title: Phospholamban inhibitory function is activated by depolymerization.
abstract: 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).
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, Charles H. Best Institute, Toronto, Ontario M5G1L6, Canada.
authors.forename: Y
authors.initials: Y
authors.lastname: Kimura
authors.forename: K
authors.initials: K
authors.lastname: Kurzydlowski
authors.forename: M
authors.initials: M
authors.lastname: Tada
authors.forename: D H
authors.initials: DH
authors.lastname: MacLennan
mesh_terms: D000818:Animals; D001704:Biopolymers; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006801:Humans; D016296:Mutagenesis; D009206:Myocardium / Q000201:enzymology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D000099211:Phospholamban
chemical_list: D001704:Biopolymers; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D004791:Enzyme Inhibitors; D000099211:Phospholamban