Functional and physical competition between phospholamban and its mutants provides insight into the molecular mechanism of gene therapy for heart failure |
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Authors: | Lockamy Elizabeth L Cornea Razvan L Karim Christine B Thomas David D |
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Affiliation: | Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55455, USA |
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Abstract: | We have used functional co-reconstitution of purified sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) with phospholamban (PLB), its inhibitor in the heart, to test the hypothesis that loss-of-function (LOF) PLB mutants (PLBM) can compete with wild-type PLB (PLBW) to relieve SERCA inhibition. Co-reconstitution at varying PLB-to-SERCA ratios was conducted using synthetic PLBW, gain-of-function mutant I40A, or LOF mutants S16E (phosphorylation mimic) or L31A. Inhibitory potency was defined as the fractional increase in KCa, measured from the Ca2+-dependence of ATPase activity. At saturating PLB, the inhibitory potency of I40A was about three times that of PLBW, while the potency of each of the LOF PLBM was about one third that of PLBW. However, there was no significant variation in the apparent SERCA affinity for these four PLB variants. When SERCA was co-reconstituted with mixtures of PLBW and LOF PLBM, inhibitory potency was reduced relative to that of PLBW alone. Furthermore, FRET between donor-labeled SERCA and acceptor-labeled PLBW was decreased by both (unlabeled) LOF PLBM. These results show that LOF PLBM can compete both physically and functionally with PLBW, provide a rational explanation for the partial success of S16E-based gene therapy in animal models of heart failure, and establish a powerful platform for designing and testing more effective PLBM targeted for gene therapy of heart failure in humans. |
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Keywords: | Abbreviations: Ca2+, divalent calcium ion Dabcyl-SE, 4-((4-(dimethylamino)phenyl)azo)-benzoic acid succinimidyl ester Fmoc, 9-fluorenylmethyloxycarbonyl FRET, fluorescence resonance energy transfer IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid Kd, dissociation constant LOF, loss of function MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy NaOH, Sodium hydroxide, pCa, &minus log[Ca2+] PKA, protein kinase A PEG&ndash PS, polyethylene glycol&ndash polystyrene (graft support) pKCa, &minus log(KCa), calcium concentration at half-maximal ATPase activity PLB, phospholamban SDS, sodium dodecyl-sulfate SERCA, sarco-endoplasmic reticulum Ca2+-ATPase SR, sarcoplasmic reticulum WT, wild-type |
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