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

Abstract

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. Equilibrium binding kinetics of RyR1/2 to FKBP12/12.6 were measured using surface plasmon resonance (Biacore). Free Ca(2+) concentration was used to modulate the open/closed conformation of RyR1/2 channels measured using [(3)H]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 [(3)H]ryanodine binding to RyR1/2 at all free Ca(2+) concentrations including PKA phosphorylated preparations. 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.