Phospholamban binds with differential affinity to calcium pump conformers

Abstract

To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca(2+)-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 Ca(2+) elevations, but these increases in Ca(2+) 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 Ca(2+) 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.

FIGURE 1.

Alternative models of PLB regulation of SERCA. A, the dissociation model (7, 9, 10). PLB dissociates from SERCA when the pump assumes the E1 (Ca²⁺-bound) conformation during systole. B, the subunit model (14). PLB remains bound throughout the catalytic cycle as a subunit of the pump.

FIGURE 2.

Adenoviral expression of Cer-SERCA and YFP-PLB in cardiac myocytes. A, confocal images of an isolated adult rabbit ventricular myocyte expressing Cer-SERCA and YFP-PLB. Both proteins were distributed in longitudinal streaks as well as striations. The striations were due to localization of both proteins at the Z-line, as indicated by counterstaining with the membrane dye FM 4-64. The overlay image shows the relative localization of Cer-SERCA, YFP-PLB, and FM 4-64. Scale bar = 10 μm. B, an overlay of Cer-SERCA and YFP-PLB images shows bright perinuclear fluorescence and an additional fraction of YFP-PLB visible at the sarcolemma (inset, arrow). Scale bar = 10 μm. C, fluorescence recovery after photobleaching (at arrow) demonstrated long range mobility of Cer-SERCA and YFP-PLB in cardiac myocytes. Scale bar = 10 μm. D, FRET from Cer-SERCA to YFP-PLB increased with protein concentration toward a maximum.

FIGURE 3.

Quantification of Ca²⁺ and FRET changes in paced cardiac myocytes. A, an average of 21 consecutive Ca²⁺ transients quantified by X-rhod-1 fluorescence (black) and the corresponding PLB-SERCA FRET ratio (red) in a paced ventricular myocyte. B, 5 μm isoproterenol increased the magnitude and decreased the width of the Ca²⁺ transient (black). We detected no change in PLB-SERCA FRET in response to beat-to-beat Ca²⁺ elevations, before or after isoproterenol. C, prolonged elevations of Ca²⁺ (black) were achieved with intervals of rapid (1-Hz) pacing after periods of rest (no stimulation). A modest decrease in FRET (red) was observed during rapid pacing. The blue lines represent the Ca²⁺ and FRET traces smoothed by 5-s adjacent averaging. D, as in C, after 5 μm isoproterenol, a small decrease in FRET (red) was observed during rapid pacing. E, averaging multiple experiments showed that rapid pacing increased mean [Ca²⁺], with a larger increase observed after the addition of 5 μm isoproterenol (iso). F, averaging multiple experiments showed that rapid pacing decreased the mean YFP/Cer ratio, and this decrease was larger after the addition of 5 μm isoproterenol. The first, second, and third pacing intervals are marked a, b, and c in C–F.

FIGURE 4.

The effect of Ca²⁺ and Tg on regulatory complex binding affinity. A, in permeabilized AAV-293 cells, FRET from Cer-SERCA to YFP-PLB increased with protein concentration toward a maximum. The concentration dependence of FRET was right-shifted by Ca²⁺ when compared with Ca²⁺-free controls. FRET concentration dependence was well described by a hyperbolic fit that yielded the maximal FRET (FRET_max) and apparent dissociation constant (K_D2). AU, arbitrary units. B, the dependence of K_D2 on [Ca²⁺] was well described by a Hill function with an EC₅₀ of 410 nm. C, FRET concentration dependence was also right-shifted by Tg in intact AAV-293 cells. D, K_D2 increased with Tg (EC₅₀ = 350 nm). E, FRET from Cer-PLB to YFP-PLB was not altered by Ca²⁺. F, FRET from Cer-PLB to YFP-PLB was not altered by Tg.

FIGURE 5.

Ca²⁺ uptake in live cells. A, wide-field fluorescence images of AAV-293 cells transfected with Cer-SERCA and YFP-PLB, loaded with Ca²⁺ indicator X-rhod-1. This field contains examples of transfected (T) and untransfected (UT) cells. B, Ca²⁺ transients were observed after the addition of extracellular ATP in untransfected cells (black), but not in cells expressing Cer-SERCA (red), suggesting Ca²⁺ uptake activity of Cer-SERCA. Ca²⁺ transients were partially restored in cells expressing both Cer-SERCA and YFP-PLB (blue). When compared with untransfected controls, Tg-releasable ER Ca²⁺ content was increased by Cer-SERCA. Cells expressing YFP-PLB (green) had reduced Ca²⁺ transients and reduced Ca²⁺ load, suggesting that exogenous PLB inhibited endogenous SERCA.

FIGURE 6.

A model of PLB binding to SERCA conformers. A, the present study suggests that PLB can bind to the E1 and E2-Tg conformations of SERCA, but with reduced affinity when compared with the E2 substate. B, a comparison of computational models of the PLB-SERCA and PLM-NKA regulatory complexes (21, 27), showing putative interactions between the regulatory peptide (red) with a groove formed by helices M2/M4/M6 (blue) or with helix M9 (green). Alternative binding sites may account for the differential binding affinities observed in the present study for Ca²⁺-free and Ca²⁺-bound SERCA.