Fluorescence and Functional Measurements of Phospholamban Mutant Competition

Abstract

Ca2+ cycling through the SR in muscle cells is largely controlled by the Ca-pump (SERCA). SERCA transports Ca2+ into the SR, allowing muscles to relax, and is inhibited by phospholamban (PLB) at submicromolar [Ca2+]. PLB inhibition can be relieved by adrenergic stimulation, leading to PLB phosphorylation. Heart Failure (HF), which contributes to 12% of US deaths, can be caused by a variety of genetic or environmental factors, but a common symptom is decreased SERCA activity. Several potential therapies for HF aim to increase SERCA activity, including overexpression of the enzyme via rAAV gene therapy. We are investigating the possibility of using LOF-PLB mutants (PLBM) as alternative gene therapy vectors to increase Ca-ATPase activity. We have used FRET and FLIM to measure quantitatively measure the ability of several PLBM to compete with WT-PLB to bind to SERCA and activate it. Experiments in living HEK cells examine the ability of PLBM to compete with WT-PLB, thus rescuing SERCA activity. Active human SERCA2a and WT-PLB, tagged with fluorescent proteins (e.g., CFP and YFP), are co-expressed in stable cell lines followed by transient co-expression with PLBM. The effects of PLBM on Ca-ATPase activity and FRET are measured to determine the mutant's ability to compete with PLBW, both physically and functionally. Optimal mutants are being tested in a rat model of heart failure.