title: The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+ Binding but Not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor.
abstract: A number of point mutations in the intracellular Ca2+-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca2+binding to CaM and impair inhibition of CaM-regulated Ca2+channels like the cardiac Ca2+release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca2+binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca2+release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca2+These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca2+binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+release by manipulating the CaM-RyR2 interaction.
authors.affiliation: From the Department of Chemistry and Bioscience, Aalborg University, 9220 Aalborg, Denmark.|the Libin Cardiovascular Institute of Alberta, the Department of Physiology and Pharmacology and the Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Mads Toft
authors.identifier: 0000-0001-6779-5109
authors.initials: MT
authors.lastname: Søndergaard
authors.affiliation: the Libin Cardiovascular Institute of Alberta, the Department of Physiology and Pharmacology and the Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Yingjie
authors.initials: Y
authors.lastname: Liu
authors.affiliation: From the Department of Chemistry and Bioscience, Aalborg University, 9220 Aalborg, Denmark.
authors.forename: Kamilla Taunsig
authors.initials: KT
authors.lastname: Larsen
authors.affiliation: the Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois 60612.
authors.forename: Alma
authors.initials: A
authors.lastname: Nani
authors.affiliation: the Libin Cardiovascular Institute of Alberta, the Department of Physiology and Pharmacology and the Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Xixi
authors.initials: X
authors.lastname: Tian
authors.affiliation: From the Department of Chemistry and Bioscience, Aalborg University, 9220 Aalborg, Denmark.
authors.forename: Christian
authors.initials: C
authors.lastname: Holt
authors.affiliation: the Libin Cardiovascular Institute of Alberta, the Department of Physiology and Pharmacology and the Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Ruiwu
authors.initials: R
authors.lastname: Wang
authors.affiliation: From the Department of Chemistry and Bioscience, Aalborg University, 9220 Aalborg, Denmark.
authors.forename: Reinhard
authors.identifier: 0000-0001-6942-5056
authors.initials: R
authors.lastname: Wimmer
authors.affiliation: the Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada, and.
authors.forename: Filip
authors.identifier: 0000-0003-2728-8537
authors.initials: F
authors.lastname: Van Petegem
authors.affiliation: the Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois 60612.
authors.forename: Michael
authors.initials: M
authors.lastname: Fill
authors.affiliation: the Libin Cardiovascular Institute of Alberta, the Department of Physiology and Pharmacology and the Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 1N4, Canada.|the Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois 60612.
authors.forename: S R Wayne
authors.identifier: 0000-0001-5808-0304
authors.initials: SR
authors.lastname: Chen
authors.affiliation: From the Department of Chemistry and Bioscience, Aalborg University, 9220 Aalborg, Denmark, mto@bio.aau.dk.
authors.forename: Michael Toft
authors.identifier: 0000-0002-1423-2481
authors.initials: MT
authors.lastname: Overgaard
mesh_terms: D019943:Amino Acid Substitution; D001145:Arrhythmias, Cardiac / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D000072417:Protein Domains; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium
keywords: CaM-F142L; arrhythmia; calcium intracellular release; calcium-binding protein; calmodulin (CaM); cardiac ryanodine receptor; protein-protein interaction; ryanodine receptor