title: In cardiomyocytes, binding of unzipping peptide activates ryanodine receptor 2 and reciprocally inhibits calmodulin binding.
abstract: BACKGROUND
  One hypothesis for elevated Ca(2+) leak through cardiac ryanodine receptors (ryanodine receptor 2 [RyR2]) in heart failure is interdomain unzipping that can enhance aberrant channel activation. A peptide (domain peptide corresponding to RyR2 residues 2460-2495 [DPc10]) corresponding to RyR2 central domain residues 2460-2495 recapitulates this arrhythmogenic RyR2 leakiness by unzipping N-terminal and central domains. Calmodulin (CaM) and FK506-binding protein (FKBP12.6) bind to RyR2 and stabilize the closed channel. Little is known about DPc10 binding to the RyR2 and how that may interact with binding (and effects) of CaM and FKBP12.6 to RyR2.
  
  
  OBJECTIVE
  To measure, directly in cardiac myocytes, the kinetics and binding affinity of DPc10 to RyR2 and how that affects RyR2 interaction with FKBP12.6 and CaM.
  
  
  RESULTS
  We used permeabilized rat ventricular myocytes and fluorescently labeled DPc10, FKBP12.6, and CaM. DPc10 access to its binding site is extremely slow in resting RyR2 but is accelerated by promoting RyR opening or unzipping (by unlabeled DPc10). RyR2-bound CaM (but not FKBP12.6) drastically slowed DPc10 binding. Conversely, DPc10 binding significantly reduced CaM (but not FKBP12.6) binding to the RyR2. Fluorescence resonance energy transfer measurements indicate that DPc10-binding and CaM-binding sites are separate and allow triangulation of the structural DPc10 binding locus on RyR2 vs FKBP12.6-binding and CaM-binding sites.
  
  
  CONCLUSIONS
  DPc10-RyR2 binding is sterically limited by the resting zipped RyR2 state. CaM binding to RyR2 stabilizes this zipped state, whereas RyR2 activation or prebound DPc10 enhances DPc10 access. DPc10-binding and CaM-binding sites are distinct but are allosterically interacting RyR2 sites. Neither DPc10 nor FKBP12.6 influences RyR2 binding of the other.
authors.affiliation: Department of Pharmacology, University of California, Davis, CA 95616, USA.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Oda
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.forename: Florentin R
authors.initials: FR
authors.lastname: Nitu
authors.forename: Bengt
authors.initials: B
authors.lastname: Svensson
authors.forename: Xiyuan
authors.initials: X
authors.lastname: Lu
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Fruen
authors.forename: Razvan L
authors.initials: RL
authors.lastname: Cornea
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006333:Heart Failure / Q000378:metabolism; D015640:Ion Channel Gating; D007700:Kinetics; D018613:Microscopy, Confocal; D008958:Models, Molecular; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000378:metabolism*; D022021:Tacrolimus Binding Proteins / Q000378:metabolism
chemical_list: D002147:Calmodulin; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; C451408:ryanodine receptor calcium release channel (2460-2495), synthetic; D022021:Tacrolimus Binding Proteins; C403484:tacrolimus binding protein 1B