PHOSPHOLAMBAN DOMAIN IB MUTATIONS INFLUENCE FUNCTIONAL INTERACTIONS WITH THE CA2-ATPASE ISOFORM OF CARDIAC SARCOPLASMIC-RETICULUM()

Alanine-scanning mutagenesis of amino acids 21-30, forming cytoplasmic domain Ib in phospholamban (PLN), revealed that mutation to Ala of As n(27), Gln(29), and Asn(30) results ins gain of inhibitory function, I n an earlier study (Kimura, Y., Kurzydlowski, E., Tada, M., and MacLen nan, D. H. (1997) J. Biol. Chem. 272, 15061-15064), gain of function i n PLN transmembrane domain II mutants was correlated with pentamer des tabilization, leading to proposals that the PLN monomer is the active inhibitory species, that dissociation of the PLN pentamer is one deter minant of PLN inhibitory function and that dissociation of the PLN car diac sarco(endo)plasmic Ca2+-ATPase isoform (SERCA2a) complex is a sec ond determinant, Because each of the new domain Ib mutants contained a normal ratio of pentamer to monomer in SDS-polyacrylamide gel electro phoresis, gain of function must have resulted from mechanisms other th an destabilization of pentameric structure. Evidence that domain Ib an d domain II mutants act through different sites and different mechanis ms was provided by a mononomeric double mutant, N30A/I40A, in which th e enhanced inhibitory function of each single mutant was additive. Evi dence for an alteration in stability of the PLN/SERCA2a heterodimer wa s obtained in a study Of double mutant N27A/N34A in which inhibitory f unction was regained by combining a gain of function, domain Ib mutati on with a loss of function domain II mutation, These results support t he proposal that PLN inhibition of SERCA2a involves, first, depolymeri zation of PLN and, second, the formation of inhibitory interactions be tween monomeric PLN and SERCA2a.