Key Words: FKBP12.6 Ⅲ FKBP12 Ⅲ Ca sparks Ⅲ binding properties Ⅲ rapamycin Ⅲ RyR2 C ardiac ryanodine receptors (RyR2) are sarcoplasmic reticulum (SR) Ca release channels, crucial in excitation-contraction coupling. 1 Dysfunctional RyR2, exhibiting enhanced Ca leak has been implicated in arrhythmogenesis and heart failure (HF). 1 Homotetrameric RyR2s have a transmembrane channel domain, and regulatory and scaffolding cytoplasmic domain.In heart, FK506 binding protein (FKBP) isoforms FKBP12 and FKBP12.6 are coexpressed and can bind RyR2 at a stoichiometry of 4 FKBP per RyR tetramer. 2,3 FKBP12 binds RyR2 with much lower affinity, but is much higher in concentration in heart than FKBP12.6. 4 Nevertheless, FKBP12 and 12.6 share 85% sequence homology and similar 3D structures. 5 Human and rat FKBP12 differ in only 3 residues, and human and rat FKBP12.6 are identical. This makes study of human FKBP function in rat myocytes reasonable.Effects of FKBPs on RyR2 activity in myocytes are controversial. Some groups reported that dissociation of FKBP12.6 from RyR2 by immunosuppressants (rapamycin or FK506) activated RyR2 channels and induced subconductance states. 6 -8 RyR2 point mutations associated with cardiac sudden death may also exhibit altered FKBP12.6 interaction, 9 and FK506 can alter resting Ca 2ϩ spark frequency (CaSpF) and SR Ca 2ϩ content. 7,8,10 FKBP12.6 overexpression also increased SR load and enhanced contraction. 11 However, others reported that FKBP12.6 removal had no effect on RyR2 activity, 3,12 and failed to observe RyR2 subconductance states in channels from FKBP12.6 knockout mice. 13,14 Some groups suggest that FKBP12 affects RyR2 differently from FKBP12.6. [15][16][17]20 Therefore, FKBP12/12.6 binding and RyR2 effects remain unclear, especially in the cardiomyocyte environment (as studied here).Altered FKBP-RyR2 interaction is a prominent hypothesis explaining increased SR Ca leak in HF via RyR2 hyperphos- Here we characterize in the myocyte environment the interaction between FKBP12/12.6 and RyR2, its functional consequences, and modulation by PKA-dependent phosphorylation. Using fluorescent FKBP12/12.6 (F-FKBP), we simultaneously assessed physical association-dissociation of FKBP12/12.6 with RyR2, and RyR2 activity (via Ca sparks) in permeabilized ventricular myocytes. We found that: (1) both FKBP12.6 and FKBP12 bound to RyR2 (K d Ϸ1 nmol/L and 200 nmol/L, respectively); (2) FKBP12.6 but not FKBP12 inhibited CaSpF; (3) the binding properties of FKBP12.6/12-RyR2 were not changed by PKA-dependent phosphorylation of RyR2; and (4) endogenous FKBP12 was Ϸ1 mol/L cytosol and FKBP12.6 Ϸ100 nmol/L cytosol. MethodsRat and mouse ventricular myocytes were isolated and permeabilized as previously described (see the Online Data Supplement, available at http://circres.ahajournals.org). 23 F-FKBP12.6 and F-FKBP12 were characterized using circular dichroism spectroscopy and ligandbinding studies (Online Figure I). 24 Consistent with previous reports, 25 F-FKBP constructs have the same secondary structure, RyR ...

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beta3-Adrenoceptor is involved in altered positive inotropic response to beta-adrenoceptor stimulation in diabetic cardiomyopathy. This effect is mediated by NOS1-derived nitric oxide in diabetic cardiomyocyte.