Cardiac sarcoplasmic-reticulum calmodulin-binding proteins. Modulation of calmodulin binding to phospholamban by phosphorylation.
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ABSTRACT:
SUBMITTER: Molla A
PROVIDER: S-EPMC1144787 | biostudies-other | 1985 Mar
REPOSITORIES: biostudies-other
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Project description:Nonmuscle cells have almost ubiquitously evolved a mechanism to detect and prevent Ca2+ store depletion-store operated calcium entry. No such mechanism has, as yet, been reported in cardiac myocytes. However, it is conceivable that such a mechanism may play an important role in cardiac Ca2+ homeostasis to ensure the availability of sufficient stored Ca2+ to maintain normal excitation contraction coupling. We present data that confirms the presence of a mechanism that is able to monitor the Ca2+ load of the SR and initiate a signaling process to accelerate Ca2+ uptake by the SR when store depletion is detected. Depletion of SR Ca2+ activates a protein kinase, the principal SR substrate of which is phospholamban. Phosphorylation of this SR protein promotes Ca2+ pump activity and therefore store refilling. Furthermore, a protein kinase activity associated with the SR that is inhibited by Ca2+ ions has been identified. We have measured lumenal [Ca2+] by using a fluorescent Ca2+ indicator and found that by initiating Ca2+ uptake and increasing Ca2+ load, we can inhibit the protein kinase activity associated with the SR. This confirms that a protein kinase, that is regulated by lumenal [Ca2+], has been identified and represents part of a previously unidentified signalling cascade. This local feedback mechanism would allow the myocyte to detect and prevent SR Ca2+ load depletion.
| S-EPMC19055 | biostudies-literature
Project description:RationaleWe previously showed that transgenic mice expressing Ca(2+)/calmodulin-dependent protein kinase II delta(C) (CaMKII-TG) develop dilated cardiomyopathy associated with increased ryanodine receptors (RyR2) phosphorylation, enhanced sarcoplasmic reticulum (SR) Ca(2+) leak and lowering of SR Ca(2+) load. We hypothesized that phospholamban (PLN) ablation would restore SR Ca(2+) load and prevent the decreased ventricular contractility, dilation and mortality seen in CaMKII-TG.ObjectiveOur objectives were to generate CaMKII-TG mice lacking PLN, determine whether the maladaptive effects of cardiac CaMKIIdelta(C) expression were corrected, and establish the mechanistic basis for these changes.Methods and resultsCaMKII-TG were crossed with PLN knockout (PLN-KO) mice to generate KO/TG mice. Myocytes from wild type (WT), CaMKII-TG, PLN-KO and KO/TG were compared. The decreased SR Ca(2+) load and twitch Ca(2+) transients seen in CaMKII-TG were normalized in KO/TG. Surprisingly the heart failure phenotype was exacerbated, as indicated by increased left ventricular dilation, decreased ventricular function, increased apoptosis and greater mortality. In KO/TG myocytes SR Ca(2+) sparks and leak were significantly increased, presumably because of the combined effects of restored SR Ca(2+) load and RyR2 phosphorylation. Mitochondrial Ca(2+) loading was increased in cardiomyocytes from KO/TG versus WT or CaMKII-TG mice and this was dependent on elevated SR Ca(2+) sparks. Cardiomyocytes from KO/TG showed poor viability, improved by inhibiting SR Ca(2+) release and mitochondrial Ca(2+) loading.ConclusionsNormalizing cardiomyocyte SR Ca(2+) loading in the face of elevated CaMKII and RyR2 phosphorylation leads to enhanced SR Ca(2+) leak and mitochondrial Ca(2+) elevation, associated with exacerbated cell death, heart failure and mortality.
| S-EPMC2818826 | biostudies-literature
Project description:Oligomeric interactions between Ca-ATPase polypeptide chains and their modulation by phospholamban (PLB) were measured in native cardiac sarcoplasmic reticulum (SR) microsomes. Progressive modification of Lys(514) with fluorescein 5-isothiocyanate (FITC), which physically blocks access to the nucleotide binding site by ATP, demonstrates that Ca-ATPase active sites function independently of one another prior to the phosphorylation of PLB. However, upon cAMP-dependent protein kinase (PKA) phosphorylation of PLB, a second-order dependence between residual enzyme activity and the fraction of active sites is observed, consistent with a dimeric functional complex. Complementary distance measurements were made using FITC or 5-iodoacetamidofluorescein (IAF) bound to Cys(674) within the N- or P-domains, respectively, to detect structural coupling within oligomeric complexes. Accompanying the phosphorylation of PLB, neighboring Ca-ATPase polypeptide chains exhibit a 4 +/- 2 A decrease in the proximity between FITC sites within the N-domain and a 9 +/- 3 A increase in the proximity between IAF sites within P-domains. Thus, the phosphorylation of PLB induces spatial rearrangements between the N- and P-domain elements of proximal Ca-ATPase polypeptide chains which restore functional interactions between neighboring polypeptide chains and, in turn, result in increased rates of catalytic turnover. These results are interpreted in terms of a structural model, calculated through optimization of shape complementarity, desolvation, and electrostatic energies, which suggests a dimeric arrangement of Ca-ATPase polypeptide chains through the proximal association of N-domains that accommodates interaction with PLB. We suggest that the phosphorylation of PLB acts to release constraints involving interdomain subunit interactions that enhance catalytically important N-domain motions.
| S-EPMC2765579 | biostudies-literature
Project description: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.
| S-EPMC6383005 | biostudies-literature
Project description:It is widely assumed that synthesis of membrane proteins, particularly in the heart, follows the classical secretory pathway with mRNA translation occurring in perinuclear regions followed by protein trafficking to sites of deployment. However, this view is based on studies conducted in less-specialized cells, and has not been experimentally addressed in cardiac myocytes. Therefore, we undertook direct experimental investigation of protein synthesis in cardiac tissue and isolated myocytes using single-molecule visualization techniques and a novel proximity-ligated in situ hybridization approach for visualizing ribosome-associated mRNA molecules for a specific protein species, indicative of translation sites. We identify here, for the first time, that the molecular machinery for membrane protein synthesis occurs throughout the cardiac myocyte, and enables distributed synthesis of membrane proteins within sub-cellular niches where the synthesized protein functions using local mRNA pools trafficked, in part, by microtubules. We also observed cell-wide distribution of membrane protein mRNA in myocardial tissue from both non-failing and hypertrophied (failing) human hearts, demonstrating an evolutionarily conserved distributed mechanism from mouse to human. Our results identify previously unanticipated aspects of local control of cardiac myocyte biology and highlight local protein synthesis in cardiac myocytes as an important potential determinant of the heart's biology in health and disease.
| S-EPMC8553722 | biostudies-literature
Project description:Phospholamban has been suggested to be a key regulator of cardiac sarcoplasmic reticulum (SR) Ca cycling and contractility and a potential therapeutic target in restoring the depressed Ca cycling in failing hearts. Our understanding of the function of phospholamban stems primarily from studies in genetically altered mouse models. To evaluate the significance of this protein in larger mammalian species, which exhibit Ca cycling properties similar to humans, we overexpressed phospholamban in adult rabbit cardiomyocytes. Adenoviral-mediated gene transfer, at high multiplicities of infection, resulted in an insignificant 1.22-fold overexpression of phospholamban. There were no effects on twitch Ca-transient amplitude or decay under basal or isoproterenol-stimulated conditions. Furthermore, the SR Ca load and Na/Ca exchanger function were not altered. These apparent differences between phospholamban overexpression in rabbit compared with previous findings in the mouse may be due to a significantly higher (1.5-fold) endogenous phospholamban-to-sarco(endo)plasmic reticulum Ca-ATPase (SERCA) 2a ratio and potential functional saturation of SERCA2a by phospholamban in rabbit cardiomyocytes. The findings suggest that important species-dependent differences in phospholamban regulation of SERCA2a occur. In larger mammals, a higher fraction of SERCA2a pumps are regulated by phospholamban, and this may influence therapeutic strategies to enhance cardiac contractility and functional cardiac reserve.
| S-EPMC2660221 | biostudies-literature
Project description: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 Ca(2+)-ATPase), pumps contractile-dependent Ca(2+) 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 Ca(2+) affinity of SERCA and attenuates contractile strength. cAMP-dependent phosphorylation of PLB reverses Ca(2+)-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 Ca(2+) in a novel detergent system employing alkyl mannosides. The structure shows PLB bound to a previously undescribed conformation of SERCA in which the Ca(2+) 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 Ca(2+) affinity and depresses cardiac contractility.
| S-EPMC3798486 | biostudies-literature
Project description: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 ((53)SFHGM) 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.
| S-EPMC4358104 | biostudies-literature
Project description:Phospholamban of isolated sarcoplasmic reticulum of cardiac and smooth muscle is phosphorylated by cyclic GMP-dependent protein kinase (G-kinase). Concomitantly, the affinity of the Ca2+ pump for Ca2+ is increased. These effects are very similar to those seen with cyclic AMP-dependent protein kinase (A-kinase). The phosphate incorporation into phospholamban and the stimulatory effects of both kinases on the Ca2+ pump are not additive, suggesting that G-kinase phosphorylates the same serine residue as A-kinase. A possible physiological role for phosphorylation of phospholamban by G-kinase is discussed.
| S-EPMC1149133 | biostudies-other
Project description:The integral membrane protein complex between phospholamban (PLN) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) regulates cardiac contractility. In the unphosphorylated form, PLN binds SERCA and inhibits Ca(2+) flux. Upon phosphorylation of PLN at Ser16, the inhibitory effect is reversed. Although structural details on both proteins are emerging from X-ray crystallography, cryo-electron microscopy, and NMR studies, the molecular mechanisms of their interactions and regulatory process are still lacking. It has been speculated that SERCA regulation depends on PLN structural transitions (order to disorder, i.e., folding/unfolding). Here, we investigated PLN conformational changes upon chemical unfolding by a combination of electron paramagnetic resonance and NMR spectroscopies, revealing that the conformational transitions involve mostly the cytoplasmic regions, with two concomitant phenomena: (1) membrane binding and folding of the amphipathic domain Ia and (2) folding/unfolding of the juxtamembrane domain Ib of PLN. Analysis of phosphorylated and unphosphorylated PLN with two phosphomimetic mutants of PLN (S16E and S16D) shows that the population of an unfolded state in domains Ia and Ib (T' state) is linearly correlated to the extent of SERCA inhibition measured by activity assays. Inhibition of SERCA is carried out by the folded ground state (T state) of the protein (PLN), while the relief of inhibition involves promotion of PLN to excited conformational states (Ser16 phosphorylated PLN). We propose that PLN population shifts (folding/unfolding) are a key regulatory mechanism for SERCA.
| S-EPMC3573877 | biostudies-literature