Tao Guo, Razvan L Cornea, Sabine Huke, Emmanuel Camors, Yi Yang, Eckard Picht, Bradley R Fruen, Donald M Bers
Department of Pharmacology, University of California, Davis, 451 Health Science Dr, Davis, CA 95616, USA.
Circulation research 2010 Jun 11FK506-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. 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. 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
Tao Guo, Razvan L Cornea, Sabine Huke, Emmanuel Camors, Yi Yang, Eckard Picht, Bradley R Fruen, Donald M Bers. Kinetics of FKBP12.6 binding to ryanodine receptors in permeabilized cardiac myocytes and effects on Ca sparks. Circulation research. 2010 Jun 11;106(11):1743-52
PMID: 20431056
View Full Text