Phospholamban phosphorylation increases the passive calcium leak from cardiac sarcoplasmic reticulum

Abstract

Phospholamban (PLN) is a 52 amino acid integral membrane protein of the sarcoplasmic reticulum (SR) that exists in both monomeric and pentameric forms. In its unphosphorylated state, PLN inhibits the SR Ca(2+) ATPase (SERCA). This inhibition is relieved when PLN is phosphorylated as a result of β-adrenergic stimulation of the heart. Consistent with some predictions from molecular models and from functional studies of PLN incorporated into planar lipid bilayers, it has also been postulated that pentameric PLN can also form ion-selective channels. Other molecular models contradict this hypothesis, however. In the work reported here, we used the Ca(2+)-sensitive fluorescent dye Fura-2, to examine the passive Ca(2+) permeability of the SR membrane in vesicles derived from cardiac ventricle. We have found that phosphorylation of PLN by protein kinase A (PKA) leads to an increase in the rate of Ca(2+) leak from Ca(2+)-loaded SR vesicles. This enhanced rate of Ca(2+) leak from the SR is also observed when SR vesicles are incubated with a PLN specific antibody (A1) that mimics phosphorylation of PLN. The ryanodine receptor blocker ruthenium red does not affect the increased rate of Ca(2+) leak from the SR after PLN phosphorylation with PKA or after exposure to A1 antibody, arguing against a possible role of ryanodine receptors in mediating the enhanced leak. Our results are consistent with the hypothesis that phosphorylated PLN forms or regulates a Ca(2+) leak pathway in cardiac SR membranes in situ.

SERCA-2a is involved in the right ventricular function following myocardial infarction in rats

“…In the present study, we examined changes in the levels of PLB Ser-16 phosphorylation with a focus on the beta-adrenergic pathway. It has been demonstrated that reduced PKA-dependent PLB phosphorylation during HF may be due to changes at various levels of the betaadrenergic signaling pathway [6,11,12]. Decreases in PLB levels and/or changes in phosphorylation can directly impact SR Ca 2+ -uptake and muscle contractility [30], which is consistent with our finding that the β-adrenergic response to isoproterenol was impaired in the HF group but not in the INF group.…”

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

“…Free energy calculations show that the energy barrier for Ca 2+ ion transport is ~40 kcal/mol, which is prohibitive in the absence of an energy source (Becucci et al , 2009). A recent paper opened up the possibility that phosphorylation at Ser16 might be responsible for ion transport or Ca 2+ leakage (Aschar-Sobbi et al , 2012), motivating our structural studies.…”

Accurate Determination of Conformational Transitions in Oligomeric Membrane Proteins

“…Indeed, the coupling between cytoplasmic and transmembrane regions has been shown to be critical in the overall regulation process of SERCA 45 . The observed enhancement in structural dynamics upon phosphorylation may be at the origin of the perturbation in the equilibrium between PLN monomers and pentamers, which is key to regulate the amount of free monomers available for SERCA binding and therefore the apparent affinity of SERCA for Ca 2+ in its physiological window 29 32 46 47 .…”