title: Extensive Ca2+ leak through K4750Q cardiac ryanodine receptors caused by cytosolic and luminal Ca2+ hypersensitivity.
abstract: Various ryanodine receptor 2 (RyR2) point mutations cause catecholamine-induced polymorphic ventricular tachycardia (CPVT), a life-threatening arrhythmia evoked by diastolic intracellular Ca2+release dysfunction. These mutations occur in essential regions of RyR2 that regulate Ca2+release. The molecular dysfunction caused by CPVT-associated RyR2 mutations as well as the functional consequences remain unresolved. Here, we study the most severe CPVT-associated RyR2 mutation (K4750Q) known to date. We define the molecular and cellular dysfunction generated by this mutation and detail how it alters RyR2 function, using Ca2+imaging, ryanodine binding, and single-channel recordings. HEK293 cells and cardiac HL-1 cells expressing RyR2-K4750Q show greatly enhanced spontaneous Ca2+oscillations. An endoplasmic reticulum-targeted Ca2+sensor, R-CEPIA1er, revealed that RyR2-K4750Q mediates excessive diastolic Ca2+leak, which dramatically reduces luminal [Ca2+]. We further show that the K4750Q mutation causes three RyR2 defects: hypersensitization to activation by cytosolic Ca2+, loss of cytosolic Ca2+/Mg2+-mediated inactivation, and hypersensitization to luminal Ca2+activation. These defects combine to kinetically stabilize RyR2-K4750Q openings, thus explaining the extensive diastolic Ca2+leak from the sarcoplasmic reticulum, frequent Ca2+waves, and severe CPVT phenotype. As the multiple concurrent defects are induced by a single point mutation, the K4750 residue likely resides at a critical structural point at which cytosolic and luminal RyR2 control input converge.
authors.affiliation: Department of Physiology, Fukuoka University School of Medicine, Fukuoka 814-0180, Japan ueharaak@fukuoka-u.ac.jp nagomik@juntendo.ac.jp.
authors.forename: Akira
authors.initials: A
authors.lastname: Uehara
authors.affiliation: Department of Pharmacology, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Murayama
authors.affiliation: Laboratory of Human Biology, Fukuoka University School of Medicine, Fukuoka 814-0180, Japan.
authors.forename: Midori
authors.initials: M
authors.lastname: Yasukochi
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL 60612.
authors.forename: Michael
authors.initials: M
authors.lastname: Fill
authors.affiliation: Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical Science, Shiga 520-2192, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Horie
authors.affiliation: Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Okamoto
authors.affiliation: Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Matsuura
authors.affiliation: Department of Cell Biology, Fukuoka University School of Medicine, Fukuoka 814-0180, Japan.
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Uehara
authors.affiliation: Department of Pathology and Applied Neurobiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Fujimoto
authors.affiliation: Department of Pharmacology, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sakurai
authors.affiliation: Department of Pharmacology, Juntendo University School of Medicine, Tokyo 113-8421, Japan ueharaak@fukuoka-u.ac.jp nagomik@juntendo.ac.jp.
authors.forename: Nagomi
authors.initials: N
authors.lastname: Kurebayashi
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D051379:Mice; D020125:Mutation, Missense; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000502:physiology; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism*
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel; C000592748:ryanodine receptor 2. mouse; D002118:Calcium