title: Dantrolene rescues aberrant N-terminus intersubunit interactions in mutant pro-arrhythmic cardiac ryanodine receptors.
abstract: OBJECTIVE
  The ryanodine receptor (RyR2) is an intracellular Ca(2+) release channel essential for cardiac excitation-contraction coupling. Abnormal RyR2 channel function results in the generation of arrhythmias and sudden cardiac death. The present study was undertaken to investigate the mechanistic basis of RyR2 dysfunction in inherited arrhythmogenic cardiac disease.
  
  
  RESULTS
  We present several lines of complementary evidence, indicating that the arrhythmia-associated L433P mutation disrupts RyR2 N-terminus self-association. A combination of yeast two-hybrid, co-immunoprecipitation, and chemical cross-linking assays collectively demonstrate that a RyR2 N-terminal fragment carrying the L433P mutation displays substantially reduced self-interaction compared with wild type. Moreover, sucrose density gradient centrifugation reveals that the L433P mutation impairs tetramerization of the full-length channel. [(3)H]Ryanodine-binding assays demonstrate that disrupted N-terminal intersubunit interactions within RyR2(L433P) confer an altered sensitivity to Ca(2+) activation. Calcium imaging of RyR2(L433P)-expressing cells reveals substantially prolonged Ca(2+) transients and reduced Ca(2+) store content indicating defective channel closure. Importantly, dantrolene treatment reverses the L433P mutation-induced impairment and restores channel function.
  
  
  CONCLUSIONS
  The N-terminus domain constitutes an important structural determinant for the functional oligomerization of RyR2. Our findings are consistent with defective N-terminus self-association as a molecular mechanism underlying RyR2 channel deregulation in inherited arrhythmogenic cardiac disease. Significantly, the therapeutic action of dantrolene may occur via the restoration of normal RyR2 N-terminal intersubunit interactions.
authors.affiliation: Wales Heart Research Institute, Institute of Molecular and Experimental Medicine, Cardiff University School of Medicine, Cardiff CF14 4XN, UK.
authors.forename: Monika
authors.initials: M
authors.lastname: Seidel
authors.affiliation: Wales Heart Research Institute, Institute of Molecular and Experimental Medicine, Cardiff University School of Medicine, Cardiff CF14 4XN, UK.
authors.forename: N Lowri
authors.initials: NL
authors.lastname: Thomas
authors.affiliation: Wales Heart Research Institute, Institute of Molecular and Experimental Medicine, Cardiff University School of Medicine, Cardiff CF14 4XN, UK.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Williams
authors.affiliation: Wales Heart Research Institute, Institute of Molecular and Experimental Medicine, Cardiff University School of Medicine, Cardiff CF14 4XN, UK.
authors.forename: F Anthony
authors.initials: FA
authors.lastname: Lai
authors.affiliation: Wales Heart Research Institute, Institute of Molecular and Experimental Medicine, Cardiff University School of Medicine, Cardiff CF14 4XN, UK zissimopouloss@cf.ac.uk.
authors.forename: Spyros
authors.initials: S
authors.lastname: Zissimopoulos
mesh_terms: D019943:Amino Acid Substitution; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D001145:Arrhythmias, Cardiac / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D019571:Arrhythmogenic Right Ventricular Dysplasia / Q000209:etiology / Q000235:genetics / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D003620:Dantrolene / Q000494:pharmacology*; D057809:HEK293 Cells; D006333:Heart Failure / Q000209:etiology / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008955:Models, Cardiovascular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017180:Tachycardia, Ventricular / Q000209:etiology / Q000235:genetics / Q000378:metabolism; D000098850:Polymorphic Catecholaminergic Ventricular Tachycardia
chemical_list: D000889:Anti-Arrhythmia Agents; D050505:Mutant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D003620:Dantrolene
keywords: arrhythmias; dantrolene; excitation–contraction coupling; oligomerization; ryanodine receptor