Reexamination of the role of the leucine/isoleucine zipper residues of phospholamban in inhibition of the Ca2+ pump of cardiac sarcoplasmic reticulum.
Phospholamban is a small phosphoprotein inhibitor of the Ca(2+)-pump in cardiac sarcoplasmic reticulum, which shows a distinct oligomeric distribution between monomers and homopentamers that are stabilized through Leu/Ile zipper interactions. A two-faced model of phospholamban inhibition of the Ca(2+)-pump was proposed, in which the Leu/Ile zipper residues located on one face of the transmembrane alpha-helix regulate the pentamer to monomer equilibrium, whereas residues on the other face of the helix bind to and inhibit the pump. Here we tested this two-faced model of phospholamban action by analyzing the functional effects of a new series of Leu/Ile zipper mutants. Pentameric stabilities of the mutants were quantified at different SDS concentrations. We show that several phospholamban mutants with hydrophobic amino acid substitutions at the Leu/Ile zipper region retain the ability to form pentamers but at the same time give the same or even stronger (i.e. L37I-PLB) inhibition of the Ca(2+)-pump than do mutants that are more completely monomeric. Steric constraints prevent the Leu/Ile zipper residues sequestered in the interior of the phospholamban pentamer from binding to the Ca(2+)-pump, leading to the conclusion that the zipper residues access the pump from the phospholamban monomer, which is the active inhibitory species. A modified model of phospholamban transmembrane domain action is proposed, in which the membrane span of the phospholamban monomer maintains contacts with the Ca(2+)-pump around most of its circumference, including residues located in the Leu/Ile zipper region.
Cornea
R L
RL
Department of Medicine and the Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Autry
J M
JM
Chen
Z
Z
Jones
L R
LR
D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D016350:Leucine Zippers; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013329:Structure-Activity Relationship; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D012967:Sodium Dodecyl Sulfate; D000099211:Phospholamban