Relative affinity of calcium pump isoforms for phospholamban quantified by fluorescence resonance energy transfer

Abstract

To investigate the regulation of SERCA1a [sarco(endo)plasmic reticulum calcium ATPase] and SERCA2a calcium pump isoforms by phospholamban (PLB), we quantified PLB-SERCA interactions by fluorescence resonance energy transfer (FRET) in live cells. For both SERCA1a and SERCA2a, FRET to PLB increased with increasing protein expression level to a maximum value corresponding to a probe separation distance of 64 A. The data indicate that the respective regulatory complexes assume the same overall quaternary conformation. However, FRET measurements also revealed that PLB has a 50% higher apparent affinity for SERCA1a relative to SERCA2a. The results suggest that despite the structural similarities of the respective regulatory complexes, there is preferential binding of PLB to SERCA1a over SERCA2a. This apparent selectivity may have implications for biochemical studies in which SERCA1a is used as a substitute for SERCA2a. It may also be an important strategic consideration for therapeutic overexpression of SERCA isoforms in cardiac muscle.

Figure 1

TIRF microscopy showed reticulate subcellular distribution of fluorescently labeled proteins. A) Cer-SERCA1a. B) YFP-PLB. C) Overlay of SERCA1a and PLB. D)Cer-SERCA2a. E) YFP-PLB F) Overlay of SERCA2a and PLB. Areas of colocalized Cer/YFP fluorescence appear white in the overlay images.

Figure 2

A) Progressive acceptor photobleaching of YFP-PLB (green) resulted in an increase in Cer-SERCA1a fluorescence (blue) indicating FRET. B) Photobleaching of YFP-PLB (green) resulted in an increase in Cer-SERCA2a fluorescence (blue). C) The relationship between the normalized fluorescence of Cer-SERCA1a and YFP-PLB during progressive photobleaching was linear, consistent with a heterodimeric regulatory complex. D) The relationship between the normalized fluorescence of Cer-SERCA1a and YFP-PLB during progressive photobleaching was also linear. E) FRET increased with protein expression level. Hyperbolic fitting showed a right-shifted concentration dependence for Cer-SERCA2a (red) compared to Cer-SERCA1a (black). F) A comparison of pooled data for Cer-SERCA1a (black) and Cer-SERCA2a (red). G) Summary of FRET_max values obtained by fitting reported as mean ± SE. H) Summary of Kd2 values obtained by fitting reported as mean ± SE. * indicates p-value <0.01. The data suggest PLB binds more avidly to SERCA1a than SERCA2a.

Figure 3

A) A model of PLB regulation of SERCA when both SERCA2a and SERCA1a are accessible. The relative distribution of PLB among the pentamer (PLB₅), monomer (PLB₁), and SERCA1a/2a regulatory complexes depends on the dissociation constants for the oligomer (Kd1) and the regulatory complexes (Kd2). B) A computational model of PLB interactions shows the concentration dependence of the distribution of PLB among oligomer (red), monomer (black), and regulatory complexes with SERCA1a (blue) and SERCA2a (green). An equimolar system is simulated; the protein concentration indicated corresponds to the measured YFP-PLB concentration (AU), as in Fig. 2E.