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

Abstract

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.

Figure 1

Oligomerization of SERCA probed with cross-linking and co-immunoprecipitation (co-IP). (A) Western blot analysis of rabbit LV myocytes. Increasing concentrations of BPM decreased the amount of monomeric SERCA (Band 1) and gave rise to two additional bands (Bands 2 and 3). (B–D) Quantification of Bands 1–3 relative to total SERCA. Values are the mean ± SD from three independent blots. (E) Preincubation with the detergent n-dodecylphosphocholine in 300 μM BPM increased the relative amount of monomeric SERCA (Band 1). (F) Quantification of (E). (G) Pretreatment of myocytes with 50 nM isoproterenol (iso) did not appreciably alter the SERCA electrophoretic pattern. (H) PCH analysis of GFP-SERCA isolated from low-molecular-weight (black) and high-molecular-weight (red) fractions revealed an increased molecular brightness (ε) for the high-molecular-weight species (fits shown in gray). Molecular brightness values are the mean ± SD from N = 3 individual gel slices (unpaired t-test, p < 0.001). (I) Co-IP of GFP-SERCA with Myc-SERCA. To see this figure in color, go online.

Figure 2

Oligomerization of SERCA quantified by FRET. (A) Fluorescent protein fusion sites for FRET experiments. (B) Average sensitized emission FRET ± SD measured from the N-terminally labeled Cer-SERCA2a to YFP (Ctrl) or YFP-SERCA2a labeled at the N-terminus (N), between amino acids 508 and 509 (509), or at the C-terminus (C). (C) Protein concentration dependence of sensitized emission FRET from Cer-SERCA2a to YFP-SERCA2a (N) (black) or negative control Cer-SERCA2a to nonfusion YFP (red). Individual cells are shown in gray, with pooled data (mean ± SE) in red or black and hyperbolic fits shown as lines. (D) Apparent dissociation constants (K_d) and maximum FRET (FRET_max) values calculated from the hyperbolic fits of FRET data in the absence (−PLB) or presence (+PLBwt) of PLB. Application of 100 μM forskolin and 100 μM 3-isobutyl-1-methylxanthine (+PLB+F+I) did not alter the apparent affinity of the dimer, nor was it altered by coexpression with nonphosphorylatable (+PLB-S16A) or phosphomimetic (+PLB-S16E) PLB mutants. Alterations to calcium (2 mM) or ATP (4 mM) did not elicit any changes. Values are the mean ± SE of N = 3 fits (there were no statistically significant differences between groups by one-way ANOVA). (E) Sensitized emission FRET from Cer-SERCA and YFP-SERCA was reduced by coexpression of nonfluorescent SERCA (data pooled and fit as in (C)). Values reflect the molar ratio of competitor to YFP-SERCA. (F) FRET_max was decreased by competition with nonfluorescent SERCA (black), but not by PLB (blue). The hyperbolic fit is shown in red. (G) Progressive acceptor photobleaching of YFP-SERCA (black) resulted in increased Cer-SERCA fluorescence (blue), indicating FRET. Values are the mean ± SE for N = 28 cells total from three independent experiments. (H) The relationship of donor/acceptor fluorescence intensities during the process of photobleaching for a construct comprising one acceptor and one donor (C32V, black) and a construct comprising two acceptors and one donor (VCV, red). Values are the mean ± SD for N = 12 cells. Lines connect the first and last data points. (I) Photobleaching of the SERCA homooligomer resulted in a linear increase in Cer intensity with decreasing YFP intensity (black), suggesting a homodimer. No FRET was observed for nonfusion control. To see this figure in color, go online.

Figure 3

Fluorescence-lifetime FRET analysis of the SERCA-PLB regulatory complex. (A) Labeling strategy for FRET from GFP-SERCA (gray) to mCherry-PLB (red). (B) Representative fluorescence decay of GFP-SERCA shortened by coexpression of mCherry-PLB, indicating FRET. Exponential tail fitting is shown in gray. (C) A single-component exponential fit of the fluorescence lifetime shows a decrease in τ with increasing protein expression. The hyperbolic model is in red. (D) FRET (calculated from the data in (C)) increased with protein expression. The hyperbolic model is in red. (E) Reduced-χ² values for one-component fits worsened as protein expression increased. The reduced χ² value was improved by a two-component fit. Linear fits appear as lines. (F) Residual plots of (B) from one- or two-component exponential decay fitting of the GFP-SERCA alone (black) or in the presence of mCherry-PLB (red). (G) The short-lifetime values from two-component exponential decays were consistent across a large range of protein expression levels. The linear fit is shown in red. (H) The relative contributions (amplitude) of the short and long lifetimes. (I) A model of the SERCA-SERCA-PLB regulatory complex in which one of the donor-labeled SERCA protomers (dotted outline) is too distant to participate in FRET with PLB. To see this figure in color, go online.