Modulation of SERCA in the chronic phase of adjuvant arthritis as a possible adaptation mechanism of redox imbalance

Adjuvant arthritis (AA) is a condition that involves systemic oxidative stress. Unexpectedly, it was found that sarcoplasmic reticulum Ca^{2 +}-ATPase (SERCA) activity was elevated in muscles of rats with AA compared to controls, suggesting possible conformational changes in the enzyme. There was no alteration in the nucleotide binding site but rather in the transmembrane domain according to the tryptophan polar/non-polar fluorescence ratio. Higher relative expression of SERCA, higher content of nitrotyrosine but no increase in phospholipid oxidation in AA SR was found. In vitro treatments of SR with HOCl showed that in AA animals SERCA activity was more susceptible to oxidative stress, but SR phospholipids were more resistant and SERCA could also be activated by phosphatidic acid. It was concluded that increased SERCA activity in AA was due to increased levels of SERCA protein and structural changes to the protein, probably induced by direct and specific oxidation involving reactive nitrogen species.     

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.     

Activating and deactivating roles of lipid bilayers on the Ca(2+)-ATPase/phospholamban complex

The physicochemical properties of the lipid bilayer shape the structure and topology of membrane proteins and regulate their biological function. Here, we investigated the functional effects of various lipid bilayer compositions on the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) in the presence and absence of its endogenous regulator, phospholamban (PLN). In the cardiac muscle, SERCA hydrolyzes one ATP molecule to translocate two Ca(2+) ions into the SR membrane per enzymatic cycle. Unphosphorylated PLN reduces SERCA's affinity for Ca(2+) and affects the enzymatic turnover. We varied bilayer thickness, headgroup, and fluidity and found that both the maximal velocity (V(max)) of the enzyme and its apparent affinity for Ca(2+) (K(Ca)) are strongly affected. Our results show that (a) SERCA's V(max) has a biphasic dependence on bilayer thickness, reaching maximum activity with 22-carbon lipid chain length, (b) phosphatidylethanolamine (PE) and phosphatidylserine (PS) increase Ca(2+) affinity, and (c) monounsaturated lipids afford higher SERCA V(max) and Ca(2+) affinity than diunsaturated lipids. The presence of PLN removes the activating effect of PE and shifts SERCA's activity profile, with a maximal activity reached in bilayers with 20-carbon lipid chain length. Our results in synthetic lipid systems compare well with those carried out in native SR lipids. Importantly, we found that specific membrane compositions closely reproduce PLN effects (V(max) and K(Ca)) found in living cells, reconciling an ongoing controversy regarding the regulatory role of PLN on SERCA function. Taken with the physiological changes occurring in the SR membrane composition, these studies underscore a possible allosteric role of the lipid bilayers on the SERCA/PLN complex.     

跨膜Ca~(2+)梯度对肌质网Ca~3+-ATP酶调节的特异性

我们曾报道跨膜Ca~(2+)梯度可通过膜脂影响肌质网Ca~(2+)-ATP 酶的构象和活性。本文就跨膜Ca~(2+)梯度对肌质网Ca~(2+)-ATP 酶的调节是否具有特异性作进一步研究。结果表明这种特异性表现在两方面:一是跨膜Ca~(2+)梯度对肌质网Ca~(2+)-ATP 酶功能的调节不能归结于跨膜Ca~(2+)浓度梯度所导致的膜电位的作用,离子载体FCCP 可消除跨膜电位但并不影响肌质网Ca~(2+)-ATP 酶的活力;二是其它二价金属离子如Sr~(2+)的跨膜梯度对肌质网Ca~(2+)-ATP 酶活力基本无影响。荧光偏振系列探剂n-AS 测定的结果表明跨膜Ca~(2+)与Sr~(2+)梯度对嵌有Ca~(2+)-ATP 酶的脂酶体的中部流动性的影响有较大差异。而Ca~(2+)-ATP 酶的Ca~(2+)结合位点正处于脂双层中部,这进一步提示膜脂参与了跨膜Ca~(2+)梯度对Ca~(2+)-ATP 酶的调节作用。     

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.     

Highly cooperative dependence of sarco/endoplasmic reticulum calcium ATPase SERCA2a pump activity on cytosolic calcium in living cells

Sarco/endoplasmic reticulum (SR/ER) Ca(2+)-ATPase (SERCA) is an intracellular Ca(2+) pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca(2+) stores is pivotal for maintaining intracellular Ca(2+) homeostasis, and disturbed SERCA activity causes many disease phenotypes, including heart failure, diabetes, cancer, and Alzheimer disease. Although SERCA activity has been described using a simple enzyme activity equation, the dynamics of SERCA activity in living cells is still unknown. To monitor SERCA activity in living cells, we constructed an enhanced CFP (ECFP)- and FlAsH-tagged SERCA2a, designated F-L577, which retains the ATP-dependent Ca(2+) pump activity. The FRET efficiency between ECFP and FlAsH of F-L577 is dependent on the conformational state of the molecule. ER luminal Ca(2+) imaging confirmed that the FRET signal changes directly reflect the Ca(2+) pump activity. Dual imaging of cytosolic Ca(2+) and the FRET signals of F-L577 in intact COS7 cells revealed that SERCA2a activity is coincident with the oscillatory cytosolic Ca(2+) concentration changes evoked by ATP stimulation. The Ca(2+) pump activity of SERCA2a in intact cells can be expressed by the Hill equation with an apparent affinity for Ca(2+) of 0.41 ± 0.0095 μm and a Hill coefficient of 5.7 ± 0.73. These results indicate that in the cellular environment the Ca(2+) dependence of ATPase activation is highly cooperative and that SERCA2a acts as a rapid switch to refill Ca(2+) stores in living cells for shaping the intracellular Ca(2+) dynamics. F-L577 will be useful for future studies on Ca(2+) signaling involving SERCA2a activity.     

Conformational changes in sarcoplasmic reticulum Ca(2+)-ATPase mutants: effect of mutations either at Ca(2+)-binding site II or at tryptophan 552 in the cytosolic domain

By analyzing, after expression in yeast and purification, the intrinsic fluorescence properties of point mutants of rabbit Ca(2+)-ATPase (SERCA1a) with alterations to amino acid residues in Ca(2+)-binding site I (E(771)), site II (E(309)), in both sites (D(800)), or in the nucleotide-binding domain (W(552)), we were able to follow the conformational changes associated with various steps in the ATPase catalytic cycle. Whereas Ca(2+) binding to purified wild-type (WT) ATPase in the absence of ATP leads to the rise in Trp fluorescence expected for the so-called E2 --> E1Ca(2) transition, the Ca(2+)-induced fluorescence rise is dramatically reduced for the E(309)Q mutant. As this purified E(309)Q mutant retains the ability to bind Ca(2+) at site I (but not at site II), we tentatively conclude that the protein reorganization induced by Ca(2+) binding at site II makes the major contribution to the overall Trp fluorescence changes observed upon Ca(2+) binding to both sites. Judging from the fluorescence response of W(552)F, similar to that of WT, these changes appear to be primarily due to membranous tryptophans, not to W(552). The same holds for the fluorescence rise observed upon phosphorylation from P(i) (the so-called E2 --> E2P transition). As for WT ATPase, Mg(2+) binding in the absence of Ca(2+) affects the fluorescence of the E(309)Q mutant, suggesting that this Mg(2+)-dependent fluorescence rise does not reflect binding of Mg(2+) to Ca(2+) sites; instead, Mg(2+) probably binds close to the catalytic site, or perhaps near transmembrane span M3, at a location recently revealed by Fe(2+)-catalyzed oxidative cleavage. Mutation of W(552) hardly affects ATP-induced fluorescence changes in the absence of Ca(2+), which are therefore mostly due to membranous Trp residues, demonstrating long-range communication between the nucleotide-binding domain and the membranous domain.     

Conformational basis of the phospholipid requirement for the activity of SR Ca(2+)-ATPase

The delipidated sarcoplasmic reticulum (SR) Ca(2+)-ATPase was reconstituted into proteoliposomes containing different phospholipids. The result demonstrated the necessity of phosphatidylcholine (PC) for optimal ATPase activity and phosphatidylethanolamine (PE) for the optimal calcium transport activity. Fluorescence intensity of Fluorescein 5-isothiocyanate (FITC)-labeled enzyme at Lys515 as well as the measurement of the distance between 5-((2-[(iodoacetyl) amino] ethyl) amino)naphthalene-1-sulphonic acid (IAEDANS) label sites (Cys674/670) and Pr3+ demonstrated a conformational change of cytoplasmic domain, consequently, leading to the variation of the enzyme function with the proteoliposomes composition. Both the intrinsic fluorescence of Trp and its dynamic quenching by HB decreased with increasing PE content, revealing the conformational change of transmembrane domain. Time-resolved fluorescence study characterized three classes of Trp residues, which showed distinctive variation with the change in phospholipid composition. The phospholipid headgroup size caused the conformational change of SR Ca(2+)-ATPase, subsequent the ATPase activity and Ca2+ uptake.     

2-Color calcium pump reveals closure of the cytoplasmic headpiece with calcium binding

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The "2-color" SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C-terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB slow the kinetics of Tg binding. FRET in ionophore-permeabilized cells was lower in EGTA than in saturating calcium for all constructs, indicating a decrease in domain separation distance with the structural transition from E2 (Ca-free) to E1 (Ca-bound). The data suggest closure of the cytoplasmic headpiece with Ca-binding. The present results provide insight into the structural dynamics of the Ca-ATPase. In addition, the 2-color SERCA constructs developed for this study may be useful for evaluating candidate small molecule regulators of Ca uptake activity.     

Phospholamban binds with differential affinity to calcium pump conformers

To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. Electrical pacing of cardiac myocytes elicited cytoplasmic Ca(2+) elevations, but these increases in Ca(2+) produced only modest changes in SERCA-PLB FRET. The data suggest that the regulatory complex is not disrupted by elevations of cytosolic calcium during cardiac contraction (systole). This conclusion was also supported by parallel experiments in heterologous cells, which showed that FRET was reduced but not abolished by calcium. Thapsigargin also elicited a small decrease in PLB-SERCA binding affinity. We propose that PLB is not displaced from SERCA by high calcium during systole, and relief of functional inhibition does not require dissociation of the regulatory complex. The observed modest reduction in the affinity of the PLB-SERCA complex with Ca(2+) or thapsigargin suggests that the binding interface is altered by SERCA conformational changes. The results are consistent with multiple modes of PLB binding or alternative binding sites.     

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.     

Characterization of the ATP-binding domain of the sarco(endo)plasmic reticulum Ca(2+)-ATPase: probing nucleotide binding by multidimensional NMR.

The skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1a) mediates muscle relaxation by pumping Ca(2+) from the cytosol to the ER/SR lumen. In efforts aimed at understanding the structural basis for the conformational changes accompanying the reaction cycle catalyzed by SERCA1a, we have studied the ATP-binding domain of SERCA1a in both nucleotide-bound and -free forms by NMR. Limited proteolysis analyses guided us to express a 28 kDa stably folded fragment containing the nucleotide-binding domain of SERCA1a spanning residues Thr357-Leu600. ATP binding activity was demonstrated for this fragment by a FITC competition assay. A nearly complete backbone resonance assignment of this 28 kDa ATP-binding fragment, in both the AMP-PNP-bound and -free forms, was obtained by means of heteronuclear multidimensional NMR techniques. NMR titration experiments with AMP-PNP revealed a confined nucleotide-binding site which coincides with a cytoplasmic pocket region identified in the crystal structure of apo-SERCA1a. These results are consistent with previous site-directed mutagenesis studies of SERCA1a.     

Capsaicin stimulates uncoupled ATP hydrolysis by the sarcoplasmic reticulum calcium pump

In muscle cells the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) couples the free energy of ATP hydrolysis to pump Ca(2+) ions from the cytoplasm to the SR lumen. In addition, SERCA plays a key role in non-shivering thermogenesis through uncoupled reactions, where ATP hydrolysis takes place without active Ca(2+) translocation. Capsaicin (CPS) is a naturally occurring vanilloid, the consumption of which is linked with increased metabolic rate and core body temperature. Here we document the stimulation by CPS of the Ca(2+)-dependent ATP hydrolysis by SERCA without effects on Ca(2+) accumulation. The stimulation by CPS was significantly dependent on the presence of a Ca(2+) gradient across the SR membrane. ATP activation assays showed that the drug reduced the nucleotide affinity at the catalytic site, whereas the affinity at the regulatory site increased. Several biochemical analyses indicated that CPS stabilizes an ADP-insensitive E(2)P-related conformation that dephosphorylates at a higher rate than the control enzyme. Under conditions where uncoupled SERCA was specifically inhibited by the treatment with fluoride, low temperatures, or dimethyl sulfoxide, CPS had no stimulatory effect on ATP hydrolysis by SERCA. It is concluded that CPS stabilizes a SERCA sub-conformation where Ca(2+) is released from the phosphorylated intermediate to the cytoplasm instead of the SR lumen, increasing ATP hydrolysis not coupled with Ca(2+) transport. To the best of our knowledge CPS is the first natural drug that augments uncoupled SERCA, presumably resulting in thermogenesis. The role of CPS as a SERCA modulator is discussed.     

Phospholamban ablation in hearts expressing the high affinity SERCA2b isoform normalizes global Ca²⁺ homeostasis but not Ca²⁺-dependent hypertrophic signaling

Cardiomyocytes from failing hearts exhibit reduced levels of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) and/or increased activity of the endogenous SERCA inhibitor phospholamban. The resulting reduction in the Ca(2+) affinity of SERCA impairs SR Ca(2+) cycling in this condition. We have previously investigated the physiological impact of increasing the Ca(2+) affinity of SERCA by substituting SERCA2a with the higher affinity SERCA2b pump. When phospholamban was also ablated, these double knockouts (DKO) exhibited a dramatic reduction in total SERCA levels, severe hypertrophy, and diastolic dysfunction. We presently examined the role of cardiomyocyte Ca(2+) homeostasis in both functional and structural remodeling in these hearts. Despite the low SERCA levels in DKO, we observed near-normal Ca(2+) homeostasis with rapid Ca(2+) reuptake even at high Ca(2+) loads and stimulation frequencies. Well-preserved global Ca(2+) homeostasis in DKO was paradoxically associated with marked activation of the Ca(2+)-dependent nuclear factor of activated T-cell-calcineurin pathway known to trigger hypertrophy. No activation of the MAP kinase signaling pathway was detected. These findings suggest that local changes in Ca(2+) homeostasis may play an important signaling role in DKO, perhaps due to reduced microdomain Ca(2+) buffering by SERCA2b. Furthermore, alterations in global Ca(2+) homeostasis can also not explain impaired in vivo diastolic function in DKO. Taken together, our results suggest that normalizing global cardiomyocyte Ca(2+) homeostasis does not necessarily protect against hypertrophy and heart failure development and that excessively increasing SERCA Ca(2+) affinity may be detrimental.     

Effects of Ser16 phosphorylation on the allosteric transitions of phospholamban/Ca(2+)-ATPase complex

Phosphorylation by protein kinase A and dephosphorylation by protein phosphatase 1 modulate the inhibitory activity of phospholamban (PLN), the endogenous regulator of the sarco(endo)plasmic reticulum calcium Ca(2+) ATPase (SERCA). This cyclic mechanism constitutes the driving force for calcium reuptake from the cytoplasm into the myocite lumen, regulating cardiac contractility. PLN undergoes a conformational transition between a relaxed (R) and tense (T) state, an equilibrium perturbed by the addition of SERCA. Here, we show that the single phosphoryl transfer at Ser16 induces a more pronounced conformational switch to the R state in phosphorylated PLN (pPLN). The binding affinity of PLN to SERCA is not affected (K(d) values for the transmembrane domains of pPLN and PLN are approximately 60 microM), supporting the hypothesis that phosphorylation at Ser16 does not dissociate PLN from SERCA. However, the binding surface and dynamics in domain Ib (residues 22-31) change substantially upon phosphorylation. Since PLN can be singly or doubly phosphorylated at Ser16 and Thr17, we propose that these sites remotely control the conformation of domain Ib. These findings constitute a paradigm for how post-translational modifications such as phosphorylation in the cytoplasmic portion of membrane proteins control intramembrane protein-protein interactions.     

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.     

Overexpression of SERCA2b in the heart leads to an increase in sarcoplasmic reticulum calcium transport function and increased cardiac contractility

The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca(2+) affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately approximately 20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calcium-dependent calcium uptake measurements showed that the maximal velocity of Ca(2+) uptake was not changed, but the apparent pump affinity for Ca(2+) (K(0.5)) was increased in SERCA2b transgenic mice (0.199 +/- 0.011 micrometer) compared with wild-type control mice (0.269 +/- 0.012 micrometer, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.     

Expression of the sarco/endoplasmic Ca(2+)-ATPase, SERCA1a, in fibroblasts induces the formation of organelle membrane arrays

Members of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) family are transmembrane proteins that are essential for the function of intracellular Ca(2+) storage organelles. We found that overexpression of avian muscle SERCA1a in transfected mouse fibroblasts led to the appearance of tubular membrane bundles that we termed plaques. These structures were generated in transfected cells when SERCA1a protein expression approached the endogenous level measured in chicken skeletal muscle. Plaque membranes had associated ribosomes and contained endoplasmic reticulum (ER) proteins. Endogenous ER protein levels were not elevated in SERCA1a-expressing cells, indicating that plaques were not generalized proliferations of ER but rather a reorganization of existing organelle membrane. Plaque formation also was observed in cells expressing a green fluorescent protein-SERCA1a fusion protein (GFP-SERCA1a). GFP-SERCA1a molecules displayed extensive lateral mobility between plaques, suggesting the presence of membrane continuities between these structures. Plaques were induced in cells expressing cDNA encoding a catalytically silent SERCA1a mutant indicating that ER redistribution was driven by a structural feature of the enzyme. SERCA1a-induced plaque formation shares some characteristics of sarcoplasmic reticulum (SR) biogenesis during muscle differentiation, and high-level SERCA1a expression in vivo may contribute to the formation of SR from ER during embryonic myogenesis.     

Ablation of sarcolipin results in atrial remodeling

Sarcolipin (SLN) is a key regulator of sarco(endo)plasmic reticulum (SR) Ca(2+)-ATPase (SERCA), and its expression is altered in diseased atrial myocardium. To determine the precise role of SLN in atrial Ca(2+) homeostasis, we developed a SLN knockout (sln-/-) mouse model and demonstrated that ablation of SLN enhances atrial SERCA pump activity. The present study is designed to determine the long-term effects of enhanced SERCA activity on atrial remodeling in the sln-/- mice. Calcium transient measurements show an increase in atrial SR Ca(2+) load and twitch Ca(2+) transients. Patch-clamping experiments demonstrate activation of the forward mode of sodium/calcium exchanger, increased L-type Ca(2+) channel activity, and prolongation of action potential duration at 90% repolarization in the atrial myocytes of sln-/- mice. Spontaneous Ca(2+) waves, delayed afterdepolarization, and triggered activities are frequent in the atrial myocytes of sln-/- mice. Furthermore, loss of SLN in atria is associated with increased interstitial fibrosis and altered expression of genes encoding collagen and other extracellular matrix proteins. Our results also show that the sln-/- mice are susceptible to atrial arrhythmias upon aging. Together, these findings indicate that ablation of SLN results in increased SERCA activity and SR Ca(2+) load, which, in turn, could cause abnormal intracellular Ca(2+) handling and atrial remodeling.     

Phospholamban expressed in slow-twitch and chronically stimulated fast-twitch muscles minimally affects calcium affinity of sarcoplasmic reticulum Ca(2+)-ATPase.

Chronic excitation, at 2 Hz for 6-7 weeks, of the predominantly fast-twitch canine latissimus dorsi muscle promoted the expression of phospholamban, a protein found in sarcoplasmic reticulum (SR) from slow-twitch and cardiac muscle but not in fast-twitch muscle. At the same time that phospholamban was expressed, there was a switch from the fast-twitch (SERCA1) to the slow-twitch (SERCA2a) Ca(2+)-ATPase isoform. Antibodies against Ca(2+)-ATPase (SERCA2a) and phospholamban were used to assess the relative amounts of the slow-twitch/cardiac isoform of the Ca(2+)-ATPase and phospholamban, which were found to be virtually the same in SR vesicles from the slow-twitch muscle, vastus intermedius; cardiac muscle; and the chronically stimulated fast-twitch muscle, latissimus dorsi. The phospholamban monoclonal antibody 2D12 was added to SR vesicles to evaluate the regulatory effect of phospholamban on calcium uptake. The antibody produced a strong stimulation of calcium uptake into cardiac SR vesicles, by increasing the apparent affinity of the Ca2+ pump for calcium by 2.8-fold. In the SR from the conditioned latissimus dorsi, however, the phospholamban antibody produced only a marginal effect on Ca2+ pump calcium affinity. These different effects of phospholamban on calcium uptake suggest that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch muscles and that phospholamban may have some other function in slow-twitch and chronically stimulated fast-twitch muscle.