An Investigation of the Phospholamban-Serca Regulatory Interaction with Fluorescence Resonance Energy Transfer

With Fluorescence Resonance Energy Transfer (FRET), we are able to detect changes in the structure and affinity of the PLB-SERCA regulatory complex in live cells. Using this approach, we have detected a high level of PLB-SERCA interaction even at Ca2+ concentrations known to fully relieve PLB inhibition of SERCA, suggesting that dissociation is not required for relief of inhibition. We also detect no real-time change in PLB-SERCA binding over the course of a single Ca2+ transient in paced myocytes. The effect of Ca2+ on the PLB-SERCA interaction is best described as a reduced affinity with no change in the structure of the complex. Even though we do not observe a change in the structure of the PLB-SERCA complex with changes in calcium concentrations, we currently hypothesize that the reduced affinity is produced by a transition of PLB into an alternative binding site. We propose that the transmembrane domain of PLB could move independently of its cytosolic domain. Our fluorescent probe is attached to the cytosolic domain of PLB, which would explain our inability to detect a change in the position of the transmembrane domain. My current work attempts to address this hypothesis by measuring the FRET between SERCA and a truncated PLB lacking the cytosolic domain