To study PLB (phospholamban) inhibition of the cardiac Ca2+ pump [SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a)], a fusion protein (SER-20G-PLB) was engineered by tethering SERCA2a with PLB through a 20-glycine residue chain, allowing the PLB tether to either bind to or dissociate from the inhibition site on SERCA2a. When expressed in insect cells, SER-20G-PLB produced active Ca2+ uptake, which was stimulated by the anti-PLB antibody, both similar to that which occurred with the control sample co-expressing WT (wild-type)-SERCA2a and WT-PLB. The KCa values of Ca2+-dependent ATPase were similar for SER-20G-PLB (0.29±0.02 μM) and for the control sample (0.30±0.02 μM), both greater than 0.17±0.01 μM for WT-SERCA2a expressed alone. Thus SER-20G-PLB retains a fully active Ca2+ pump, but its apparent Ca2+ affinity was decreased intrinsically by tethered PLB at a 1:1 molar stoichiometry. Like WT-PLB, SER-20G-PLB ran as both monomers and homo-pentamers on SDS/PAGE. As Ca2+ concentrations increase from 0 to the micromolar range, the proportion of non-inhibiting pentamers increased from 32% to 52%, suggesting that Ca2+ activation of the pump completely dissociates the PLB tether from the inhibition site on SERCA2a, with concurrent association of PLB pentamers. Collectively, the regulation of SERCA2a is achieved through the Ca2+-dependent equilibria involving PLB association and dissociation from SERCA2a, and assembling and disassembling of SER-20G-PLB pentamers.

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