PHOSPHOLAMBAN INHIBITORY FUNCTION IS ACTIVATED BY DEPOLYMERIZATION

Phospholamban (PLN), a homopentameric, integral membrane protein, reve rsibly inhibits cardiac sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) a ctivity through intramembrane interactions, Here, alanine-scanning mut agenesis of the PLN transmembrane sequence was used to identify two fu nctional domains on opposite faces of the transmembrane helix. Mutatio ns in one face diminish inhibitory interactions with transmembrane seq uences of SERCA2a, but have relatively little effect on the pentameric state, while mutations in the other face activate inhibitory interact ions 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 int eraction site lies on the helical face which is not involved in pentam er 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 PL N 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 concentra tion of this inhibited complex is determined by the dissociation const ant for the PLN pentamer (which is mutation-sensitive) and by the diss ociation constant for the PLN/SERCA2a heterodimer (which is likely to be mutation-sensitive).