This study explores the functional relationship between protein kinase A (PKA) phosphorylation and binding of FK506 binding proteins (FKBP12/12.6) to the ryanodine receptor (RyR). The results show that the affinity of RyR1/2 binding to FKBP12/12.6 is greater when the channel is closed versus open. Phosphorylation and the drug K201 reduced this affinity by increasing the dissociation rate, shifting RyR to an open state. The findings support a model where phosphorylation and K201 similarly alter RyR conformation and regulate FKBP12/12.6 binding.

1. 11/15/13 A mechanism of ryanodine receptor modulation ... [Cardiovasc Res. 2010] - PubMed - NCBI
www.ncbi.nlm.nih.gov/pubmed/19661110 1/1
AIMS:
METHODS AND RESULTS:
CONCLUSION:
Cardiovasc Res. 2010 Jan 1;85(1):68-78. doi: 10.1093/cvr/cvp273.
A mechanism of ryanodine receptor modulation by FKBP12/12.6, protein
kinase A, and K201.
Blayney LM, Jones JL, Griffiths J, Lai FA.
Department of Medicine - Cardiology, Wales Heart Research Institute, Cardiff University, Heath Park, Cardiff CF14
4XN, UK. blayney@cf.ac.uk
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.
PMID: 19661110 [PubMed - indexed for MEDLINE] Free full text
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