Förster Transfer Recovery Reveals That Phospholamban Exchanges Slowly From Pentamers but Rapidly From the SERCA Regulatory Complex

• DOI: 10.1161/circresaha.107.159947
• Publication Date: 2007-11-02T00:49:04Z
• Authors: Seth L. Robia; Kenneth S. Campbell; Eileen M. Kelly; Zhanjia Hou; Deborah L. Winters; David D. Thomas
• Keywords: SERCA; Phospholamban; Yellow fluorescent protein; Bimolecular fluorescence complementation

Phospholamban (PLB) or the sarcoplasmic reticulum Ca 2+ –ATPase (SERCA) were fused to cyan fluorescent protein (CFP) and coexpressed with PLB yellow (YFP). The expressed fluorescently tagged proteins imaged using epifluorescence total internal reflection fluorescence microscopy. YFP was selectively bleached by a focused laser beam. CFP at targeted site increased after photobleaching, indicating resonance energy transfer between CFP-SERCA/CFP-PLB YFP-PLB. donor relaxed back toward baseline as result of diffusion exchange YFP-PLB for unbleached YFP-PLB, which restored transfer. Requenching donors, termed Förster recovery (FTR), quantified an index rate subunit from PLB:SERCA PLB:PLB membrane complexes. regulatory complex rapid, showing diffusion-limited FTR (τ=1.4 second). Conversely, oligomeric complexes found be stable on much longer time scale. Despite free lateral in membrane, they showed no over 80 seconds. Mutation position 40 isoleucine alanine (I40A-PLB) did not abolish transfer, but destabilization apparent (τ=8.4 seconds). Oligomers I40A-PLB stabilized oxidative crosslinking transmembrane cysteines diamide. We conclude that exchanges rapidly its SERCA pump, whereas is slow does occur scale cardiac cycle.