Effects of Phospholamban Transmembrane Mutants on the Calcium Affinity, Maximal Activity, and Cooperativity of the Sarcoplasmic Reticulum Calcium Pump

Abstract

Regulation of the SERCA calcium pump by phospholamban (PLB) is largely due to interactions between their respective transmembrane domains. In spite of numerous mutagenesis and kinetic studies, we still do not have a clear mechanistic picture of how PLB influences the calcium transport cycle of SERCA. Herein, we have created alanine mutants for each residue in the transmembrane domain of PLB, we have co-reconstituted these mutants with SERCA into proteoliposomes, and we have performed kinetic simulations of the calcium-dependent ATPase activity isotherms. The PLB transmembrane mutants had a variable effect on the calcium affinity, maximal activity, and cooperativity of SERCA, such that a range of values was observed. Kinetic simulations using a well-established reaction scheme for SERCA then allowed us to correlate the effects on SERCA activity with changes in the reaction scheme rate constants. Only three steps in the reaction scheme were affected by the presence of PLB, namely, binding of the first calcium ion, a subsequent conformational change in SERCA, and binding of the second calcium ion. The ability of wild-type and mutant forms of PLB to alter the apparent calcium affinity of SERCA correlated with a decreased rate of binding of the second calcium ion. In addition, the ability of wild-type and mutant forms of PLB to alter the maximal activity of SERCA correlated with a change in the forward rate constant for the slow conformational change in SERCA following binding of the first calcium ion.

Hydrophobic Imbalance in the Cytoplasmic Domain of Phospholamban Is a Determinant for Lethal Dilated Cardiomyopathy

“…SERCA calcium binding occurs as cooperative steps linked by a conformational transition (ECa 7 EЈCa). PLN slows this conformational change via an effect on B rev , thereby reducing the K Ca of SERCA (28,29). For wild-type PLN, phosphorylated PLN, R9C, and R14del, we observed effects on all three calciumbinding steps, particularly A for , B rev , and C for (29).…”

“…Reconstitution versus Cardiac SR-The proteoliposomes used herein contain both SERCA1a and PLN oriented with their cytoplasmic domains on the external surface of the vesicles at lipid/protein ratios similar to those of cardiac SR membranes (19,29). Although there may be subtle differences in the interaction of PLN with SERCA1a versus SERCA2a (31), the skeletal muscle isoform is well established as a surrogate for structural and functional studies of the complex (for examples, see Refs.…”

“…PLN slows this conformational change via an effect on B rev , thereby reducing the K Ca of SERCA (28,29). For wild-type PLN, phosphorylated PLN, R9C, and R14del, we observed effects on all three calciumbinding steps, particularly A for , B rev , and C for (29). The primary effect was on B rev , yet a decrease in the forward rate constant for binding of the second calcium ion (C for ) contributed to SERCA inhibition.…”

“…Kinetic Simulations-Reaction rate simulations have been described for the transport cycle of SERCA (27,28), and we have previously used this approach to understand SERCA inhibition by PLN mutants (29). As before, we performed a global non-linear regression fit of the model to each plot of SERCA ATPase activity versus calcium concentration using Dynafit (Biokin Inc., Pullman, WA).…”

Highly Cooperative Dependence of Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) 2a Pump Activity on Cytosolic Calcium in Living Cells

“…These results suggest that factors such as Ca 2ϩ -dependent SERCA binding proteins, which can be excluded by the permeabilization treatment, are involved in the modulation of the transition rate from the E2 state to the E1 state for the generation of the high cooperativity in living cells. In fact, there is a report that an interacting modulator, such as phospholamban, alters the cooperativity of Ca 2ϩ binding to SERCA (45). The mechanism of the highly cooperative dependence of SERCA2a pump activity on cytosolic Ca 2ϩ is an interesting issue to be clarified in future studies.…”