The effects of mutation on the regulatory properties of phospholamban in co-reconstituted membranes

Reconstitution into proteoliposomes is a powerful method for studying calcium transport in a chemically pure membrane environment. By use of this approach, we have studied the regulation of Ca2+ ATPase by phospholamban (PLB) as a function of calcium concentration and PLB mutation. Coreconstitution of PLB and Ca2+-ATPase revealed the expected effects of PLB on the apparent calcium affinity of Ca2+-ATPase (K-ca) and unexpected effects of PLB on maximal activity (V-max). Wild-type PLB, six loss-of-function mutants (L7A, R9E, 112A, N34A, 138A, L42A), and three gain-of-function mutants (N27A, L37A, and 140A) were evaluated for their effects on K-ca and V-max. With the loss-of-function mutants, their ability to shift K-Ca correlated with their ability to increase V-max,. A total loss-of-function mutant, N34A, had no effect on KCa of the calcium pump and produced only a marginal increase in V(ma)x near-wild-type mutant, I12A, significantly altered both K-Ca and V-max of the calcium pump. With the gain-of-function mutants, their ability to shift KC, did not correlate with their ability to increase V-max The super-shifting mutants N27A, L37A, and 140A produced a large shift in KCa of the calcium pump; however, L37A decreased V-max while N27A and 140A increased V a,. For wild-type PLB, phosphorylation completely reversed the effect on K-Ca but had no effect on V-max,. We conclude that PLB increases V-max of Ca2+-ATPase, and that the magnitude of this effect is sensitive to mutation. The mutation sensitivity of PLB Asn(34) and Leu(37) identifies a region of the protein that is responsible for this regulatory property.