Low temperature molecular adaptation of the skeletal muscle sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA 1) in the wood frog (Rana sylvatica)

We have compared the primary sequence and enzymatic properties of the sarcoplasmic reticulum Ca(2+)-ATPases from a cold-tolerant frog Rana sylvatica with those of a closely related cold-intolerant frog, Rana clamitans. Sarcoplasmic reticulum isolated from leg muscles of both species contains a major protein ( approximately 100 kDa) that reacts with a monoclonal antibody against sarco(endo)plasmic reticulum Ca(2+)-ATPase type 1 (SERCA1). The apparent molecular mass of R. sylvatica SERCA1 is 115 kDa, whereas that of R. clamitans is 105 kDa. However, the deduced amino acid sequences obtained from cDNAs do not indicate a difference in molecular weight, thus suggesting post-translational protein modification of R. sylvatica SERCA1. Comparison of the temperature dependence of both ATP hydrolysis and Ca(2+) transport indicates that R. sylvatica SERCA1 exhibits significantly lower activation energy below 20 degrees C and an approximately 2-fold greater Ca(2+)-ATPase activity near 0 degrees C. Furthermore, R. sylvatica SERCA1 exhibits simple Michaelis-Menten kinetics with ATP and Ca(2+) as opposed to the two-site ATP kinetics and positive cooperativity with Ca(2+) observed for R. clamitans and mammalian SERCA1s. Cooperativity has been linked to protein-protein interaction in SERCA1, and this property may be altered in R. sylvatica SERCA1. Primary sequence comparison shows that R. sylvatica SERCA1 exhibits seven unique amino acid substitutions, three of which are in the ATP binding domain. We also report for the first time the presence of alternative splicing in the frog, resulting in isoforms SERCA1a and SERCA1b. Thus, it appears that the low temperature muscle contractility of R. sylvatica can be explained partially by significant functional and structural differences in SERCA1.     

Regulation of sarco-endoplasmic reticulum Ca(2+)-ATPases during platelet-derived growth factor-induced smooth muscle cell proliferation.

The role of the sarco-endoplasmic reticulum Ca(2+)-ATPases (SERCA) in the regulation of cell proliferation by Ca2+ was investigated by testing the effect of platelet-derived growth factor (PDGF) on cultured pig aorta smooth muscle cells. For this purpose, the PDGF-mediated rise in the Ca2+ concentration was first examined for its ability to induce the formation of prostaglandins from the specific membrane enzyme, cyclooxygenase. In parallel experiments, similar conditions (10 ng/ml PDGF for 24 h) were used to investigate the smooth muscle cell membrane SERCA2 isoforms. Total SERCA2 activity rose by 472% as reflected by their specific formation of phosphorylated intermediate (E approximately P). This rise correlated with an increase in the amount of SERCA2 proteins (100 kDa) as shown by Western blotting. With isoform-specific anti-SERCA2-a and anti-SERCA2-b antibodies, we demonstrated that the increase in total SERCA2 proteins concerned the minor isoform SERCA2-a, which rose 10-fold, whereas SERCA2-b proteins were not affected. Lastly, Northern blotting using riboprobes showed that PDGF treatment increased the SERCA2-a mRNA species by 82%, and concomitantly decreased the SERCA2-b mRNA by 28%, as a result of isoform switching. We conclude that up-regulation of the SERCA2-type Ca(2+)-ATPases occurs in PDGF-treated smooth muscle cells, which suggests that this enzymatic system plays an essential part in cell proliferation.     

Store operated Ca2+ entry dependent contraction of coronary artery smooth muscle: inhibition by peroxide pretreatment

The sarco/endoplasmic reticulum (SER) Ca(2+) pool is refilled by the SER Ca(2+) pump (SERCA) using cytosolic Ca(2+) and/or extracellular Ca(2+) entering the cell. The effects of the SERCA pump inhibitor cyclopiazonic acid (CPA) were studied in pig coronary artery smooth muscle using two protocols. In protocol A, the SERCA pump was inhibited by adding CPA to cells/tissues in Ca(2+)-containing solution, whereas in protocol B, CPA was added to cells/tissues in Ca(2+)-free solution, followed by reintroduction of extracellular Ca(2+). Addition of CPA increased cytosolic Ca(2+) in cultured smooth muscle cells and elicited contraction in de-endothelialized coronary arteries in both protocols. Based on pharmacological experiments, the CPA-induced contraction of de-endothelialized arteries in protocol B resulted from store operated Ca(2+) entry (SOCE). Reactive oxygen species such as peroxides are known to damage the SERCA pump in this tissue. Consistently, CPA-induced contractions were decreased in arteries pre-treated with hydrogen peroxide in protocol A. However, this pretreatment also decreased the force of contraction due to SOCE in protocol B, suggesting that it closed SOCE. We propose that the closure of SOCE triggered by exposure to reactive oxygen species may be a protective mechanism, so that Ca(2+) entry by this pathway is disallowed when SERCA is damaged in pathologies such as ischemia-reperfusion.     

Thyroid hormones differentially regulate the distribution of rabbit skeletal muscle Ca(2+)-ATPase (SERCA) isoforms in light and heavy sarcoplasmic reticulum

The sarcoplasmic reticulum (SR) is composed of two fractions, the heavy fraction that contains proteins involved in Ca2+ release, and the light fraction enriched in Ca(2+)-ATPase (SERCA), an enzyme responsible for Ca2+ transport from the cytosol to the lumen of SR. It is known that in red muscle thyroid hormones regulate the expression of SERCA 1 and SERCA 2 isoforms. Here we show the effects of thyroid hormone on SERCA expression and distribution in light and heavy SR fractions from rabbit white and red muscles. In hyperthyroid red muscle there is an increase of SERCA 1 and a decrease of SERCA 2 expression. This is far more pronounced in the heavy than in the light SR fraction. As a result, the rates of Ca(2+)- ATPase activity and Ca(2+)-uptake by the heavy vesicles are increased. In hypothyroidism we observed a decrease in SERCA 1 and no changes in the amount of SERCA 2 expressed. This promoted a decrease of both Ca(2+)-uptake and Ca(2+)-ATPase activity. While the major differences in hyperthyroidism were found in the heavy SR fraction, the effects of hypothyroidism were restricted to light SR fraction. In white muscle we did not observe any significant changes in either hypo- or hyperthyroidism in both SR fractions. Thus, the regulation of SERCA isoforms by thyroid hormones is not only muscle specific but also varies depending on the subcellular compartment analyzed. These changes might correspond to the molecular basis of the altered contraction and relaxation rates detected in thyroid dysfunction.     

Physical interactions between phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPases are dissociated by elevated Ca2+, but not by phospholamban phosphorylation, vanadate, or thapsigargin, and are enhanced by ATP

Previous co-immunoprecipitation studies (Asahi, M., Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 32855-32862) revealed that physical interactions between phospholamban (PLN) and the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA1a) were retained, even with PLN monoclonal antibody 1D11 bound to an epitope lying between PLN residues 7 and 17. Because the 1D11 antibody relieves inhibitory interaction between the two proteins, it was of interest to determine whether PLN phosphorylation or elevation of Ca(2+), which also relieves inhibitory interactions between PLN and SERCA, would disrupt physical interactions. Co-immunoprecipitation was measured in the presence of increasing concentrations of Ca(2+) or after phosphorylation of PLN by protein kinase A. Physical interactions were dissociated by elevated Ca(2+) but not by PLN phosphorylation. The addition of ATP enhanced interactions between PLN and SERCA. The further addition of vanadate and thapsigargin, both of which stabilize the E(2) conformation, did not diminish binding of PLN to SERCA. These data suggest that physical interactions between PLN and SERCA are stable when SERCA is in the Ca(2+)-free E(2) conformation but not when it is in the E(1) conformation and that phosphorylation of PLN does not dissociate physical interactions between PLN and SERCA.     

Sarcolipin inhibits polymerization of phospholamban to induce superinhibition of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs)

Sarcolipin (SLN), a regulator of the sarco(endo)plasmic reticulum Ca(2+)-ATPase of fast-twitch skeletal muscle (SERCA1a), is also expressed in cardiac and slow-twitch skeletal muscles where phospholamban (PLN) and SERCA2a are expressed. Co-expression in HEK-293 cells of SLN tagged N-terminally with a FLAG epitope (NF-SLN), PLN, and SERCAs followed by measurement of the Ca(2+) dependence of Ca(2+) transport activity in isolated microsomal fractions showed that NF-SLN can reduce the apparent Ca(2+) affinity of both SERCA1a (DeltaK(Ca) = -0.22 +/- 0.01 pCa units) and SERCA2a (DeltaK(Ca) = -0.37 +/- 0.04 pCa units). When SERCA1a or SERCA2a were co-expressed with both NF-SLN and PLN, inhibition was synergistic, reducing DeltaK(Ca) by about -1.0 pCa units. Co-immunoprecipitation showed that NF-SLN increased the binding of PLN to SERCA, whereas PLN did not increase the binding of NF-SLN to SERCA. Elevated Ca(2+) dissociates both PLN and NF-SLN from their complexes with both SERCA1a and SERCA2a, but NF-SLN induced resistance to Ca(2+) dissociation of the PLN.SERCA complex. Co-immunoprecipitation of PLN and NF-SLN without SERCA showed that NF-SLN binds directly to PLN and that NF-SLN inhibits the formation of PLN pentamers. Thus the ability of NF-SLN to elevate the content of PLN monomers can account, at least in part, for the superinhibitory effects of NF-SLN in the presence of PLN.     

Distribution of the intracellular Ca(2+)-ATPase isoform 2b in pig brain subcellular fractions and cross-reaction with a monoclonal antibody raised against the enzyme isoform

The presence and distribution of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) isoform 2b in microsomes and other subcellular fractions isolated from pig brain has been demonstrated by the combined use of a specific antibody raised against the SERCA2b isoform and ATP phosphorylation experiments. All subcellular fractions show an approximately 110 kDa phosphorylated protein, the band intensity being stronger in microsomes. Preliminary treatment of the samples with trypsin generates two phosphorylated fragments of about 57 and 33 kDa in the presence of Ca(2+). The observed fragments are typical trypsinized products of the SERCA2b isoform. The monoclonal antibody Y/1F4 raised against the sarcoplasmic reticulum Ca(2+)-ATPase (isoform 1) binds to the 110 kDa band in membranes isolated from brain. The binding was stronger in microsomes than in other fractions. Furthermore, this antibody also recognizes a clear band at around 115 kDa. This band is always stronger in plasma membrane than in synaptosomes or microsomes and is unaffected by trypsin. Phosphorylation studies in the absence of Ca(2+) suggest that the 115 kDa protein is not a Ca(2+)-ATPase.     

Thapsigargin decreases the Na(+)- Ca(2+) exchanger mediated Ca(2+) entry in pig coronary artery smooth muscle

Na(+)- Ca(2+) exchanger (NCX) has been proposed to play a role in refilling the sarco/endoplasmic reticulum (SER) Ca(2+) pool along with the SER Ca(2+) pump (SERCA). Here, SERCA inhibitor thapsigargin was used to determine the effects of SER Ca(2+) depletion on NCX-SERCA interactions in smooth muscle cells cultured from pig coronary artery. The cells were Na(+)-loaded and then placed in either a Na(+)-containing or in a Na(+)-substituted solution. Subsequently, the difference in Ca(2+) entry between the two groups was examined and defined as the NCX mediated Ca(2+) entry. The NCX mediated Ca(2+) entry in the smooth muscle cells was monitored using two methods: Ca(2+)sensitive fluorescence dye Fluo-4 and radioactive Ca(2+). Ca(2+)-entry was greater in the Na(+)-substituted cells than in the Na(+)-containing cells when measured by either method. This difference was established to be NCX-mediated as it was sensitive to the NCX inhibitors. Thapsigargin diminished the NCX mediated Ca(2+) entry as determined by either method. Immunofluorescence confocal microscopy was used to determine the co-localization of NCX1 and subsarcolemmal SERCA2 in the cells incubated in the Na(+)-substituted solution with or without thapsigargin. SER Ca(2+) depletion with thapsigargin increased the co-localization between NCX1 and the subsarcolemmal SERCA2. Thus, inhibition of SERCA2 leads to blockade of constant Ca(2+) entry through NCX1 and also increases proximity between NCX1 and SERCA2. This blockade of Ca(2+) entry may protect the cells against Ca(2+)-overload during ischemia-reperfusion when SERCA2 is known to be damaged.     

Tracing cytoplasmic Ca(2+) ion and water access points in the Ca(2+)-ATPase

Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) transports two Ca(2+) ions across the membrane of the sarco(endo)plasmic reticulum against the concentration gradient, harvesting the required energy by hydrolyzing one ATP molecule during each transport cycle. Although SERCA is one of the best structurally characterized membrane transporters, it is still largely unknown how the transported Ca(2+) ions reach their transmembrane binding sites in SERCA from the cytoplasmic side. Here, we performed extended all-atom molecular dynamics simulations of SERCA. The calculated electrostatic potential of the protein reveals a putative mechanism by which cations may be attracted to and bind to the Ca(2+)-free state of the transporter. Additional molecular dynamics simulations performed on a Ca(2+)-bound state of SERCA reveal a water-filled pathway that may be used by the Ca(2+) ions to reach their buried binding sites from the cytoplasm. Finally, several residues that are involved in attracting and guiding the cations toward the possible entry channel are identified. The results point to a single Ca(2+) entry site close to the kinked part of the first transmembrane helix, in a region loaded with negatively charged residues. From this point, a water pathway outlines a putative Ca(2+) translocation pathway toward the transmembrane ion-binding sites.     

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.     

Mechanism of reversal of phospholamban inhibition of the cardiac Ca2+-ATPase by protein kinase A and by anti-phospholamban monoclonal antibody 2D12

Our model of phospholamban (PLB) regulation of the cardiac Ca(2+)-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca(2+)-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca(2+)-bound state. PLB and Ca(2+) binding to SERCA2a are mutually exclusive, and PLB inhibition of SERCA2a is manifested as a decreased apparent affinity of SERCA2a for Ca(2+). Here we extend this model to explain the reversal of SERCA2a inhibition that occurs after phosphorylation of PLB at Ser(16) by protein kinase A (PKA) and after binding of the anti-PLB monoclonal antibody 2D12, which recognizes residues 7-13 of PLB. Site-specific cysteine variants of PLB were co-expressed with SERCA2a, and the effects of PKA phosphorylation and 2D12 on Ca(2+)-ATPase activity and cross-linking to SERCA2a were monitored. In Ca(2+)-ATPase assays, PKA phosphorylation and 2D12 partially and completely reversed SERCA2a inhibition by decreasing K(Ca) values for enzyme activation, respectively. In cross-linking assays, cross-linking of PKA-phosphorylated PLB to SERCA2a was inhibited at only two of eight sites when conducted in the absence of Ca(2+) favoring E2. However, at a subsaturating Ca(2+) concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. K(Ca) values for cross-linking inhibition were decreased nearly 2-fold at all sites by PLB phosphorylation, demonstrating that phosphorylated PLB binds more weakly to SERCA2a than dephosphorylated PLB. In parallel assays, 2D12 blocked PLB cross-linking to SERCA2a at all eight sites regardless of Ca(2+) concentration. Our results demonstrate that 2D12 restores maximal Ca(2+)-ATPase activity by physically disrupting the binding interaction between PLB and SERCA2a. Phosphorylation of PLB by PKA weakens the binding interaction between PLB and SERCA2a (yielding more PLB-free SERCA2a molecules at intermediate Ca(2+) concentrations), only partially restoring Ca(2+) affinity and Ca(2+)-ATPase activity.     

Proton paths in the sarcoplasmic reticulum Ca(2+) -ATPase

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) pumps Ca(2+) and countertransport protons. Proton pathways in the Ca(2+) bound and Ca(2+)-free states are suggested based on an analysis of crystal structures to which water molecules were added. The pathways are indicated by chains of water molecules that interact favorably with the protein. In the Ca(2+) bound state Ca(2)E1, one of the proposed Ca(2+) entry paths is suggested to operate additionally or alternatively as proton pathway. In analogs of the ADP-insensitive phosphoenzyme E2P and in the Ca(2+)-free state E2, the proton path leads between transmembrane helices M5 to M8 from the lumenal side of the protein to the Ca(2+) binding residues Glu-771, Asp-800 and Glu-908. The proton path is different from suggested Ca(2+) dissociation pathways. We suggest that separate proton and Ca(2+) pathways enable rapid (partial) neutralization of the empty cation binding sites. For this reason, transient protonation of empty cation binding sites and separate pathways for different ions are advantageous for P-type ATPases in general.     

Regulation of RYR1 activity by Ca(2+) and calmodulin

The skeletal muscle calcium release channel (RYR1) is a Ca(2+)-binding protein that is regulated by another Ca(2+)-binding protein, calmodulin. The functional consequences of calmodulin's interaction with RYR1 are dependent on Ca(2+) concentration. At nanomolar Ca(2+) concentrations, calmodulin is an activator, but at micromolar Ca(2+) concentrations, calmodulin is an inhibitor of RYR1. This raises the question of whether the Ca(2+)-dependent effects of calmodulin on RYR1 function are due to Ca(2+) binding to calmodulin, RYR1, or both. To distinguish the effects of Ca(2+) binding to calmodulin from those of Ca(2+) binding to RYR1, a mutant calmodulin that cannot bind Ca(2+) was used to evaluate the effects of Ca(2+)-free calmodulin on Ca(2+)-bound RYR1. We demonstrate that Ca(2+)-free calmodulin enhances the affinity of RYR1 for Ca(2+) while Ca(2+) binding to calmodulin converts calmodulin from an activator to an inhibitor. Furthermore, Ca(2+) binding to RYR1 enhances its affinity for both Ca(2+)-free and Ca(2+)-bound calmodulin.     

Comparing the structure and dynamics of phospholamban pentamer in its unphosphorylated and pseudo-phosphorylated states

Human phospholamban (PLN), a 30 kDa homopentamer in the sarcoplasmic reticulum (SR) membrane, controls the magnitude of heart muscle contraction and relaxation by regulating the calcium pumping activity of the SR Ca(2+)-ATPase (SERCA). When PLN is not phosphorylated, it binds and inhibits SERCA. Phosphorylation of PLN at S16 or T17 releases such inhibitory effect. It remains a matter of debate whether phosphorylation perturbs the structure of PLN, which in turn affects its interaction with SERCA. Here we examine by NMR spectroscopy the structure and dynamics of PLN pentamer with a physiologically relevant, phosphorylation-mimicking mutation, S16E. Based on extensive NMR data, including NOEs, dipolar couplings, and solvent exchange of backbone amides, we conclude that the phosphorylation-mimicking mutation does not perturb the pentamer structure. However, (15)N R(1) and R(2) relaxation rates and (15)N((1)H) NOEs suggest subtle differences in the dynamics of the extramembrane portion of the protein.     

Targeted disruption of the ATP2A1 gene encoding the sarco(endo)plasmic reticulum Ca2+ ATPase isoform 1 (SERCA1) impairs diaphragm function and is lethal in neonatal mice

Mutations in the ATP2A1 gene, encoding isoform 1 of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1), are one cause of Brody disease, characterized in humans by exercise-induced contraction of fast twitch (type II) skeletal muscle fibers. In an attempt to create a model for Brody disease, the mouse ATP2A1 gene was targeted to generate a SERCA1-null mutant mouse line. In contrast to humans, term SERCA1-null mice had progressive cyanosis and gasping respiration and succumbed from respiratory failure shortly after birth. The percentage of affected homozygote SERCA1(-/-) mice was consistent with predicted Mendelian inheritance. A survey of multiple organs from 10-, 15-, and 18-day embryos revealed no morphological abnormalities, but analysis of the lungs in term mice revealed diffuse congestion and epithelial hypercellularity and studies of the diaphragm muscle revealed prominent hypercontracted regions in scattered fibers and increased fiber size variability. The V(max) of Ca(2+) transport activity in mutant diaphragm and skeletal muscle was reduced by 80% compared with wild-type muscle, and the contractile response to electrical stimulation under physiological conditions was reduced dramatically in mutant diaphragm muscle. No compensatory responses were detected in analysis of mRNAs encoding other Ca(2+) handling proteins or of protein levels. Expression of ATP2A1 is largely restricted to type II fibers, which predominate in normal mouse diaphragm. The absence of SERCA1 in type II fibers, and the absence of compensatory increases in other Ca(2+) handling proteins, coupled with the marked increase in contractile function required of the diaphragm muscle to support postnatal respiration, can account for respiratory failure in term SERCA1-null mice.     

Rabbit sarcoplasmic reticulum Ca(2+)-ATPase replaces yeast PMC1 and PMR1 Ca(2+)-ATPases for cell viability and calcineurin-dependent regulation of calcium tolerance

SERCA1a, the fast-twitch skeletal muscle isoform of sarco(endo)plasmic reticulum Ca(2+)-ATPase, was expressed in yeast using the promoter of the plasma membrane H(+)-ATPase. In the yeast Saccharomyces cerevisiae, the Golgi PMR1 Ca(2+)-ATPase and the vacuole PMC1 Ca(2+)-ATPase function together in Ca2+ sequestration and Ca2+ tolerance. SERCA1a expression restored growth of pmc1 mutants in media containing high Ca2+ concentrations, consistent with increased Ca2+ uptake in an internal compartment. SERCA1a expression also prevented synthetic lethality of pmr1 pmc1 double mutants on standard media. Electron microscopy and subcellular fractionation analysis showed that SERCA1a was localized in intracellular membranes derived from the endoplasmic reticulum. Finally, we found that SERCA1a ATPase activity expressed in yeast was regulated by calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase. This result indicates that calcineurin contributes to calcium homeostasis by modulating the ATPase activity of Ca2+ pumps localized in intra-cellular compartments.     

Inhibition by 4-hydroxynonenal (HNE) of Ca2+ transport by SERCA1a: low concentrations of HNE open protein-mediated leaks in the membrane

Exposure of sarcoplasmic reticulum membranes to 4-hydroxy-2-nonenal (HNE) resulted in inhibition of the maximal ATPase activity and Ca(2+) transport ability of SERCA1a, the Ca(2+) pump in these membranes. The concomitant presence of ATP significantly protected SERCA1a ATPase activity from inhibition. ATP binding and phosphoenzyme formation from ATP were reduced after treatment with HNE, whereas Ca(2+) binding to the high-affinity sites was altered to a lower extent. HNE reacted with SH groups, some of which were identified by MALDI-TOF mass spectrometry, and competition studies with FITC indicated that HNE also reacted with Lys(515) within the nucleotide binding pocket of SERCA1a. A remarkable fact was that both the steady-state ability of SR vesicles to sequester Ca(2+) and the ATPase activity of SR membranes in the absence of added ionophore or detergent were sensitive to concentrations of HNE much smaller than those that affected the maximal ATPase activity of SERCA1a. This was due to an increase in the passive permeability of HNE-treated SR vesicles to Ca(2+), an increase in permeability that did not arise from alteration of the lipid component of these vesicles. Judging from immunodetection with an anti-HNE antibody, this HNE-dependent increase in permeability probably arose from modification of proteins of about 150-160kDa, present in very low abundance in longitudinal SR membranes (and in slightly larger abundance in SR terminal cisternae). HNE-induced promotion, via these proteins, of Ca(2+) leakage pathways might be involved in the general toxic effects of HNE.     

Complex effects of ryanodine on the sarcoplasmic reticulum Ca2+ levels in smooth muscle cells

We have studied the effects of ryanodine and inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) with thapsigargin, on both [Ca(2+)](i) and the sarcoplasmic reticulum (SR) Ca(2+) level during caffeine-induced Ca(2+) release in single smooth muscle cells. Incubation with 10 microM ryanodine did not inhibit the first caffeine-induced [Ca(2+)](i) response, although it abolished the [Ca(2+)](i) response to a second application of caffeine. To assess whether ryanodine was inducing a permanent depletion of the internal Ca(2+) stores, we measured the SR Ca(2+) level with Mag-Fura-2. The magnitude of the caffeine-induced reduction in the SR Ca(2+) level was not augmented by incubating cells with 1 microM ryanodine. Moreover, on removal of caffeine, the SR Ca(2+) levels partially recovered in 61% of the cells due to the activity of thapsigargin-sensitive SERCA pumps. Unexpectedly, 10 microM ryanodine instead of inducing complete depletion of SR Ca(2+) stores markedly reduced the caffeine-induced SR Ca(2+) response. It was necessary to previously inhibit SERCA pumps with thapsigargin for ryanodine to be able to induce caffeine-triggered permanent depletion of SR Ca(2+) stores. These data suggest that the effect of ryanodine on smooth muscle SR Ca(2+) stores was markedly affected by the activity of SERCA pumps. Our data highlight the importance of directly measuring SR Ca(2+) levels to determine the effect of ryanodine on the internal Ca(2+) stores.     

Solid-state NMR and functional measurements indicate that the conserved tyrosine residues of sarcolipin are involved directly in the inhibition of SERCA1

The transmembrane protein sarcolipin regulates calcium storage in the sarcoplasmic reticulum of skeletal and cardiac muscle cells by modulating the activity of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs). The highly conserved C-terminal region ((27)RSYQY-COOH) of sarcolipin helps to target the protein to the sarcoplasmic reticulum membrane and may also participate in the regulatory interaction between sarcolipin and SERCA. Here we used solid-state NMR measurements of local protein dynamics to illuminate the direct interaction between the Tyr(29) and Tyr(31) side groups of sarcolipin and skeletal muscle Ca(2+)-ATPase (SERCA1a) embedded in dioleoylphosphatidylcholine bilayers. Further solid-state NMR experiments together with functional measurements on SERCA1a in the presence of NAc-RSYQY, a peptide representing the conserved region of sarcolipin, suggest that the peptide binds to the same site as the parent protein at the luminal face of SERCA1a, where it reduces V(max) for calcium transport and inhibits ATP hydrolysis with an IC(50) of approximately 200 microM. The inhibitory effect of NAc-RSYQY is remarkably sequence-specific, with the native aromatic residues being essential for optimal inhibitory activity. This combination of physical and functional measurements highlights the importance of aromatic and polar residues in the C-terminal region of sarcolipin for regulating calcium cycling and muscle contractility.     

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.