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 Ca(2+)-ATPase by phospholamban (PLB) as a function of calcium concentration and PLB mutation. Co-reconstitution of PLB and Ca(2+)-ATPase revealed the expected effects of PLB on the apparent calcium affinity of Ca(2+)-ATPase (K(Ca)) and unexpected effects of PLB on maximal activity (V(max)). Wild-type PLB, six loss-of-function mutants (L7A, R9E, I12A, N34A, I38A, L42A), and three gain-of-function mutants (N27A, L37A, and I40A) 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 K(Ca) of the calcium pump and produced only a marginal increase in V(max). A 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 K(Ca) did not correlate with their ability to increase V(max). The "super-shifting" mutants N27A, L37A, and I40A produced a large shift in K(Ca) of the calcium pump; however, L37A decreased V(max), while N27A and I40A increased V(max). 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 Ca(2+)-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.

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.

The sequence of phospholamban (PLB) is practically invariant among mammalian species. The hydrophobic transmembrane domain has 10 leucine and 8 isoleucine residues. Two roles have been proposed for the leucines; one subset stabilizes PLB oligomers, while a second subset physically interacts with SERCA. On the basis of the sequence of the PLB transmembrane domain, we chemically synthesized a series of peptides and tested their ability to regulate SERCA in reconstituted membranes. In all, eight peptides were studied: a peptide corresponding to the null-cysteine transmembrane domain of PLB (TM-Ala-PLB), two polyleucine peptides (Leu18 and Leu24), polyalanine peptides containing 4, 7, and 12 leucine residues (Leu4, Leu7, and Leu12, respectively), and a polyalanine peptide containing the 9 leucine residues present in the transmembrane domain of PLB with and without the essential Asn34 residue (Asn1Leu9 and Leu9, respectively). With the exception of Leu18, co-reconstitution of the peptides revealed effects on the apparent calcium affinity of SERCA. The TM-Ala-PLB peptide possessed approximately 70% of the inhibitory function of wild-type PLB. The remaining peptides exhibited significant inhibitory activity decreasing in the following order: Leu12, Leu9, Leu24, Leu7, and Leu4. Replacing Asn34 of PLB in the Leu9 peptide resulted in superinhibition of SERCA. On the basis of these observations, we conclude that a partial requirement for SERCA inhibition is met by a simple hydrophobic surface on a transmembrane alpha-helix. In addition, the superinhibition observed for the Asn34-containing peptide suggests that the model peptides mimic the inhibitory properties of PLB. A model is presented in which surface complementarity around key amino acid positions is enhanced in the interaction with SERCA.

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