Ryanodine receptor binding to FKBP12 is modulated by channel activation state.
Ryanodine receptor (RyR) Ca2+ release channels undergo a conformational change between the open and closed states. Its protein modulator, FK506 binding protein 12 (FKBP12), stabilises the channel gating between the four subunits that surround a central Ca2+-conducting pore. To understand the interdependence of RyR and FKBP12 binding, physiological and pharmacological agents were used to modulate the RyR open/closed state. ELISA sandwich binding assays showed that FKBP12 binding was dependent on the free Ca2+ and was lower at 1-10 microM free Ca2+ compared with 1 mM EGTA and 1 mM Ca2+, and this effect was enhanced by the inclusion of 1 mM ATP. Ruthenium red increased the binding of FKBP12. [3H]Ryanodine binding confirmed that 1 mM EGTA, 1 mM Ca2+ and 1 microM ruthenium red closed the channel, whereas 1 microM free Ca2+, 1 microM free Ca2+ + 1 mM ATP, or 10 mM caffeine opened it. These binding conditions were used in surface plasmon resonance studies to measure equilibrium binding kinetics. The affinity constant KA was significantly greater for the closed than the open channel, a change mediated by a decreased dissociation rate constant, kd. The results show that surface plasmon resonance is a powerful technique that can measure differences in RyR1 equilibrium binding kinetics with FKBP12.
Jones
Jonathan-Lee
JL
Wales Heart Research Institute, Department of Cardiology, School of Medicine, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK.
Reynolds
Deborah F
DF
Lai
F Anthony
FA
Blayney
Lynda M
LM
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D012430:Ruthenium Red / Q000494:pharmacology; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D022061:Tacrolimus Binding Protein 1A / Q000172:deficiency / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012430:Ruthenium Red; D012433:Ryanodine; D002110:Caffeine; D022061:Tacrolimus Binding Protein 1A