Alanine-scanning mutagenesis of amino acids 21-30, forming cytoplasmic domain Ib 
in phospholamban (PLN), revealed that mutation to Ala of Asn27, Gln29, and Asn30 
results in gain of inhibitory function. In an earlier study (Kimura, Y., 
Kurzydlowski, K., Tada, M. , and MacLennan, D. H. (1997) J. Biol. Chem. 272, 
15061-15064), gain of function in PLN transmembrane domain II mutants was 
correlated with pentamer destabilization, leading to proposals that the PLN 
monomer is the active inhibitory species, that dissociation of the PLN pentamer 
is one determinant of PLN inhibitory function and that dissociation of the 
PLN.cardiac sarco(endo)plasmic Ca2+-ATPase isoform (SERCA2a) complex is a second 
determinant. Because each of the new domain Ib mutants contained a normal ratio 
of pentamer to monomer in SDS-polyacrylamide gel electrophoresis, gain of 
function must have resulted from mechanisms other than destabilization of 
pentameric structure. Evidence that domain Ib and domain II mutants act through 
different sites and different mechanisms was provided by a monomeric double 
mutant, N30A/I40A, in which the enhanced inhibitory function of each single 
mutant was additive. Evidence for an alteration in stability of the PLN/SERCA2a 
heterodimer was obtained in a study of double mutant N27A/N34A in which 
inhibitory function was regained by combining a gain of function, domain Ib 
mutation with a loss of function domain II mutation. These results support the 
proposal that PLN inhibition of SERCA2a involves, first, depolymerization of PLN 
and, second, the formation of inhibitory interactions between monomeric PLN and 
SERCA2a.