Regulation of the Sarcoplasmic Reticulum Calcium Pump by Divergent Phospholamban Isoforms in Zebrafish

The sarcoplasmic reticulum calcium pump (SERCA) is regulated by the small integral membrane proteins phospholamban (PLN) and sarcolipin (SLN). These regulators have homologous transmembrane regions, yet they differ in their cytoplasmic and luminal domains. Although the sequences of PLN and SLN are practically invariant among mammals, they vary in fish. Zebrafish (zf) appear to harbor multiple PLN isoforms, one of which contains 18 sequence variations and a unique luminal extension. Characterization of this isoform (zfPLN) revealed that SERCA inhibition and reversal by phosphorylation were comparable with human PLN. To understand the sequence variations in zfPLN, chimeras were created by transferring the N terminus, linker, and C terminus of zfPLN onto human PLN. A chimera containing the N-terminal domain resulted in a mild loss of function, whereas a chimera containing the linker domain resulted in a gain of function. This latter effect was due to changes in basic residues in the linker region of PLN. Removing the unique luminal domain of zfPLN (⁵³SFHGM) resulted in loss of function, whereas adding this domain to human PLN had a minimal effect on SERCA inhibition. We conclude that the luminal extension contributes to SERCA inhibition but only in the context of zfPLN. Although this domain is distinct from the SLN luminal tail, zfPLN appears to use a hybrid PLN-SLN inhibitory mechanism. Importantly, the different zebrafish PLN isoforms raise the interesting possibility that sarcoplasmic reticulum calcium handling and cardiac contractility may be regulated by the differential expression of PLN functional variants.     

The Structural Basis for Phospholamban Inhibition of the Calcium Pump in Sarcoplasmic Reticulum

P-type ATPases are a large family of enzymes that actively transport ions across biological membranes by interconverting between high (E1) and low (E2) ion-affinity states; these transmembrane transporters carry out critical processes in nearly all forms of life. In striated muscle, the archetype P-type ATPase, SERCA (sarco(endo)plasmic reticulum Ca²⁺-ATPase), pumps contractile-dependent Ca²⁺ ions into the lumen of sarcoplasmic reticulum, which initiates myocyte relaxation and refills the sarcoplasmic reticulum in preparation for the next contraction. In cardiac muscle, SERCA is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the Ca²⁺ affinity of SERCA and attenuates contractile strength. cAMP-dependent phosphorylation of PLB reverses Ca²⁺-ATPase inhibition with powerful contractile effects. Here we present the long sought crystal structure of the PLB-SERCA complex at 2.8-Å resolution. The structure was solved in the absence of Ca²⁺ in a novel detergent system employing alkyl mannosides. The structure shows PLB bound to a previously undescribed conformation of SERCA in which the Ca²⁺ binding sites are collapsed and devoid of divalent cations (E2-PLB). This new structure represents one of the key unsolved conformational states of SERCA and provides a structural explanation for how dephosphorylated PLB decreases Ca²⁺ affinity and depresses cardiac contractility.     

The Phospholamban Pentamer Alters Function of the Sarcoplasmic Reticulum Calcium Pump SERCA

The interaction of phospholamban (PLN) with the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump is a major regulatory axis in cardiac muscle contractility. The prevailing model involves reversible inhibition of SERCA by monomeric PLN and storage of PLN as an inactive pentamer. However, this paradigm has been challenged by studies demonstrating that PLN remains associated with SERCA and that the PLN pentamer is required for the regulation of cardiac contractility. We have previously used two-dimensional (2D) crystallization and electron microscopy to study the interaction between SERCA and PLN. To further understand this interaction, we compared small helical crystals and large 2D crystals of SERCA in the absence and presence of PLN. In both crystal forms, SERCA molecules are organized into identical antiparallel dimer ribbons. The dimer ribbons pack together with distinct crystal contacts in the helical versus large 2D crystals, which allow PLN differential access to potential sites of interaction with SERCA. Nonetheless, we show that a PLN oligomer interacts with SERCA in a similar manner in both crystal forms. In the 2D crystals, a PLN pentamer interacts with transmembrane segments M3 of SERCA and participates in a crystal contact that bridges neighboring SERCA dimer ribbons. In the helical crystals, an oligomeric form of PLN also interacts with M3 of SERCA, though the PLN oligomer straddles a SERCA-SERCA crystal contact. We conclude that the pentameric form of PLN interacts with M3 of SERCA and that it plays a distinct structural and functional role in SERCA regulation. The interaction of the pentamer places the cytoplasmic domains of PLN at the membrane surface proximal to the calcium entry funnel of SERCA. This interaction may cause localized perturbation of the membrane bilayer as a mechanism for increasing the turnover rate of SERCA.     

Relative Affinity of Calcium Pump Isoforms for Phospholamban Quantified by Fluorescence Resonance Energy Transfer

To investigate the regulation of SERCA1a [sarco(endo)plasmic reticulum calcium ATPase] and SERCA2a calcium pump isoforms by phospholamban (PLB), we quantified PLB-SERCA interactions by fluorescence resonance energy transfer (FRET) in live cells. For both SERCA1a and SERCA2a, FRET to PLB increased with increasing protein expression level to a maximum value corresponding to a probe separation distance of 64 Å. The data indicate that the respective regulatory complexes assume the same overall quaternary conformation. However, FRET measurements also revealed that PLB has a 50% higher apparent affinity for SERCA1a relative to SERCA2a. The results suggest that despite the structural similarities of the respective regulatory complexes, there is preferential binding of PLB to SERCA1a over SERCA2a. This apparent selectivity may have implications for biochemical studies in which SERCA1a is used as a substitute for SERCA2a. It may also be an important strategic consideration for therapeutic overexpression of SERCA isoforms in cardiac muscle.     

Structural Dynamics and Topology of Phosphorylated Phospholamban Homopentamer Reveal Its Role in the Regulation of Calcium Transport

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Doxorubicin Induces Sarcoplasmic Reticulum Calcium Regulation Dysfunction via the Decrease of SERCA2 and Phospholamban Expressions in Rats

This study aims to explore the changes in calcium regulation in the sarcoplasmic reticulum (SR) during doxorubicin (DOX) treatment. Sprague–Dawley rats were treated with intravenous DOX (1.5 mg/kg) twice weekly for 12 treatments. The hemodynamic changes, myocardial oxidative stress, levels of cardiac toxicity markers, and calcium handling of the myocardial SR were observed. When the accumulation of DOX reached 12 mg/kg, (1) heart weight, left ventricular mass, and lung congestion increased significantly, and ascites appeared; (2) SBP, DBP, MAP, +dP/dt, ?dP/dt, and LVSP decreased significantly, and LVEDP increased (p < 0.01); (3) the iNOS activity and MDA and NO concentrations significantly increased, while the SOD decreased (p < 0.05 or 0.01); (4) the serum level of the AST, LDH CPK, cTnI, and BNP increased significantly (p < 0.01); (5) during DOX treatment, the rat SR Ca²⁺ absorption function and Ca²⁺-stimulated ATPase activity declined dramatically, as did the SERCA2 and phospholamban levels (p < 0.01). As expected, all these changes became evident with DOX accumulation in vivo (p < 0.05 or 0.01). In conclusion, DOX induces SR calcium regulation dysfunction via the decrease of SERCA2 and phospholamban expressions in rats.     

Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations

Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein’s constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery—the fact that the two sites involved influence one another in a symmetrical manner—can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest.     

Phospholamban C-terminal Residues Are Critical Determinants of the Structure and Function of the Calcium ATPase Regulatory Complex

To determine the structural and regulatory role of the C-terminal residues of phospholamban (PLB) in the membranes of living cells, we fused fluorescent protein tags to PLB and sarco/endoplasmic reticulum calcium ATPase (SERCA). Alanine substitution of PLB C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB, suggesting a change in quaternary conformation of PLB pentamer and SERCA-PLB regulatory complex. Val to Ala substitution at position 49 (V49A) had particularly large effects on PLB pentamer structure and PLB-SERCA regulatory complex conformation, increasing and decreasing probe separation distance, respectively. We also quantified a decrease in oligomerization affinity, an increase in binding affinity of V49A-PLB for SERCA, and a gain of inhibitory function as quantified by calcium-dependent ATPase activity. Notably, deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. C-terminal truncations also resulted in progressive loss of PLB-PLB FRET due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the binding affinity of truncated PLB for SERCA and loss of inhibitory potency. However, despite decreased SERCA-PLB binding, intermolecular FRET for Val⁴⁹-stop (V49X) truncation mutant was paradoxically increased as a result of an 11.3-Å decrease in the distance between donor and acceptor fluorophores. We conclude that PLB C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLB C terminus is an important determinant of the quaternary structure of the SERCA regulatory complex.     

肾脏发育的分子调控机制

随着分子生物学技术的迅猛发展和广泛应用,参与肾脏发育过程的新基因相继被发现,肾脏发育过程中复杂的分子信号调控机制也得到进一步的研究,为阐明肾脏疾病的发病机制及从基因水平开展治疗提供了新的思路。文章对肾脏发育的3个阶段,即输尿管芽的发生和分支形成、生后肾原基的早期上皮性分化、肾小球血管球的发生和发育的分子信号调控研究进展进行了总结,主要涉及多种转录因子、生长因子及细胞因子,同时细胞外基质和黏附分子也参与其调控。     

Mechanisms for Intracellular Calcium Regulation in Heart : I. Stopped-Flow Measurements of Ca++ Uptake by Cardiac Mitochondria

Initial velocities of energy-dependent Ca⁺⁺ uptake were measured by stopped-flow and dual-wavelength techniques in mitochondria isolated from hearts of rats, guinea pigs, squirrels, pigeons, and frogs. The rate of Ca⁺⁺ uptake by rat heart mitochondria was 0.05 nmol/mg/s at 5 µM Ca⁺⁺ and increased sigmoidally to 8 nmol/mg/s at 200 µM Ca⁺⁺. A Hill plot of the data yields a straight line with slope n of 2, indicating a cooperativity for Ca⁺⁺ transport in cardiac mitochondria. Comparable rates of Ca⁺⁺ uptake and sigmoidal plots were obtained with mitochondria from other mammalian hearts. On the other hand, the rates of Ca⁺⁺ uptake by frog heart mitochondria were higher at any Ca⁺⁺ concentrations. The half-maximal rate of Ca⁺⁺ transport was observed at 30, 60, 72, 87, 92 µM Ca⁺⁺ for cardiac mitochondria from frog, squirrel, pigeon, guinea pig, and rat, respectively. The sigmoidicity and the high apparent K_m render mitochondrial Ca⁺⁺ uptake slow below 10 µM. At these concentrations the rate of Ca⁺⁺ uptake by cardiac mitochondria in vitro and the amount of mitochondria present in the heart are not consistent with the amount of Ca⁺⁺ to be sequestered in vivo during heart relaxation. Therefore, it appears that, at least in mammalian hearts, the energy-linked transport of Ca⁺⁺ by mitochondria is inadequate for regulating the beat-to-beat Ca⁺⁺ cycle. The results obtained and the proposed cooperativity for mitochondrial Ca⁺⁺ uptake are discussed in terms of physiological regulation of intracellular Ca⁺⁺ homeostasis in cardiac cells.     

Structural Significance of the Plasma Membrane Calcium Pump Oligomerization

The oligomerization of the plasma membrane calcium pump (PMCA) in phospholipid/detergent micelles was evaluated using a combined spectroscopic and kinetic approach and related to the enzyme stability. Energy transfer between fluorescein-5′-isothiocyanate and eosin-5′-isothiocyanate attached to different PMCA molecules was used to determine the dissociation constant of dimeric PMCA (140 ± 50 nM at 25°C) and characterize the time course of dimerization. The enzyme thermal stability at different dimer/monomer ratios was evaluated, quantifying the kinetic coefficient of thermal inactivation. This coefficient decreases with PMCA concentration, becoming approximately constant beyond 300 nM. Thermal treatment leads to the formation of inactive monomers that associate only with native monomers. These mixed dimers are formed with a kinetic coefficient that is half that determined for the native dimers. We proposed a model for PMCA thermal inactivation that considers the equilibria among dimers, monomers, and mixed dimers, and the inactivation of the last two species through irreversible steps. The numerical resolution of the differential equations describing this model fitted to the experimental data allowed the determination of the model coefficients. This analysis shows that thermal inactivation occurs through the denaturation of the monomer, which lifetime is 25 min at 44°C. The obtained results suggest that PMCA dimerization constitutes a mechanism of self protection against spontaneous denaturation.     

Molecular Characterization of the Glycated Plasma Membrane Calcium Pump

We have previously demonstrated (Diabetes 39:707–711, 1990) that in vitro glycation of the red cell Ca²⁺ pump diminishes the Ca²⁺-ATPase activity of the enzyme up to 50%. Such effect is due to the reaction of glucose with lysine residues of the Ca²⁺ pump (Biochem. J. 293:369–375, 1993). The aim of this work was to determine whether the effect of glucose is due to a full inactivation of a fraction of the total population of Ca²⁺ pump, or to a partial inactivation of all the molecules. Glycation decreased the V _max for the ATPase activity leaving unaffected the apparent affinities for Ca²⁺, calmodulin or ATP. The apparent turnover was identical in both, the glycated and the native enzyme. Glycation decreased the V _max for the ATP-dependent but not for the calmodulin-activated phosphatase activities. Concomitantly with the inhibition, up to 6.5% of the lysine residues were randomly glycated. The probabilistic analysis of the relation between the enzyme activity and the fraction of nonmodified residues indicates that only one Lys residue is responsible for the inhibition. We suggest that glucose decreases the Ca²⁺-ATPase activity by reacting with one essential Lys residue probably located in the vicinity of the catalytic site, which results in the full inactivation of the enzyme. Thus, Ca²⁺-ATPase activity measured in erythrocyte membranes or purified enzyme preparations preincubated with glucose depends on the remaining enzyme molecules in which the essential Lys residue stays unglycated. Received: 9 March 1999/Revised: 11 May 1999     

Ca2+ Binding to Site I of the Cardiac Ca2+ Pump Is Sufficient to Dissociate Phospholamban

Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca²⁺-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca²⁺. Recent cross-linking studies have suggested that PLB binding and Ca²⁺ binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca²⁺-ATPase, preventing formation of E1, the conformation that binds two Ca²⁺ (at sites I and II) with high affinity and is required for ATP hydrolysis. Here we determined whether Ca²⁺ binding to site I, site II, or both sites is sufficient to dissociate PLB from the Ca²⁺ pump. Seven SERCA2a mutants with amino acid substitutions at Ca²⁺-binding site I (E770Q, T798A, and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q) were made, and the effects of Ca²⁺ on N30C-PLB cross-linking to Lys³²⁸ of SERCA2a were measured. In agreement with earlier reports with the skeletal muscle Ca²⁺-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP in the presence of Ca²⁺; however, all were phosphorylatable by P_i to form E2P. Ca²⁺ inhibition of E2P formation was observed only in SERCA2a mutants retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB and each Ca²⁺-ATPase mutant was observed in the absence of Ca²⁺. Importantly, however, micromolar Ca²⁺ inhibited PLB cross-linking only to mutants retaining a functional Ca²⁺-binding site I. The dynamic equilibrium between Ca²⁺ pumps and N30C-PLB was retained by all mutants, demonstrating normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody. From these results we conclude that site I is the key Ca²⁺-binding site regulating the physical association between PLB and SERCA2a.     

细胞外Ca^2＋内流入胞质的机制

细胞外Ｃａ＾２＋主要是通过塌压依赖性Ｃａ＾２＋通道和钙池耗竭依赖性Ｃａ＾２＋通道而内流的。前者主要见于电兴奋细胞,这一过程比较清楚;后者主要见非兴奋细胞,情况远较复杂：外来信号激活内贮钙池,钙池在释放Ｃａ＾２＋同时通过目前尚不清楚的途径将直接或间接传至质膜Ｃａ＾２＋通道,而诱发Ｃａ＾２＋内流。     

Epigenetic Mechanisms of Blood-Pressure Regulation

The role of epigenetic mechanisms involved in blood-pressure regulation has been reviewed. It is known that some periods in early pre- and postnatal ontogenesis are very sensitive to some environmental and endogenous influences. These periods are characterized as highly vulnerable to the formation of a complex of epigenetic changes that may determine the trajectory of the further formation of physiological systems involved in the blood-pressure regulation. Early life influences on these systems may predispose an individual to the development of hypertensive disease in further life. In some cases, the transmission of epigenetic changes to the next generations may resolve the contradiction between the high heritability of arterial hypertensive disease and the low total contribution of polymorphic DNA variants in the population variability of blood pressure values.     

Regulation Mechanisms of Stomatal Oscillation

Stomata function as the gates between the plant and the atmospheric environment. Stomatal movement, including stomatal opening and closing, controls CO2 absorption as the raw material for photosynthesis and water loss through transpiration. How to reduce water loss and maintain enough CO2 absorption has been an interesting research topic for some time. Simple stomatal opening may elevate CO2 absorption,but, in the meantime, promote the water loss, whereas simple closing of stomatal pores may reduce both water loss and CO2 absorption, resulting in impairment of plant photosynthesis. Both processes are not economical to the plant. As a special rhythmic stomatal movement that usually occurs at smaller stomatal apertures, stomatal oscillation can keep CO2 absorption at a sufficient level and reduce water loss at the same time, suggesting a potential improvement in water use efficiency. Stomatal oscillation is usually found after a sudden change in one environmental factor in relatively constant environments. Many environmental stimuli can induce stomatal oscillation. It appears that, at the physiological level, feedback controls are involved in stomatal oscillation. At the cellular level, possibly two different patterns exist: (i) a quicker responsive pattern; and (ii) a slower response. Both involve water potential changes and water channel regulation, but the mechanisms of regulation of the two patterns are different. Some evidence suggests that the regulation of water channels may play a vital and primary role in stomatal oscillation. The present review summarizes studies on stomatal oscillation and concludes with some discussion regarding the mechanisms of regulation of stomatal oscillation.     

Regulation Mechanisms of Stomatal Oscillation

Stomata function as the gates between the plant and the atmospheric environment. Stomatal movement, including stomatal opening and closing, controls CO2 absorption as the raw material for photosynthesis and water loss through transpiration. How to reduce water loss and maintain enough CO2 absorption has been an interesting research topic for some time. Simple stomatal opening may elevate CO2 absorption, but, in the meantime, promote the water loss, whereas simple closing of stomatal pores may reduce both water loss and CO2 absorption, resulting in impairment of plant photosynthesis. Both processes are not economical to the plant. As a special rhythmic stomatal movement that usually occurs at smaller stomatal apertures, stomatal oscillation can keep CO2 absorption at a sufficient level and reduce water loss at the same time, suggesting a potential improvement in water use efficiency. Stomatal oscillation is usually found after a sudden change in one environmental factor in relatively constant environments. Many environmental stimuli can induce stomatal oscillation. It appears that, at the physiological level, feedback controls are involved in stomatal oscillation. At the cellular level, possibly two different patterns exist： （i） a quicker responsive pattern; and （ii） a slower response. Both involve water potential changes and water channel regulation, but the mechanisms of regulation of the two patterns are different. Some evidence suggests that the regulation of water channels may play a vital and primary role in stomatal oscillation. The present review summarizes studies on stomatal oscillation and concludes with some discussion regarding the mechanisms of regulation of stomatal oscillation.     

细胞内贮存钙释放的机制

细胞内贮存钙的释放主要由１,４,５－三磷酸肌醇（ＩＰ３）受体系统和ｒｙａｎｏｄｉｎｅ受体系统调控。前通过ＩＰ３与其受体结合后,诱发细胞内钙释放;后通过复杂的机制调节环腺苷二磷酸核糖含量,由ｃＡＤＰＲ直接或间接作用于ｒｙａｎｏｄｉｎｅ受体,进而启动由Ｃａ＾２＋诱发的Ｃａ＾２＋释放机制。上述两系统之间相互作用,共同调节细胞内贮存钙的释放。