Anionic phospholipids decrease the rate of slippage on the Ca2+-ATPase of sarcoplasmic reticulum

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 solidstate 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 ϳ200 M. 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.Normal physiological control of muscle contractility depends on the strict regulation of intracellular calcium cycling (1). Muscle relaxation is coupled to the reuptake of Ca 2ϩ by the sarcoplasmic reticulum (SR), 2 driven by the sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA), a 110-kDa membraneembedded ion pump that maintains a 10,000-fold Ca 2ϩ concentration gradient across the SR membrane (2). The cardiac isoform of SERCA (SERCA2a) associates reversibly with the pentameric transmembrane protein phospholamban (PLB) in the SR membrane resulting in a transitory inhibition of calcium transport into the SR, which is essential for efficient muscle contraction. Inhibition of SERCA2a is relieved during muscle relaxation as a result of phosphorylation of PLB by cAMP-dependent protein kinases in response to ␤-adrenergic stimulation.Sarcolipin (SLN) is a 31-amino acid PLB homologue that co-expresses and co-localizes with the fast twitch skeletal muscle (SERCA1a) isoform of Ca 2ϩ -ATPase and, to a lesser extent, with SERCA2a in cardiac cells (3-5). SLN causes a decrease in the apparent affinity of SERCA enzymes for calcium, but its effect on V max is less clear, with some studies noting an inhibitory effect and others showing a stimulatory response (6, 7). Expression of SLN in cardiac tissue is low in humans and predominates in the atria, whereas PLB levels are higher and localized in the ventricles, but there is mounting evidence that SLN plays a role in regulating calcium cycling in healthy and compromised heart muscle (10 -13). Co-expression of SERCA2a with SLN and PLB enhances the inhibition of calcium transport by S...

Section: Resultsmentioning

confidence: 99%

Solid-state NMR and Functional Measurements Indicate That the Conserved Tyrosine Residues of Sarcolipin Are Involved Directly in the Inhibition of SERCA1

Hughes

Clayton

Kitmitto

et al. 2007

“…Two reconstitution compositions were examined, one with a lipid/ PLB/Ca 2ϩ -ATPase ratio of 500:20:1 and one with a lipid/PLB/ Ca 2ϩ -ATPase ratio of 160:10:1. Fast-twitch skeletal muscle (SERCA1) Ca 2ϩ -ATPase was reconstituted alone or with synthetic null-cysteine PLB into unsaturated lipid (DOPC) membranes using an adaptation of a previously described procedure (29). SERCA1 was examined instead of the cardiac isoform because skeletal muscle readily yields sufficient (milligram) quantities of enzyme for NMR analyses.…”

Section: Resultsmentioning

confidence: 99%

“…Co-reconstitution of Ca 2ϩ -ATPase with PLB-Co-reconstitution of Ca 2ϩ ATPase and PLB were carried out by an adaptation of a previous method (8,29). Initial molar ratios of DOPC/PLB/Ca 2ϩ -ATPase were 500:20:1 or 160:10:1.…”

Section: Methodsmentioning

confidence: 99%

“…Reconstitution of Ca 2ϩ -ATPase-SERCA1 was reconstituted into dioleoyl-phosphatidylcholine (DOPC) membranes using an adaptation of methods described previously (8,29) but with protein concentrations suitable for NMR analysis. Briefly, DOPC in chloroform was dried to a thin film with argon before drying further under vacuum and rehydrating with 1-2 ml of 10 mM Tris, 0.25 M sucrose buffer, pH 7.5, containing 6 mg/ml ␤OG.…”

Section: Methodsmentioning

confidence: 99%

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Solid-state NMR Reveals Structural Changes in Phospholamban Accompanying the Functional Regulation of Ca2+-ATPase

Hughes

Middleton

2003

Calcium transport across the sarcoplasmic reticulum of cardiac myocytes is regulated by a reversible inhibitory interaction between the Ca 2؉ -ATPase and the small transmembrane protein phospholamban (PLB). A nullcysteine analogue of PLB, containing isotope labels in the transmembrane domain or cytoplasmic domain, was reconstituted into membranes in the absence and presence of the SERCA1 isoform of Ca 2؉ -ATPase for structural investigation by cross-polarization magic-angle spinning (CP-MAS) NMR. PLB lowered the maximal hydrolytic activity of SERCA1 and its affinity for calcium in membrane preparations suitable for structural analysis by NMR. switches from an ␣-helix in pure lipid membranes to a more extended structure in the presence of SERCA1, which may reflect local structural distortions which change the orientations of the transmembrane and cytoplasmic domains. These results suggest that Ca 2؉ -ATPase has a long-range effect on the structure of PLB around residue 25, which promotes the functional association of the two proteins. Phospholamban (PLB)1 is a 52-amino acid membrane-spanning protein, which is expressed predominantly in the sarcoplasmic reticulum of cardiac myocytes (1). The primary physiological function of PLB is to regulate the active transport of calcium ions into the sarcoplasmic reticulum lumen via an inhibitory association with SERCA2a, the cardiac isoform of Ca 2ϩ -ATPase (2, 3). PLB is believed to bind to the calcium-free conformation of SERCA2a (4), and exerts its inhibitory action by reducing the apparent calcium affinity of the enzyme (5). In response to ␤-adrenergic stimulation, PLB is phosphorylated at Ser 16 and Thr 17 by cAMP-dependent kinase and calcium/calmodulin-dependent kinase, respectively. PLB phosphorylation relieves SERCA2a inhibition, which increases the rate of calcium uptake into the sarcoplasmic reticulum and results in the accelerated relaxation of cardiac muscle (2). Mechanistic failures in the reversibility of SERCA2a inhibition, or overexpression of PLB relative to SERCA2a, are linked to the disruption of calcium homeostasis in cardiac cells and may contribute to cardiovascular disorders such as congestive heart failure (6).The exact nature of the interaction between PLB and SERCA enzymes is a subject of debate. PLB readily self-associates to form a homopentamer within the lipid bilayer (7,8), with a dynamic equilibrium believed to exist between the oligomeric and monomeric state of the protein (9). Most evidence suggests that it is the monomeric form of PLB that binds to and inhibits SERCA, with the pentamer acting as a reservoir from which monomers dissociate (5, 8, 10 -12). In reconstitution studies, the molar stoichiometry of PLB to SERCA required for maximal regulation of calcium transport is between 3:1 and 15:1 (12, 13), whereas a fluorescence energy transfer study has suggested that two PLB monomers interact with two Ca 2ϩ ATPase molecules to form a heterodimer (14). In the latter study, it was found that both PLB molecules must be phosphorylated in order t...

“…Removal of detergent by the addition of SM 2 Bio-Beads led to the formation of tightly sealed vesicles containing the Ca 2ϩ -ATPase (26). The reconstituted vesicles are impermeable to ATP so that only outwardly facing ATPase molecules hydrolyze ATP added to the external medium.…”

mentioning

confidence: 99%

The Importance of Carboxyl Groups on the Lumenal Side of the Membrane for the Function of the Ca2+-ATPase of Sarcoplasmic Reticulum

Webb

Khan

East

et al. 2000

Self Cite

The conventional model for transport of Ca 2؉ by the Ca 2؉ -ATPase of skeletal muscle sarcoplasmic reticulum (SR) involves a pair of binding sites for Ca 2؉ that change upon phosphorylation of the ATPase from being high affinity and exposed to the cytoplasm to being low affinity and exposed to the lumen. However, a number of recent experiments suggest that in fact transport involves two separate pairs of binding sites for Ca