Two-dimensional solid-state NMR reveals two topologies of sarcolipin in oriented lipid bilayers

Sarcolipin (SLN), a 31 amino acid integral membrane protein, regulates SERCA1a and SERCA2a, two isoforms of the sarco(endo)plasmic Ca-ATPase, by lowering their apparent Ca(2+) affinity and thereby enabling muscle relaxation. SLN is expressed in both fast-twitch and slow-twitch muscle fibers with significant expression levels also found in the cardiac muscle. SLN shares approximately 30% identity with the transmembrane domain of phospholamban (PLN), and recent solution NMR studies carried out in detergent micelles indicate that the two polypeptides bind to SERCA in a similar manner. Previous 1D solid-state NMR experiments on selectively (15)N-labeled sites showed that SLN crosses the lipid bilayer with an orientation nearly parallel to the bilayer normal. With a view toward the characterization of SLN structure and its interactions with both lipids and SERCA, herein we report our initial structural and topological assignments of SLN in mechanically oriented DOPC/DOPE lipid bilayers as mapped by 2D (15)N PISEMA experiments. The PISEMA spectra obtained on uniformly (15)N-labeled protein as well as (15)N-Leu, (15)N-Ile and (15)N-Val map the secondary structure of SLN and, simultaneously, reveal that SLN exists in two distinct topologies. Both the major and the minor populations assume an orientation with the helix axis tilted by approximately 23 degrees with respect to the lipid bilayer normal, but vary in the rotation angle about the helix axis by approximately 5 degrees . The existence of the multiple populations in model membranes may be a significant requirement for SLN interaction with SERCA.     

Measurement and modeling of Ca2+ waves in isolated rabbit ventricular cardiomyocytes

The time course and magnitude of the Ca(2+) fluxes underlying spontaneous Ca(2+) waves in single permeabilized ventricular cardiomyocytes were derived from confocal Fluo-5F fluorescence signals. Peak flux rates via the sarcoplasmic reticulum (SR) release channel (RyR2) and the SR Ca(2+) ATPase (SERCA) were not constant across a range of cellular [Ca(2+)] values. The Ca(2+) affinity (K(mf)) and maximum turnover rate (V(max)) of SERCA and the peak permeability of the RyR2-mediated Ca(2+) release pathway increased at higher cellular [Ca(2+)] loads. This information was used to create a computational model of the Ca(2+) wave, which predicted the time course and frequency dependence of Ca(2+) waves over a range of cellular Ca(2+) loads. Incubation of cardiomyocytes with the Ca(2+) calmodulin (CaM) kinase inhibitor autocamtide-2-related inhibitory peptide (300 nM, 30 mins) significantly reduced the frequency of the Ca(2+) waves at high Ca(2+) loads. Analysis of the Ca(2+) fluxes suggests that inhibition of CaM kinase prevented the increases in SERCA V(max) and peak RyR2 release flux observed at high cellular [Ca(2+)]. These data support the view that modification of activity of SERCA and RyR2 via a CaM kinase sensitive process occurs at higher cellular Ca(2+) loads to increase the maximum frequency of spontaneous Ca(2+) waves.     

Regulation of sarcoplasmic reticulum Ca2+ reuptake in porcine airway smooth muscle

Regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in airway smooth muscle (ASM) during agonist stimulation involves sarcoplasmic reticulum (SR) Ca(2+) release and reuptake. The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is key to replenishment of SR Ca(2+) stores. We examined regulation of SERCA in porcine ASM: our hypothesis was that the regulatory protein phospholamban (PLN) and the calmodulin (CaM)-CaM kinase (CaMKII) pathway (both of which are known to regulate SERCA in cardiac muscle) play a role. In porcine ASM microsomes, we examined the expression and extent of PLN phosphorylation after pharmacological inhibition of CaM (with W-7) vs. CaMKII (with KN-62/KN-93) and found that PLN is phosphorylated by CaMKII. In parallel experiments using enzymatically dissociated single ASM cells loaded with the Ca(2+) indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [Ca(2+)](i) responses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [Ca(2+)](i) transients to 1 microM ACh, as did W-7 and KN-62. The two inhibitors additionally slowed reuptake in the absence of PLN. In other cells, preexposure to W-7 or KN-62 did not prevent initiation of ACh-induced [Ca(2+)](i) oscillations (which were previously shown to result from repetitive SR Ca(2+) release/reuptake). However, when ACh-induced [Ca(2+)](i) oscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca(2+)](i) transients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [Ca(2+)](i) oscillations in some cells. Preexposure to W-7 or KN-62 did not affect caffeine-induced SR Ca(2+) release, indicating that ryanodine receptor channels were not directly inhibited. These data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR Ca(2+) reuptake, potentially through altered PLN phosphorylation.     

Molecular dynamics simulation of cation-phospholipid clustering in phospholipid bilayers: possible role in stalk formation during membrane fusion

In this study, we performed all-atom long-timescale molecular dynamics simulations of phospholipid bilayers incorporating three different proportions of negatively charged lipids in the presence of K(+), Mg(2+), and Ca(2+) ions to systemically determine how membrane properties are affected by cations and lipid compositions. Our simulations revealed that the binding affinity of Ca(2+) ions with lipids is significantly stronger than that of K(+) and Mg(2+) ions, regardless of the composition of the lipid bilayer. The binding of Ca(2+) ions to the lipids resulted in bilayers having smaller lateral areas, greater thicknesses, greater order, and slower rotation of their lipid head groups, relative to those of corresponding K(+)- and Mg(2+)-containing systems. The Ca(2+) ions bind preferentially to the phosphate groups of the lipids. The complexes formed between the cations and the lipids further assembled to form various multiple-cation-centered clusters in the presence of anionic lipids and at higher ionic strength-most notably for Ca(2+). The formation of cation-lipid complexes and clusters dehydrated and neutralized the anionic lipids, creating a more-hydrophobic environment suitable for membrane aggregation. We propose that the formation of Ca(2+)-phospholipid clusters across apposed lipid bilayers can work as a "cation glue" to adhere apposed membranes together, providing an adequate configuration for stalk formation during membrane fusion.     

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.     

Transmembrane helix M6 in sarco(endo)plasmic reticulum Ca(2+)-ATPase forms a functional interaction site with phospholamban. Evidence for physical interactions at other sites.

In an earlier study (Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1997) J. Biol. Chem. 272, 15061-15064), mutation of amino acids on one face of the phospholamban (PLN) transmembrane helix led to loss of PLN inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) molecules. This helical face was proposed to form a site of PLN interaction with a transmembrane helix in SERCA molecules. To determine whether predicted transmembrane helices M4, M5, M6, or M8 in SERCA1a interact with PLN, SERCA1a mutants were co-expressed with wild-type PLN and effects on Ca(2+) dependence of Ca(2+) transport were measured. Wild-type inhibitory interactions shifted apparent Ca(2+) affinity of SERCA1a by an average of -0.34 pCa units, but four of the seven mutations in M4 led to a more inhibitory shift in apparent Ca(2+) affinity, averaging -0.53 pCa units. Seven mutations in M5 led to an average shift of -0.32 pCa units and seven mutations in M8 led to an average shift of -0.30 pCa units. Among 11 mutations in M6, 1, Q791A, increased the inhibitory shift (-0.59 pCa units) and 5, V795A (-0.11), L802A (-0.07), L802V (-0.04), T805A (-0.11), and F809A (-0.12), reduced the inhibitory shift, consistent with the view that Val(795), Leu(802), Thr(805), and Phe(809), located on one face of a predicted M6 helix, form a site in SERCA1a for interaction with PLN. Those mutations in M4, M6, or M8 of SERCA1a that enhanced PLN inhibitory function did not enhance PLN physical association with SERCA1a, but mutants V795A and L802A in M6, which decreased PLN inhibitory function, decreased physical association, as measured by co-immunoprecipitation. In related studies, those PLN mutants that gained inhibitory function also increased levels of co-immunoprecipitation of wild-type SERCA1a and those that lost inhibitory function also reduced association, correlating functional interaction sites with physical interaction sites. Thus, both functional and physical data confirm that PLN interacts with M6 SERCA1a.     

Decreased cardiac SERCA2 expression, SR Ca uptake, and contractile function in hypothyroidism are attenuated in SERCA2 overexpressing transgenic rats

The sarco/endoplasmic reticulum (SR) Ca(2+)-ATPase SERCA2a has a key role in controlling cardiac contraction and relaxation. In hypothyroidism, decreased expression of the thyroid hormone (TH)-responsive SERCA2 gene contributes to slowed SR Ca(2+) reuptake and relaxation. We investigated whether cardiac expression of a TH-insensitive SERCA2a cDNA minigene can rescue SR Ca(2+) handling and contractile function in female SERCA2a-transgenic rats (TG) with experimental hypothyroidism. Wild-type rats (WT) and TG were rendered hypothyroid by 6-N-propyl-2-thiouracil treatment for 6 wk; control rats received no treatment. In vivo measured left ventricular (LV) hemodynamic parameters were compared with SERCA2a expression and function in LV tissue. Hypothyroidism decreased LV peak systolic pressure, dP/dt(max), and dP/dt(min) in both WT and TG. However, loss of function was less in TG. Thus slowed relaxation in hypothyroidism was found to be 1.5-fold faster in TG compared with WT (P < 0.05). In parallel, a 1.4-fold higher V(max) value of homogenate SR Ca(2+) uptake was observed in hypothyroid TG (P < 0.05 vs. hypothyroid WT), and the hypothyroidism-caused decline of LV SERCA2a mRNA expression in TG by -24% was markedly less than the decrease of -49% in WT (P < 0.05). A linear relationship was observed between the SERCA2a/PLB mRNA ratio values and the V(max) values of SR Ca(2+) uptake when the respective data of all experimental groups were plotted together (r = 0.90). The data show that expression of the TH-insensitive SERCA2a minigene compensates for loss of expressional activity of the TH-responsive native SERCA2a gene in the female hypothyroid rat heart. However, SR Ca(2+) uptake and in vivo heart function were only partially rescued.     

Abnormal sarcoplasmic reticulum Ca2+-sequestering properties in skeletal muscle in chronic obstructive pulmonary disease

The objective of this study was to investigate the hypothesis that alterations in sarcoplasmic reticulum (SR) Ca(2+)-cycling properties would occur in skeletal muscle in patients with moderate to severe chronic obstructive pulmonary disease (COPD). To investigate this hypothesis, tissue samples were obtained from the vastus lateralis of 8 patients with COPD [age 65.6 +/- 3.2 yr; forced expiratory volume in 1 s (FEV(1))/forced vital capacity (FVC) = 44 +/- 2%; mean +/- SE] and 10 healthy age-matched controls (CON, age 67.5 +/- 2.5 yr; FEV(1)/FVC = 77 +/- 2%), and homogenates were analyzed for a wide range of SR properties. Compared with CON, COPD displayed (in mumol.g protein(-1).min(-1)) a 16% lower maximal Ca(2+)-ATPase activity [maximal velocity (V(max)), 158 +/- 10 vs. 133 +/- 7, P < 0.05] and a 17% lower Ca(2+) uptake (4.65 +/- 0.039 vs. 3.85 +/- 0.26, P < 0.05) that occurred in the absence of differences in Ca(2+) release. The lower V(max) in COPD was also accompanied by an 11% lower (P < 0.05) Ca(2+) sensitivity, as measured by the Hill coefficient (defined as the relationship between Ca(2+)-ATPase activity and free cytosolic Ca(2+) concentration for 10-90% V(max)). For the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoforms, SERCA1a was 16% higher (P < 0.05) and SERCA2a was 14% lower (P < 0.05) in COPD. It is concluded that moderate to severe COPD results in abnormalities in SR Ca(2+)-ATPase properties that cannot be explained by changes in the SERCA isoform phenotypes. The reduced catalytic properties of SERCA in COPD suggest a disturbance in Ca(2+) cycling, possibly resulting in impairment in Ca(2+)-mediated mechanical function and/or second messenger regulated processes.     

Structural dynamics and topology of phospholamban in oriented lipid bilayers using multidimensional solid-state NMR

Phospholamban (PLN), a single-pass membrane protein, regulates heart muscle contraction and relaxation by reversible inhibition of the sarco(endo)plasmic reticulum Ca-ATPase (SERCA). Studies in detergent micelles and oriented lipid bilayers have shown that in its monomeric form PLN adopts a dynamic L shape (bent or T state) that is in conformational equilibrium with a more dynamic R state. In this paper, we use solid-state NMR on both uniformly and selectively labeled PLN to refine our initial studies, describing the topology and dynamics of PLN in oriented lipid bilayers. Two-dimensional PISEMA (polarization inversion spin exchange at the magic angle) experiments carried out in DOPC/DOPE mixed lipid bilayers reveal a tilt angle of the transmembrane domain with respect to the static magnetic field, of 21 +/- 2 degrees and, at the same time, map the rotation angle of the transmembrane domain with respect to the bilayer. PISEMA spectra obtained with selectively labeled samples show that the cytoplasmic domain of PLN is helical and makes an angle of 93 +/- 6 degrees with respect to the bilayer normal. In addition, using samples tilted by 90 degrees , we find that the transmembrane domain of PLN undergoes fast long-axial rotational diffusion about the bilayer normal with the cytoplasmic domain undergoing this motion and other complex dynamics, scaling the values of chemical shift anisotropy. While this dynamic was anticipated by previous solution NMR relaxation studies in micelles, these measurements in the anisotropic lipid environment reveal new dynamic and conformational features encoded in the free protein that might be crucial for SERCA recognition and subsequent inhibition.     

SERCA2a, phospholamban, sarcolipin, and ryanodine receptors gene expression in children with congenital heart defects

In animal models of conotruncal heart defects, an abnormal calcium sensitivity of the contractile apparatus and a depressed L-type calcium current have been described. Sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) is a membrane protein that catalyzes the ATP-dependent transport of Ca(2+) from the cytosol to the SR. The activity of SERCA is inhibited by phospholamban (PLN) and sarcolipin (SLN), and all these proteins participate in maintaining the normal intracellular calcium handling. Ryanodine receptors (RyRs) are the major SR calcium-release channels required for excitation-contraction coupling in skeletal and cardiac muscle. Our objective was to evaluate SERCA2a (i.e., the SERCA cardiac isoform), PLN, SLN, and RyR2 (i.e., the RyR isoform enriched in the heart) gene expression in myocardial tissue of patients affected by tetralogy of Fallot (TOF), a conotruncal heart defect. The gene expression of target genes was assessed semiquantitatively by RT-PCR using the calsequestrin (CASQ, a housekeeping gene) RNA as internal standard in the atrial myocardium of 23 pediatric patients undergoing surgical correction of TOF, in 10 age-matched patients with ventricular septal defect (VSD) and in 13 age-matched children with atrial septal defect (ASD). We observed a significantly lower expression of PLN and SLN in TOF patients, while there was no difference between the expression of SERCA2a and RyR2 in TOF and VSD. These data suggest a complex mechanism aimed to enhance the intracellular Ca(2+) reserve in children affected by tetralogy of Fallot.     

Atomic view of calcium-induced clustering of phosphatidylserine in mixed lipid bilayers

Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca(2+) ions. While Ca(2+) is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established. In this study, combining magic angle spinning (MAS) solid-state NMR (SSNMR) measurements of isotopically labeled serine headgroups in mixed lipid bilayers with molecular dynamics (MD) simulations of PS lipid bilayers in the presence of different counterions, we provide site-resolved insights into the effects of Ca(2+) on the structure and dynamics of lipid bilayers. Ca(2+)-induced conformational changes of PS in mixed bilayers are observed in both liposomes and Nanodiscs, a nanoscale membrane mimetic of bilayer patches. Site-resolved multidimensional correlation SSNMR spectra of bilayers containing (13)C,(15)N-labeled PS demonstrate that Ca(2+) ions promote two major PS headgroup conformations, which are well resolved in two-dimensional (13)C-(13)C, (15)N-(13)C, and (31)P-(13)C spectra. The results of MD simulations performed on PS lipid bilayers in the presence or absence of Ca(2+) provide an atomic view of the conformational effects underlying the observed spectra.     

Transmembrane helix 11 is a genuine regulator of the endoplasmic reticulum Ca2+ pump and acts as a functional parallel of β-subunit on α-Na+,K+-ATPase

The housekeeping sarco(endo)plasmic reticulum Ca(2+) ATPase SERCA2b transports Ca(2+) across the endoplasmic reticulum membrane maintaining a vital Ca(2+) gradient. Compared with the muscle-specific isoforms SERCA2a and SERCA1a, SERCA2b houses an 11th transmembrane segment (TM11) and a short luminal extension (LE) at its C terminus (2b-tail). The 2b-tail imposes a 2-fold higher apparent Ca(2+) affinity and lower V(max). Previously, we assumed that LE is the sole functional region of the 2b-tail and that TM11 is a passive element providing an additional membrane passage. However, here we show that peptides corresponding to the TM11 region specifically modulate the activity of the homologous SERCA1a in co-reconstituted proteoliposomes and mimic the 2b-tail effect (i.e. lower V(max) and higher Ca(2+) affinity). Using truncated 2b-tail variants we document that TM11 regulates SERCA1a independently from LE, confirming that TM11 is a second, previously unrecognized functional region of the 2b-tail. A phylogenetic analysis further indicates that TM11 is the oldest and most conserved feature of the 2b-tail, found in the SERCA pump of all Bilateria, whereas LE is only present in Nematoda and vertebrates. Considering remarkable similarities with the Na(+),K(+)-ATPase α-β interaction, we now propose a model for interaction of TM11 with TM7 and TM10 in the anchoring subdomain of the Ca(2+) pump. This model involves a TM11-induced helix bending of TM7. In conclusion, more than just a passive structural feature, TM11 acts as a genuine regulator of Ca(2+) transport through interaction with the pump.     

Effect of calcium and magnesium on phosphatidylserine membranes: experiments and all-atomic simulations

It is known that phosphatidylserine (PS(-)) lipids have a very similar affinity for Ca(2+) and Mg(2+) cations, as revealed by electrokinetic and stability experiments. However, despite this similar affinity, experimental evidence shows that the presence of Ca(2+) or Mg(2+) induces very different aggregation behavior for PS(-) liposomes as characterized by their fractal dimensions. Also, turbidity measurements confirm substantial differences in aggregation behavior depending on the presence of Ca(2+) or Mg(2+) cations. These puzzling results suggest that although these two cations have a similar affinity for PS(-) lipids, they induce substantial structural differences in lipid bilayers containing each of these cations. In other words, these cations have strong ion-specific effects on the structure of PS(-) membranes. This interpretation is supported by all-atomic molecular-dynamics simulations showing that Ca(2+) and Mg(2+) cations have different binding sites and induce different membrane hydration. We show that although both ions are incorporated deep into the hydrophilic region of the membrane, they have different positions and configurations at the membrane. Absorbed Ca(2+) cations present a peak at a distance ~2 nm from the center of the lipid bilayer, and their most probable binding configuration involves two oxygen atoms from each of the charged moieties of the PS molecule (phosphate and carboxyl groups). In contrast, the distribution of absorbed Mg(2+) cations has two different peaks, located a few angstroms before and after the Ca(2+) peak. The most probable configurations (corresponding to these two peaks) involve binding to two oxygen atoms from carboxyl groups (the most superficial binding peak) or two oxygen atoms from phosphate groups (the most internal peak). Moreover, simulations also show differences in the hydration structure of the membrane: we obtained a hydration of 7.5 and 9 water molecules per lipid in simulations with Ca(2+) and Mg(2+), respectively.     

Mechanisms underlying increases in SR Ca2+-ATPase activity after exercise in rat skeletal muscle

Prolonged exercise followed by a brief period of reduced activity has been shown to result in an overshoot in maximal sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity [maximal velocity (V(max))] in rat locomoter muscles (Ferrington DA, Reijneveld JC, B?r PR, and Bigelow DJ. Biochim Biophys Acta 1279: 203-213, 1996). To investigate the functional significance and underlying mechanisms for the increase in V(max), we analyzed Ca(2+)-ATPase activity and Ca(2+) uptake in SR vesicles from the fast rat gastrocnemius muscles after prolonged running (RUN) and after prolonged running plus 45 min of low-intensity activity (RUN+) or no activity (REC45) and compared them with controls (Con). Although no differences were observed between RUN and Con, both V(max) and Ca(2+) uptake were higher (P < 0.05) by 43 and 63%, respectively, in RUN+ and by 35 and 34%, respectively, in REC45. The increase in V(max) was accompanied by increases (P < 0.05) in the phosphorylated enzyme intermediate measured by [gamma-(32)P]ATP. No differences between groups for each condition were found for the fluorescent probes FITC and (N-cyclohexyl-N(1)-dimethylamino-alpha-naphthyl)carbodiimide, competitive inhibitors of the nucleotide-binding and Ca(2+)-binding sites on the enzyme, respectively. Similarly, no differences for the Ca(2+)-ATPase were observed between groups in nitrotyrosine and phosphoserine residues, a measure of nitrosylation and phosphorylation states, respectively. Western blots indicated no changes in relative isoform content of sarcoendoplasmic reticulum (SERCA)1 and SERCA2a. It is concluded that the increase in V(max) of the Ca(2+)-ATPase observed in recovery is not the result of changes in enzyme nitroslyation or phosphorylation, changes in ATP and Ca(2+)-binding affinity, or changes in protein content of the Ca(2+)-ATPase.     

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.     

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.     

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.     

Probing ground and excited states of phospholamban in model and native lipid membranes by magic angle spinning NMR spectroscopy

In this paper, we analyzed the ground and excited states of phospholamban (PLN), a membrane protein that regulates sarcoplasmic reticulum calcium ATPase (SERCA), in different membrane mimetic environments. Previously, we proposed that the conformational equilibria of PLN are central to SERCA regulation. Here, we show that these equilibria detected in micelles and bicelles are also present in native sarcoplasmic reticulum lipid membranes as probed by MAS solid-state NMR. Importantly, we found that the kinetics of conformational exchange and the extent of ground and excited states in detergent micelles and lipid bilayers are different, revealing a possible role of the membrane composition on the allosteric regulation of SERCA. Since the extent of excited states is directly correlated to SERCA inhibition, these findings open up the exciting possibility that calcium transport in the heart can be controlled by the lipid bilayer composition. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Muscle sarcoplasmic reticulum calcium regulation in humans during consecutive days of exercise and recovery.

The study investigated the hypothesis that three consecutive days of prolonged cycle exercise would result in a sustained reduction in the Ca(2+)-cycling properties of the vastus lateralis in the absence of changes in the sarcoplasmic (endoplasmic) reticulum Ca(2+)-ATPase (SERCA) protein. Tissue samples were obtained at preexercise (Pre) and postexercise (Post) on day 1 (E1) and day 3 (E3) and during recovery day 1 (R1), day 2 (R2), and day 3 (R3) in 12 active but untrained volunteers (age 19.2 +/- 0.27 yr; mean +/- SE) and analyzed for changes (nmol.mg protein(-1).min(-1)) in maximal Ca(2+)-ATPase activity (V(max)), Ca(2+) uptake and Ca(2+) release (phase 1 and phase 2), and SERCA isoform expression (SERCA1a and SERCA2a). At E1, reductions (P < 0.05) from Pre to Post in V(max) (150 +/- 7 vs. 121 +/- 7), Ca(2+) uptake (7.79 +/- 0.28 vs. 5.71 +/- 0.33), and both phases of Ca(2+) release (phase 1, 20.3 +/- 1.3 vs. 15.2 +/- 1.1; phase 2, 7.70 +/- 0.60 vs. 4.99 +/- 0.48) were found. In contrast to V(max), which recovered at Pre E3 and then remained stable at Post E3 and throughout recovery, Ca(2+) uptake remained depressed (P < 0.05) at E3 Pre and Post and at R1 as did phase 2 of Ca(2+) release. Exercise resulted in an increase (P < 0.05) in SERCA1a (14% at R2) but not SERCA2a. It is concluded that rapidly adapting mechanisms protect V(max) following the onset of regular exercise but not Ca(2+) uptake and Ca(2+) release.     

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.