Ca2+ Binding to Site I of the Cardiac Ca2+ Pump (SERCA2a) is Sufficient to Dissociate Phospholamban (PLB)

Abstract

Our model of mutually exclusive binding of PLB and Ca2+ to SERCA2a suggests that the Ca2+-bound form of SERCA2a (E1) cannot interact with PLB. However, it is unclear whether Ca2+ binding to site I, site II, or both sites of SERCA2a is sufficient to dissociate PLB. To investigate this, we made several SERCA2a mutants: mutants lacking Ca2+ binding site I (E770Q or T798A), Ca2+ binding site II (E309Q or N795A), or both sites (D799N, or E309Q, E770Q double mutant). When individually expressed in insect cell microsomes, all these mutants failed to transport Ca2+, but were readily phosphorylated by Pi to form E2∼P (measured in Ca2+-free buffer favoring formation of E2, the low Ca2+ affinity conformation). Ca2+ inhibition of E2∼P formation was maintained with all site II mutants, but was lost with site I mutants, demonstrating that Ca2+ binding to site I is sufficient to prevent E2∼P formation. On the other hand, N30C-PLB cross-linked strongly to all Ca2+ binding site mutants, including those lacking either site I, site II, or both sites, thus demonstrating that PLB binds preferentially to E2, the Ca2+-free state of SERCA2a. 10 μM Ca2+ blocked cross-linking of N30C-PLB to site II mutants, yielding KCa values of 1.25 ± 0.3 μM for E309Q, and 0.32 ± 0.03 μM for N795A, compared to 0.44 ± 0.04 μM for WT-SERCA2a. However, Ca2+ had no effect on cross-linking of N30C-PLB to SERCA2a with site I mutants, even at Ca2+ concentrations of 100 μM or higher. These results demonstrate that Ca2+ binding site I of SERCA2a is the key Ca2+-binding site regulating PLB association and dissociation.