)-derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca2+ levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to preformed or enzymatically generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrent with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca2+, to HOSCN or HOCl resulted in a time- and concentration-dependent increase in intracellular Ca2+ under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca2+ pumps/channels, completely attenuated the increase in intracellular Ca2+ consistent with a critical role for SERCA in maintaining endothelial cell Ca2+ homeostasis. Angiotensin II pretreatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca2+ levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers because of their higher SCN− levels.

Slowed relaxation and preserved maximal force in soleus muscles of mice with targeted disruption of theSerca2gene in skeletal muscle

Sarcoplasmic reticulum Ca(2+) ATPases (SERCAs) play a major role in muscle contractility by pumping Ca(2+) from the cytosol into the sarcoplasmic reticulum (SR) Ca(2+) store, allowing muscle relaxation and refilling of the SR with releasable Ca(2+). Decreased SERCA function has been shown to result in impaired muscle function and disease in human and animal models. In this study, we present a new mouse model with targeted disruption of the Serca2 gene in skeletal muscle (skKO) to investigate the functional consequences of reduced SERCA2 expression in skeletal muscle. SkKO mice were viable and basic muscle structure was intact. SERCA2 abundance was reduced in multiple muscles, and by as much as 95% in soleus muscle, having the highest content of slow-twitch fibres (40%). The Ca(2+) uptake rate was significantly reduced in SR vesicles in total homogenates. We did not find any compensatory increase in SERCA1 or SERCA3 abundance, or altered expression of several other Ca(2+)-handling proteins. Ultrastructural analysis revealed generally well-preserved muscle morphology, but a reduced volume of the longitudinal SR. In contracting soleus muscle in vitro preparations, skKO muscles were able to fully relax, but with a significantly slowed relaxation time compared to controls. Surprisingly, the maximal force and contraction rate were preserved, suggesting that skKO slow-twitch fibres may be able to contribute to the total muscle force despite loss of SERCA2 protein. Thus it is possible that SERCA-independent mechanisms can contribute to muscle contractile function.

FRET Detection of Calmodulin Binding to the Cardiac RyR2 Calcium Release Channel

Calmodulin (CaM) binding to the type 2 ryanodine receptor (RyR2) regulates Ca release from the cardiac sarcoplasmic reticulum (SR). However, the structural basis of CaM regulation of the RyR2 is poorly defined, and the presence of other potential CaM binding partners in cardiac myocytes complicates resolution of CaM's regulatory interactions with RyR2. Here, we show that a fluorescence-resonance-energy-transfer (FRET)-based approach can effectively resolve RyR2 CaM binding, both in isolated SR membrane vesicles and in permeabilized ventricular myocytes. A small FRET donor was targeted to the RyR2 cytoplasmic assembly via fluorescent labeling of the FKBP12.6 subunit. Acceptor fluorophore was attached at discrete positions within either the N- or the C-lobe of CaM. FRET between FKBP12.6 and CaM bound to SR vesicles indicated CaM binding at a single high-affinity site within 60 Å of FKBP12.6. Micromolar Ca increased the apparent affinity of CaM binding and slowed CaM dissociation, but did not significantly affect maximal FRET efficiency at saturating CaM. FRET was strongest when the acceptor was attached at either of two positions within CaM's N-lobe versus sites in CaM's C-lobe, providing CaM orientation information. In permeabilized ventricular myocytes, FKBP12.6 and CaM colocalized to Z-lines, and the efficiency of energy transfer to both the N- and C-lobes of CaM was comparable to that observed in SR vesicle experiments. Results also indicate that both the location and orientation of CaM binding on the RyR2 are very similar to the skeletal muscle RyR1 isoform. Specific binding of CaM to functional RyR2 channels in the cardiac myocyte environment can be monitored using FKBP biosensors and FRET.

Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molybdate

Over the last few decades there has been increasing interest in oxometalate and polyoxometalate applications to medicine and pharmacology. This interest arose, at least in part, due to the properties of these classes of compounds as anti-cancer, anti-diabetic agents, and also for treatment of neurodegenerative diseases, among others. However, our understanding of the mechanism of action would be improved if biological models could be used to clarify potential toxicological effects in main cellular processes. Sarcoplasmic reticulum (SR) vesicles, containing a large amount of Ca 2 + -ATPase, an enzyme that accumulates calcium by active transport using ATP, have been suggested as a useful model to study the effects of oxometalates on calcium homeostasis. In the present article, it is shown that decavanadate, decaniobate, vanadate, tungstate and molybdate, all inhibited SR Ca 2+-ATPase, with the following IC 50 values: 15, 35, 50, 400 μM and 45 mM, respectively. Decaniobate (Nb 10), is the strongest P-type enzyme inhibitor, after decavanadate (V 10). Atomic-absorption spectroscopy (AAS) analysis, indicates that decavanadate binds to the protein with a 1:1 decavanadate:Ca 2 + -ATPase stoichiometry. Furthermore, V 10 binds with similar extension to all the protein conformations, which occur during calcium translocation by active transport, namely E1, E1P, E2 and E2P, as analysed by AAS. In contrast, it was confirmed that the binding of monomeric vanadate (H 2VO 4 2 − ; V 1) to the calcium pump is favoured only for the E2 and E2P conformations of the ATPase, whereas no significant amount of vanadate is bound to the E1 and E1P conformations. Scatchard plot analysis, confirmed a 1:1 ratio for decavanadate–Ca 2 + -ATPase, with a dissociation constant, k d of 1 μM − 1 . The interaction of decavanadate V 10O 28 6 − (V 10) with Ca 2 + -ATPase is prevented by the isostructural and isoelectronic decaniobate Nb 10O 28 6 − (Nb 10), whereas no significant effects were detected with ATP or with heparin, a known competitive ATP binding molecule, suggesting that V 10 binds non-competitively, with respect to ATP, to the protein. Finally, it was shown that decaniobate inhibits SR Ca 2 + -ATPase activity in a non competitive type of inhibition, with respect to ATP. Taken together, these data demonstrate that decameric niobate and vanadate species are stronger inhibitors of the SR calcium ATPase than simple monomeric vanadate, tungstate and molybdate oxometalates, thus affecting calcium homeostasis, cell signalling and cell bioenergetics, as well many other cellular processes. The ability of these oxometalates to act either as phosphate analogues, as a transition-state analogue in enzyme-catalysed phosphoryl group transfer processes and as potentially nucleotide-dependent enzymes modulators or inhibitors, suggests that different oxometalates may reveal different mechanistic preferences in these classes of enzymes.

Kinetics of Luminal Proton Binding to the SR Ca-ATPase

An open membrane preparation containing SR Ca-ATPase was prepared from sarcoplasmic-reticulum vesicles to study the ion binding kinetics in the P-E2 conformation. Because Ca2+ and H+ binding are electrogenic reactions, fluorescent styryl dyes could be used to determine changes in the binding site occupation in equilibrium titration experiments and time-resolved relaxation processes triggered by a pH jump. By photo release from caged proton the pH of the electrolyte could be decreased in a step of 0.1 pH units by a single ultraviolet-laser flash. Analysis of the pH-jump induced relaxation process in the P-E2 conformation showed that three Ca-ATPase-specific processes could be identified, fast H+ binding (τ < 100 μs) and pH-insensitive conformational relaxations after the release of the Ca2+ ion (τ ∼160 ms), and a slow process (τ ∼3.4 s) whose origin could not be unambiguously revealed. The Ca2+-binding affinity in the P-E2 conformation was reduced with increasing pH, a behavior that can be explained by a reversible transition of the empty P-E2 state to an inactivated state of the ion pump. All findings are interpreted in the framework of the Post-Albers pump cycle introduced previously, supplemented by an additional transition to an inhibited state of the ion pump.