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.

Figure 1

Single molecule fluorescence spectroscopy of two-color SERCA. (A) CY-SERCA comprises a Cerulean FRET donor fused to the N-terminus of SERCA (gray) in the actuator, or A-domain, and an intrasequence YFP inserted in the nucleotide-binding, or N-domain (16). (B) RG-SERCA is an analogous construct composed of a TagRFP acceptor fused to the N-terminus in the A-domain and GFP (green) donor inserted in the N-domain. Changes in SERCA conformation alter the distance between the N- and A-domains, changing intramolecular FRET efficiency. (C) Fluorescence bursts from single molecules of detergent-solubilized two-color SERCA. (D) Histogram of the fluorescence lifetime measurements obtained from two-color RG-SERCA (red) or control GFP-SERCA (black). (E) Histogram of FRET efficiency calculated from the lifetime measurements shown in panel D. Gaussian fit revealed subpopulations consistent with four discrete conformations of SERCA. (F) CY-SERCA also exhibited four discrete FRET states. The reduced FRET of these states (compared to panel E) was consistent with a shorter R₀ for the Cer-YFP FRET pair. (G) RG-SERCA single-molecule FRET time trajectory. (Horizontal lines indicate States I–IV (orange through green) identified by Gaussian analysis, as in panel E). The pump is observed to transiently sample the low FRET state (at arrow), before returning to higher FRET conformations, suggesting that State I does not represent denatured protein. (H) Analysis of dwell time for SERCA-sampling States I–IV revealed a biphasic distribution of dwell times characterized by fast (80 μs) and slow (690 μs) kinetics. To see this figure in color, go online.

Figure 2

Two-color SERCA expressed in adult ventricular cardiac myocytes. (A) Localization of RG-SERCA GFP fluorescence in adult ventricular myocytes. (B) Localization of RG-SERCA TagRFP fluorescence. (C) FRET distribution of two-color SERCA in electrically paced myocytes, during the relaxation phase (diastole). (D) Systole (during the contraction phase). (E) Diastole, plus isoproterenol. (F) Systole, plus isoproterenol. (G) Summary of FRET subpopulations during diastole before (solid bars) or after addition of isoproterenol (striped bars). (H) Systole, before (solid bars) or after addition of isoproterenol (striped bars). (I) FRET distribution after addition of Tg. (J) Summary of data in panel I. Panels C–F and I are representative data obtained from individual cells; each histogram integrates multiple consecutive contraction/relaxation cycles. Panels G, H, and J are mean ± SE of all data. To see this figure in color, go online.

Figure 3

Expression of two-color SERCA in AAV-293 cells. (A) Localization of RG-SERCA GFP fluorescence in AAV-293 cells. (B) Localization of RG-SERCA TagRFP fluorescence. (C) FRET distribution of two-color SERCA and nonphosphorylatable PLB in intact cells. (D) As in panel C, plus ionophore. (E) Two-color SERCA plus phosphomimetic PLB. (F) As in panel E, plus ionophore. (G) Summary of FRET subpopulations from intact cells expressing two-color SERCA and nonphosphorylatable PLB (solid bars) or phosphomimetic PLB (striped bars). (H) Plus ionophore, with nonphosphorylatable PLB (solid bars) or phosphomimetic PLB (striped bars). (I) FRET distribution after addition of Tg. (J) Summary of I. Panels C–F and I are representative data obtained from individual cells. Panels G, H, and J are mean ± SE of all data. To see this figure in color, go online.

Figure 4

Comparison of FRET histogram Gaussian fit width versus center position data obtained from cardiac myocytes. (A) States I–IV for RG-SERCA showed that the population Gaussian width decreased with increasing population Gaussian center value. This relationship was not detectably altered by Tg (triangles) or iso (squares) compared to control (circles). The data suggest that low FRET (open) conformations are more dynamically disordered than high FRET (solid) structures. (B) Average RG-SERCA peak parameters (black) compared with CY-SERCA (blue). The shorter R₀ of the Cer/YFP pair yielded decreased FRET (decreased center values), but similar Gaussian widths for each state. Data are mean ± SE. Proposed novel conformation of SERCA: (C) open E2 (Ca-free) SERCA, characterized by a dynamically disordered cytoplasmic headpiece; and (D) proposed closed E1 (Ca-bound) SERCA, with a rigidly ordered headpiece. The conformation represents a unique E1 state that is stabilized by phosphorylated phospholamban. To see this figure in color, go online.