A Mutation Associated with DCM Increases Phospholamban Oligomerization and Decreases SERCA-Binding, but Does Not Change Phopholamban Tertiary Strucuture or Phosphorylation by PKA

Abstract

To better understand the pathological mechanism of a human dilated cardiomyopathy phospholamban (PLB) mutation (R9C), we investigated the effects of this mutation on PLB structure and regulatory interactions. Notably, we observed efficient phosphorylation of R9C-PLB by PKA in vitro, and nuclear magnetic resonance (NMR) spectroscopy showed no change in R9C-PLB structure compared to WT. To test R9C-PLB binding interactions in live cells, PLB was expressed as cyan and yellow fluorescent protein (CFP/YFP) fusions in AAV-293 cells, and PLB oligomerization and SERCA-binding were quantified by fluorescence resonance energy transfer (FRET). 100 micromolar H2O2 applied to the cells induced a rapid quench of CFP-R9C-PLB fluorescence and a concomitant increase in YFP-R9C-PLB fluorescence, indicating an increase in intraoligomeric FRET after oxidation. FRET enhancement after peroxide addition was not observed for CFP/YFP-WT-PLB. To test whether the FRET increase was due to increased oligomerization or a quaternary conformation change, we measured intraoligomeric FRET in a population of cells expressing a wide range of R9C-PLB protein concentrations. FRET dependence on concentration yielded oligomer intrinsic FRET efficiency (FRETmax) and relative dissociation constant (KD). Compared to WT, R9C-PLB had a decreased KD and increased FRETmax, indicating an increased oligomerization affinity and more compact oligomer structure, respectively. The enhanced oligomerization of R9C-PLB was matched by a decrease in SERCA-binding compared to WT. Overall the data suggest a new mechanism by which the R9C mutation may exert a pathological effect: decreased SERCA regulation and increased oligomerization, as consequences of increased sensitivity of R9C-PLB to oxidation.