Phosphorylated Phospholamban Stabilizes a Compact Conformation of the Cardiac Calcium-ATPase

Abstract

The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics. The goals of this work were to quantify the direction and magnitude of SERCA motions as the pump performs work in live cardiac myocytes, and to identify structural determinants of SERCA regulation by phospholamban. We measured intramolecular fluorescence resonance energy transfer (FRET) between fluorescent proteins fused to SERCA cytoplasmic domains. We detected four discrete structural substates for SERCA expressed in cardiac muscle cells. The relative populations of these discrete states oscillated with electrical pacing. Low FRET states were most populated in low Ca (diastole), and were indicative of an open, disordered structure for SERCA in the E2 (Ca-free) enzymatic substate. High FRET states increased with Ca (systole), suggesting rigidly closed conformations for the E1 (Ca-bound) enzymatic substates. Notably, a special compact E1 state was observed after treatment with β-adrenergic agonist or with coexpression of phosphomimetic mutants of phospholamban. The data suggest that SERCA calcium binding induces the pump to undergo a transition from an open, dynamic conformation to a closed, ordered structure. Phosphorylated phospholamban stabilizes a unique conformation of SERCA that is characterized by a compact architecture.

Acute Inotropic and Lusitropic Effects of Cardiomyopathic R9C Mutation of Phospholamban

“…1C) as observed previously for fluorescently labeled wild-type (WT)-PLB (32). The fluorescence pattern is consistent with localization in the sarcoplasmic reticulum and is similar to that previously observed for fluorescently labeled SERCA2a (32,37). R9C mutation is within the epitope region for the PLB-2D12 antibody (between amino acid residues 9 and 17 of canine PLB), and we observed that this mutation abolishes 2D12 reactivity (Fig.…”

“…Cardiomyocytes were incubated at 37°C for 1 h, and CFP-PLB and/or YFP-PLB adenoviruses were added at a multiplicity of infection of 1000 as described previously (37). Cardiomyocytes were then paced for 48 h in culture using a C-Pace EP Pacer (IonOptix, Milton, MA) set to 10 V with a frequency of 0.1 Hz, with 5-ms pulse duration (32,37).…”

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

“…Alternatively, the putative misregistration of the PLB TM domain in the SERCA regulatory cleft may result in a noninhibitory interaction. We and others have previously provided evidence that PLB and SERCA can interact in a noninhibitory complex in the presence of Ca 2ϩ (29,62,63) or after PLB phosphorylation (64). This physiological relief of inhibition also alters the affinity and structure of the PLB-SERCA complex (29,(62)(63)(64).…”

ATP–Binding Cassette Transporter Structure Changes Detected by Intramolecular Fluorescence Energy Transfer for High-Throughput Screening

“…AMP-PCP, adenylylmethylenediphosphonate. et al, 2012;Pallikkuth et al, 2013). Moreover, substrate binding may not induce an all-or-nothing switch to a new conformation but rather shift the balance of an equilibrium of multiple structures.…”

“…Specifically, a conformational change that brings the nucleotidebinding domains closer together was expected to increase FRET from a basal level to a new higher level, reflecting the closer proximity of the fluorescent tags. We have previously used this approach to monitor structural dynamics of a calcium transporter within the native environment of the cell membrane (Hou et al, 2012;Pallikkuth et al, 2013). This strategy may be of general utility for investigating the conformational changes of transporter proteins in the native environment of the live cell.…”