Abstract 17233: Characterization of Reduced Binding of the PLB-SERCA Regulatory Complex Induced by Calcium and Thapsigargin
Phospholamban (PLB) is the key regulator of sarco/endoplasmic reticulum calcium ATPase (SERCA), and understanding the mechanisms of regulation requires full understanding the PLB-SERCA binding interaction. We hypothesized that the enzymatic structural conformations of SERCA have different affinities for binding PLB. To evaluate our hypothesis, we measured PLB-SERCA binding in the presence of thapsigargin (TG) and calcium, which are thought to stabilize structural conformations of SERCA with low affinity for PLB. To quantify binding, we measured Fluorescence Resonance Energy Transfer (FRET) between fluorescently tagged fusion proteins expressed in AAV-293 cells or myocytes. Both TG and calcium decreased FRET between SERCA and PLB, suggesting altered binding. By measuring the dependence of FRET on protein expression levels, we found that the decrease was due to a reduced affinity, but PLB-SERCA binding was not completely abolished. No significant change in the distance between our probes was detected, suggesting that the structure of the regulatory complex was not greatly altered by TG or calcium. The dose responses of TG and calcium showed that binding affinity decreased with increasing ligand concentration up to a maximal level. This observation suggests that PLB can bind to SERCA already bound by TG or calcium, indicating that these binding events are not mutually exclusive. Our data also suggest that total protein concentration is a critical determinant of PLB-SERCA binding. We conclude that TG and calcium bound SERCA conformations have a reduced affinity for PLB, but are not incompatible with PLB binding as some previous studies have suggested.
- © 2010 by American Heart Association, Inc.