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 ≈1 μmol/L (similar to [RyR]), whereas [FKBP12.6] is ≤150 nmol/L.

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.