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First published online 21 September 2005
doi: 10.1242/jcs.02582

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Ryanodine receptor binding to FKBP12 is modulated by channel activation state

Wales Heart Research Institute, Department of Cardiology, School of Medicine, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK

^* Author for correspondence (e-mail: blayney{at}cf.ac.uk)

Accepted 13 July 2005

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 µM 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 µM ruthenium red closed the channel, whereas 1 µM free Ca²⁺, 1 µM 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 K_A was significantly greater for the closed than the open channel, a change mediated by a decreased dissociation rate constant, k_d. The results show that surface plasmon resonance is a powerful technique that can measure differences in RyR1 equilibrium binding kinetics with FKBP12.

Key words: Ryanodine receptor, FKBP12, Surface plasmon resonance, Channel activation state