Defining the molecular components of calcium transport regulation in a reconstituted membrane system

Using a chemically defined reconstitution system, we performed a systematic study of key factors in the regulation of the Ca-ATPase by phospholamban (PLB). We varied both the lipid/protein and PLB/Ca-ATPase ratios, determined the effects of PLB phosphorylation, and compared the regulatory effects of several PLB mutants, as a function of Ca concentration. The reconstitution system allowed us to determine accurately not only the PLB effects on K-ca (Ca concentration at half-maximal activity) of the Ca-ATPase, but also the effects on V-max (maximal activity). Wild-type PLB (WT-PLB) and two gain-of-function mutants, N27A-PLB and I40A-PLB, showed not only the previously reported increase in K-Ca, but also an increase in V-max. Specifically, V-max increases linearly with the intramembrane PLB concentration, and is approximately doubled when the sample composition approaches that of cardiac SR. Upon phosphorylation of PLB at Ser-16, the K-Ca effects were almost completely reversed for WT- and N27A-PLB but were only partially reversed for 140A-PLB. Phosphorylation induced a V-max increase for WT-PLB, and a V-max decrease for N27A- and I40A-PLB. We conclude that PLB and PLB phosphorylation affect V-max as well as K-Ca, and that the magnitude of both effects is sensitive to the PLB concentration in the membrane.