title: Kinetics of FKBP12.6 binding to ryanodine receptors in permeabilized cardiac myocytes and effects on Ca sparks.
abstract: BACKGROUND
  FK506-binding proteins FKBP12.6 and FKBP12 are associated with cardiac ryanodine receptors (RyR2), and cAMP-dependent protein kinase A (PKA)-dependent phosphorylation of RyR2 was proposed to interrupt FKBP12.6-RyR2 association and activate RyR2. However, the function of FKBP12.6/12 and role of PKA phosphorylation in cardiac myocytes are controversial.
  
  
  OBJECTIVE
  To directly measure in situ binding of FKBP12.6/12 to RyR2 in ventricular myocytes, with simultaneous Ca sparks measurements as a RyR2 functional index.
  
  
  RESULTS
  We used permeabilized rat and mouse ventricular myocytes, and fluorescently-labeled FKBP12.6/12. Both FKBP12.6 and FKBP12 concentrate at Z-lines, consistent with RyR2 and Ca spark initiation sites. However, only FKBP12.6 inhibits resting RyR2 activity. Assessment of fluorescent FKBP binding in myocyte revealed a high FKBP12.6-RyR2 affinity (K(d)=0.7+/-0.1 nmol/L) and much lower FKBP12-RyR2 affinity (K(d)=206+/-70 nmol/L). Fluorescence recovery after photobleach confirmed this K(d) difference and showed that it is mediated by k(off). RyR2 phosphorylation by PKA did not alter binding kinetics or affinity of FKBP12.6/12 for RyR2. Using quantitative immunoblots, we determined endogenous [FKBP12] in intact myocytes is approximately 1 micromol/L (similar to [RyR]), whereas [FKBP12.6] is <or=150 nmol/L.
  
  
  CONCLUSIONS
  Only 10% to 20% of endogenous myocyte RyR2s have FKBP12.6 associated, but virtually all myocyte FKBP12.6 is RyR2-bound (because of very high affinity). FKBP12.6 but not FKBP12 inhibits basal RyR2 activity. PKA-dependent RyR2 phosphorylation has no significant effect on binding of either FKBP12 or 12.6 to RyR2 in myocytes.
authors.affiliation: Department of Pharmacology, University of California, Davis, 451 Health Science Dr, Davis, CA 95616, USA.
authors.forename: Tao
authors.initials: T
authors.lastname: Guo
authors.forename: Razvan L
authors.initials: RL
authors.lastname: Cornea
authors.forename: Sabine
authors.initials: S
authors.lastname: Huke
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Camors
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.forename: Eckard
authors.initials: E
authors.lastname: Picht
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Fruen
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
mesh_terms: D000818:Animals; D015153:Blotting, Western; D020013:Calcium Signaling / Q000187:drug effects; D002463:Cell Membrane Permeability; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D006352:Heart Ventricles / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D013552:Swine; D022061:Tacrolimus Binding Protein 1A / Q000235:genetics / Q000378:metabolism*; D022021:Tacrolimus Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019837:Ryanodine Receptor Calcium Release Channel; D020123:Sirolimus; D022061:Tacrolimus Binding Protein 1A; D022021:Tacrolimus Binding Proteins; C509259:FKBP1B protein, mouse; C403484:tacrolimus binding protein 1B