Kinetics of FKBP12.6 binding to ryanodine receptors in permeabilized cardiac myocytes and effects on Ca sparks.

Guo T, Cornea RL, Huke S, Camors E, Yang Y, Picht E, Fruen BR, Bers DM
Circ Res. 2010 106 (11): 1743-52

PMID: 20431056 · PMCID: PMC2895429 · DOI:10.1161/CIRCRESAHA.110.219816

RATIONALE - FK506-binding proteins FKBP12.6 and FKBP12 are associated with cardiac ryanodine receptors (RyR2), and cAMP-dependent protein kinase A (PKA)-dependent phosphorylation of RyR2 was proposed to interrupt FKBP12.6-RyR2 association and activate RyR2. However, the function of FKBP12.6/12 and role of PKA phosphorylation in cardiac myocytes are controversial.

OBJECTIVE - To directly measure in situ binding of FKBP12.6/12 to RyR2 in ventricular myocytes, with simultaneous Ca sparks measurements as a RyR2 functional index.

METHODS AND RESULTS - We used permeabilized rat and mouse ventricular myocytes, and fluorescently-labeled FKBP12.6/12. Both FKBP12.6 and FKBP12 concentrate at Z-lines, consistent with RyR2 and Ca spark initiation sites. However, only FKBP12.6 inhibits resting RyR2 activity. Assessment of fluorescent FKBP binding in myocyte revealed a high FKBP12.6-RyR2 affinity (K(d)=0.7+/-0.1 nmol/L) and much lower FKBP12-RyR2 affinity (K(d)=206+/-70 nmol/L). Fluorescence recovery after photobleach confirmed this K(d) difference and showed that it is mediated by k(off). RyR2 phosphorylation by PKA did not alter binding kinetics or affinity of FKBP12.6/12 for RyR2. Using quantitative immunoblots, we determined endogenous [FKBP12] in intact myocytes is approximately 1 micromol/L (similar to [RyR]), whereas [FKBP12.6] is
CONCLUSIONS - Only 10% to 20% of endogenous myocyte RyR2s have FKBP12.6 associated, but virtually all myocyte FKBP12.6 is RyR2-bound (because of very high affinity). FKBP12.6 but not FKBP12 inhibits basal RyR2 activity. PKA-dependent RyR2 phosphorylation has no significant effect on binding of either FKBP12 or 12.6 to RyR2 in myocytes.

MeSH Terms (26)

Animals Blotting, Western Calcium Signaling Cell Membrane Permeability Circular Dichroism Cyclic AMP Cyclic AMP-Dependent Protein Kinases Fluorescence Recovery After Photobleaching Heart Ventricles Humans Kinetics Mice Mice, Knockout Microscopy, Confocal Mutagenesis, Site-Directed Mutation Myocytes, Cardiac Phosphorylation Protein Binding Rats Ryanodine Receptor Calcium Release Channel Sarcoplasmic Reticulum Sirolimus Swine Tacrolimus Binding Protein 1A Tacrolimus Binding Proteins

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