title: The H29D Mutation Does Not Enhance Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor.
abstract: The N-terminal domain of the cardiac ryanodine receptor (RyR2) harbors a large number of naturally occurring mutations that are associated with stress-induced ventricular tachyarrhythmia and sudden death. Nearly all these disease-associated N-terminal mutations are located at domain interfaces or buried within domains. Mutations at these locations would alter domain-domain interactions or the stability/folding of domains. Recently, a novel RyR2 mutation H29D associated with ventricular arrhythmia at rest was found to enhance the activation of single RyR2 channels by diastolic levels of cytosolic Ca2+. Unlike other N-terminal disease-associated mutations, the H29D mutation is located on the surface of the N-terminal domain. It is unclear how this surface-exposed H29D mutation that does not appear to interact with other parts of the RyR2 structure could alter the intrinsic properties of the channel. Here we carried out detailed functional characterization of the RyR2-H29D mutant at the molecular and cellular levels. We found that the H29D mutation has no effect on the basal level or the Ca2+ dependent activation of [3H]ryanodine binding to RyR2, the cytosolic Ca2+ activation of single RyR2 channels, or the cytosolic Ca2+- or caffeine-induced Ca2+ release in HEK293 cells. In addition, the H29D mutation does not alter the propensity for spontaneous Ca2+ release or the thresholds for Ca2+ release activation or termination. Furthermore, the H29D mutation does not have significant impact on the thermal stability of the N-terminal region (residues 1-547) of RyR2. Collectively, our data show that the H29D mutation exerts little or no effect on the function of RyR2 or on the folding stability of the N-terminal region. Thus, our results provide no evidence that the H29D mutation enhances the cytosolic Ca2+ activation of RyR2.
authors.affiliation: Libin Cardiovascular Institute of Alberta, Department of Physiology & Pharmacology, University of Calgary, Calgary, Alberta, Canada T2N 4N1; Libin Cardiovascular Institute of Alberta, Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
authors.forename: Zhichao
authors.initials: Z
authors.lastname: Xiao
authors.affiliation: Libin Cardiovascular Institute of Alberta, Department of Physiology & Pharmacology, University of Calgary, Calgary, Alberta, Canada T2N 4N1; Libin Cardiovascular Institute of Alberta, Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
authors.forename: Wenting
authors.initials: W
authors.lastname: Guo
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, Canada V6T 1Z3.
authors.forename: Siobhan M Wong King
authors.initials: SM
authors.lastname: Yuen
authors.affiliation: Libin Cardiovascular Institute of Alberta, Department of Physiology & Pharmacology, University of Calgary, Calgary, Alberta, Canada T2N 4N1; Libin Cardiovascular Institute of Alberta, Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
authors.forename: Ruiwu
authors.initials: R
authors.lastname: Wang
authors.affiliation: Libin Cardiovascular Institute of Alberta, Department of Physiology & Pharmacology, University of Calgary, Calgary, Alberta, Canada T2N 4N1; Libin Cardiovascular Institute of Alberta, Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, Canada V6T 1Z3.
authors.forename: Filip
authors.initials: F
authors.lastname: Van Petegem
authors.affiliation: Libin Cardiovascular Institute of Alberta, Department of Physiology & Pharmacology, University of Calgary, Calgary, Alberta, Canada T2N 4N1; Libin Cardiovascular Institute of Alberta, Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
authors.forename: S R Wayne
authors.initials: SR
authors.lastname: Chen
mesh_terms: D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D003600:Cytosol / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D055550:Protein Stability / Q000187:drug effects; D017434:Protein Structure, Tertiary; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature; D014316:Tritium / Q000378:metabolism
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel; D014316:Tritium; D012433:Ryanodine; D002110:Caffeine; D002118:Calcium