Daniel Blackwell

Phospholamban binds with differential affinity to calcium pump conformers

To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. Electrical pacing of cardiac myocytes elicited cytoplasmic Ca2+ elevations, but these increases in Ca2+ produced only modest changes in SERCA-PLB FRET. The data suggest that the regulatory complex is not disrupted by elevations of cytosolic calcium during cardiac contraction (systole). This conclusion was also supported by parallel experiments in heterologous cells, which showed that FRET was reduced but not abolished by calcium. Thapsigargin also elicited a small decrease in PLB-SERCA binding affinity. We propose that PLB is not displaced from SERCA by high calcium during systole, and relief of functional inhibition does not require dissociation of the regulatory complex. The observed modest reduction in the affinity of the PLB-SERCA complex with Ca2+ or thapsigargin suggests that the binding interface is altered by SERCA conformational changes. The results are consistent with multiple modes of PLB binding or alternative binding sites.

2-Color Calcium Pump Reveals Closure of the Cytoplasmic Headpiece with Calcium Binding

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The “2-color” SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C-terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB slow the kinetics of Tg binding. FRET in ionophore-permeabilized cells was lower in EGTA than in saturating calcium for all constructs, indicating a decrease in domain separation distance with the structural transition from E2 (Ca-free) to E1 (Ca-bound). The data suggest closure of the cytoplasmic headpiece with Ca-binding. The present results provide insight into the structural dynamics of the Ca-ATPase. In addition, the 2-color SERCA constructs developed for this study may be useful for evaluating candidate small molecule regulators of Ca uptake activity.

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

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.

Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer

The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation.

Phosphorylated Phospholamban Stabilizes a Distinct Compact Conformation of Sarco(endo)Plasmic Reticulum Ca-ATPase

Sarco(endo)plasmic reticulum Ca-ATPase (SERCA) is inhibited by the 52 amino acid protein phospholamban (PLB). Inhibition of SERCA is relieved when PLB is phosphorylated by protein kinase A (PKA). To better understand the mechanism of this regulatory mechanism on SERCA structure, we designed a “2-color” SERCA labeled with donor and acceptor fluorescent proteins in the A- and N-domain. Intramolecular fluorescence resonance energy transfer (FRET) between these two domains was quantified as an index of SERCA conformation. Fluorescence lifetime distribution analysis (FLDA) showed that SERCA sampled three discrete conformations in the absence of PLB or in the presence of non-phosphorylatable PLB (S16A). Co-transfection of SERCA with phosphomimetic PLB (S16E) revealed an additional state characterized by a highly compact conformation. This unique state was only observed under conditions of high intracellular calcium. The data support the hypothesis that PLB remains bound to SERCA after phosphorylation by PKA, inducing the pump to assume a very compact E1 conformation.

Conformational Changes of SERCA Revealed by Intramolecular FRET

The first structures of the sarcoendoplasmic reticulum ATPase (SERCA) determined by X-ray crystallography suggested that pump undergoes a large conformational change during catalytic cycling. The transition from the E1 (Ca-bound) state to the E2 (Ca-free) state was predicted to decrease inter-domain separation distance with closure of the SERCA cytoplasmic headpiece. To test this model, we fused Cerulean to the A-domain and YFP to the N or P or TM-domain of SERCA2a. These “2-color” SERCA constructs were expressed in AAV cells, and SERCA structure transitions were detected by changes in intramolecular fluorescence resonance energy transfer (FRET). FRET decreased with thapsigargin for the two N-domain fusion sites (residues 510, 577), while the P- (residue 610) and TM- domain (C-terminus) fusions showed increased FRET with thapsigargin. Unexpectedly, FRET in permeabilized cells was higher in Ca [10 μM] than in EGTA for all constructs, suggesting an increase in domain separation distance with the E1 to E2 transition. These observations were supported by parallel experiments in fluorescence lifetime distribution (FLD) analysis. FLD resolved two broad distributions of fluorescence lifetimes for 2-color SERCA expressed in live cardiac myocytes, consistent with two major FRET states. The relative populations of these states oscillated with electrical pacing, favoring the high FRET (short distance) state during systole (contraction) and the low FRET (long distance) state during diastole (relaxation). We expect 2-color SERCA constructs to be useful for exploring the magnitude, direction, and kinetics of calcium pump conformational changes. They may also be useful for screening candidate compounds for modulation of SERCA pump structure and function.