title: Role of Cys³⁶⁰² in the function and regulation of the cardiac ryanodine receptor.
abstract: The cardiac Ca²⁺ release channel [ryanodine receptor type 2 (RyR2)] is modulated by thiol reactive agents, but the molecular basis of RyR2 modulation by thiol reagents is poorly understood. Cys³⁶³⁵ in the skeletal muscle RyR1 is one of the most hyper-reactive thiols and is important for the redox and calmodulin (CaM) regulation of the RyR1 channel. However, little is known about the role of the corresponding cysteine residue in RyR2 (Cys³⁶⁰²) in the function and regulation of the RyR2 channel. In the present study, we assessed the impact of mutating Cys³⁶⁰² (C³⁶⁰²A) on store overload-induced Ca²⁺ release (SOICR) and the regulation of RyR2 by thiol reagents and CaM. We found that the C³⁶⁰²A mutation suppressed SOICR by raising the activation threshold and delayed the termination of Ca²⁺ release by reducing the termination threshold. As a result, C³⁶⁰²A markedly increased the fractional Ca²⁺ release. Furthermore, the C³⁶⁰²A mutation diminished the inhibitory effect of N-ethylmaleimide on Ca²⁺ release, but it had no effect on the stimulatory action of 4,4'-dithiodipyridine (DTDP) on Ca²⁺ release. In addition, Cys³⁶⁰² mutations (C³⁶⁰²A or C³⁶⁰²R) did not abolish the effect of CaM on Ca²⁺-release termination. Therefore, RyR2-Cys³⁶⁰² is a major site mediating the action of thiol alkylating agent N-ethylmaleimide, but not the action of the oxidant DTDP. Our data also indicate that residue Cys³⁶⁰² plays an important role in the activation and termination of Ca²⁺ release, but it is not essential for CaM regulation of RyR2.
authors.affiliation: *The Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: Tao
authors.initials: T
authors.lastname: Mi
authors.affiliation: *The Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: Zhichao
authors.initials: Z
authors.lastname: Xiao
authors.affiliation: *The Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: Wenting
authors.initials: W
authors.lastname: Guo
authors.affiliation: *The Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: Yijun
authors.initials: Y
authors.lastname: Tang
authors.affiliation: *The Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: Florian
authors.initials: F
authors.lastname: Hiess
authors.affiliation: *The Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: Jianmin
authors.initials: J
authors.lastname: Xiao
authors.affiliation: *The Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: Yundi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: †Department of Physiology and Heart Otago, University of Otago, Dunedin 9045, New Zealand.
authors.forename: Joe Z
authors.initials: JZ
authors.lastname: Zhang
authors.affiliation: *The Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: *The Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: Ruiwu
authors.initials: R
authors.lastname: Wang
authors.affiliation: †Department of Physiology and Heart Otago, University of Otago, Dunedin 9045, New Zealand.
authors.forename: Peter P
authors.initials: PP
authors.lastname: Jones
authors.affiliation: *The Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: S R Wayne
authors.initials: SR
authors.lastname: Chen
mesh_terms: D000478:Alkylation / Q000187:drug effects; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D054730:Protein Interaction Domains and Motifs; D011725:Pyridines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D004220:Disulfides; D010446:Peptide Fragments; D011725:Pyridines; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D013439:Sulfhydryl Reagents; C000592748:ryanodine receptor 2. mouse; C007542:4,4'-dipyridyl disulfide; D003545:Cysteine; D005033:Ethylmaleimide