Ryanodine Receptor Phosphorylation by Protein Kinase a Alters the Affinity for FKBP12

Abstract

The Ryanodine Receptor (RyR) ion channel cells plays a vital part in muscle cell Excitation Contraction Coupling by regulating sarcoplasmic reticulum Ca2+ release - a process modulated by interaction with FK506 Binding Protein 12 (FKBP12). Pathological hyperphosphorylation of RyR1 by Protein Kinase A (PKA) is reported by some to cause the loss of FKBP12 in excessive exercise and in muscle fatigue reported in heart failure. The physiological relationship between FKBP12 and phosphorylation in channel regulation is uncertain. We have shown previously, using Surface Plasmon Resonance (SPR) that the open channel has less affinity (measured as KA) for FKBP12, than the closed channel. In this study the effect of phosphorylation on the binding of RyR1 to FKBP12 in the open or closed state was tested. Phosphorylation by PKA was measured by Western blot. SPR results showed that KA, for FKBP12 of RyR1 in the closed states (EGTA) was significantly reduced by both phosphorylation and K201 to the values of the open state of the channel (1μM Ca2+). This resulted from an increase in kd, with the rate of association (ka) remaining constant. In [3H]Ryanodine binding assays, FKBP12 and K201individually decreased the open probability (Po) of the channel by about 40% whether the channel was phosphorylated or not and there was no additive effect. Phosphorylation had no effect on the Ca2+ dose response curve whereas K201 significantly reduced the Po. Both phosphorylation and K201 reversed the effects of Mg2+ inhibition. The results are explained through a model which proposes that both phosphorylation and K201 acted similarly to change the conformation of RyR1, but K201 stabilised the conformation whereas phosphorylation facilitated a subsequent molecular event that triggered channel opening.