A mechanism of ryanodine receptor modulation by FKBP12/12.6, protein kinase A, and K201

Aims Our objective was to explore the functional interdependence of protein kinase A (PKA) phosphorylation with binding of modulatory FK506 binding proteins (FKBP12/12.6) to the ryanodine receptor (RyR). RyR type 1 or type 2 was prepared from rabbit skeletal muscle or pig cardiac muscle, respectively. In heart failure, RyR2 dysfunction is implicated in fatal arrhythmia and RyR1 dysfunction is associated with muscle fatigue. A controversial underlying mechanism of RyR1/2 dysfunction is proposed to be hyperphosphorylation of RyR1/2 by PKA, causing loss of FKBP12/12.6 binding that is reversible by the experimental inhibitory drug K201 (JTV519). Phosphorylation is also a trigger for fatal arrhythmia in catecholaminergic polymorphic ventricular tachycardia associated with point mutations in RyR2. Methods and results Equilibrium binding kinetics of RyR1/2 to FKBP12/12.6 were measured using surface plasmon resonance (Biacore). Free Ca2+ concentration was used to modulate the open/closed conformation of RyR1/2 channels measured using [H-3] ryanodine binding assays. The affinity constant-K-A, for RyR1/2 binding to FKBP12/12.6, was significantly greater for the closed compared with the open conformation. The effect of phosphorylation or K201 was to reduce the K-A of the closed conformation by increasing the rate of dissociation k(d). K201 reduced [H-3] ryanodine binding to RyR1/2 at all free Ca2+ concentrations including PKA phosphorylated preparations. Conclusion The results are explained through a model proposing that phosphorylation and K201 acted similarly to change the conformation of RyR1/2 and regulate FKBP12/12.6 binding. K201 stabilized the conformation, whereas phosphorylation facilitated a subsequent molecular event that might increase the rate of an open/closed conformational transition.