title: L30A Mutation of Phospholemman Mimics Effects of Cardiac Glycosides in Isolated Cardiomyocytes.
abstract: To determine if mutations introduced into phospholemman (PLM) could increase the level of PLM-Na,K-ATPase (NKA) binding, we performed scanning mutagenesis of the transmembrane domain of PLM and measured Förster resonance energy transfer (FRET) between each mutant and NKA. We observed an increased level of binding to NKA for several PLM mutants compared to that of the wild type (WT), including L27A, L30A, and I32A. In isolated cardiomyocytes, overexpression of WT PLM increased the amplitude of the Ca2+transient compared to the GFP control. The Ca2+transient amplitude was further increased by L30A PLM overexpression. The L30A mutation also delayed Ca2+extrusion and increased the duration of cardiomyocyte contraction. This mimics aspects of the effect of cardiac glycosides, which are known to increase contractility through inhibition of NKA. No significant differences between WT and L30A PLM-expressing myocytes were observed after treatment with isoproterenol, suggesting that the superinhibitory effects of L30A are reversible with β-adrenergic stimulation. We also observed a decrease in the extent of PLM tetramerization with L30A compared to WT using FRET, suggesting that L30 is an important residue for mediating PLM-PLM binding. Molecular dynamics simulations revealed that the potential energy of the L30A tetramer is greater than that of the WT, and that the transmembrane α helix is distorted by the mutation. The results identify PLM residue L30 as an important determinant of PLM tetramerization and of functional inhibition of NKA by PLM.
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago , Maywood, Illinois 60153, United States.
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Himes
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago , Maywood, Illinois 60153, United States.
authors.forename: Nikolai
authors.initials: N
authors.lastname: Smolin
authors.affiliation: School of Life and Medical Sciences, University of Hertfordshire , Hatfield, U.K.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kukol
authors.affiliation: Department of Pharmacology, The University of California , Davis, California 95616, United States.
authors.forename: Julie
authors.initials: J
authors.lastname: Bossuyt
authors.affiliation: Department of Pharmacology, The University of California , Davis, California 95616, United States.
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago , Maywood, Illinois 60153, United States.
authors.forename: Seth L
authors.initials: SL
authors.lastname: Robia
mesh_terms: D000818:Animals; D002301:Cardiac Glycosides / Q000494:pharmacology*; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D008565:Membrane Proteins / Q000235:genetics*; D056004:Molecular Dynamics Simulation; D009154:Mutation; D032383:Myocytes, Cardiac / Q000187:drug effects*; D010750:Phosphoproteins / Q000235:genetics*; D011817:Rabbits
chemical_list: D002301:Cardiac Glycosides; D008565:Membrane Proteins; D010750:Phosphoproteins; C069141:phospholemman