Phospholamban (PLN), a homopentameric, integral membrane protein, reversibly 
inhibits cardiac sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) activity through 
intramembrane interactions. Here, alanine-scanning mutagenesis of the PLN 
transmembrane sequence was used to identify two functional domains on opposite 
faces of the transmembrane helix. Mutations in one face diminish inhibitory 
interactions with transmembrane sequences of SERCA2a, but have relatively little 
effect on the pentameric state, while mutations in the other face activate 
inhibitory interactions and enhance monomer formation. Double mutants are 
monomeric, but loss of inhibitory function is dominant over activation of 
inhibitory function. These observations support the proposal that the SERCA2a 
interaction site lies on the helical face which is not involved in pentamer 
formation. Four highly inhibitory mutants are effectively devoid of pentamer, 
suggesting that pentameric PLN represents a less active or inactive reservoir 
that dissociates to provide inhibitory monomeric PLN subunits. A model is 
presented in which the degree of PLN inhibition of SERCA2a activity is 
ultimately determined by the concentration of the inhibited PLN monomer.SERCA2a 
heterodimeric complex. The concentration of this inhibited complex is determined 
by the dissociation constant for the PLN pentamer (which is mutation-sensitive) 
and by the dissociation constant for the PLN/SERCA2a heterodimer (which is 
likely to be mutation-sensitive).