Kinetics of FKBP12.6 Binding to Ryanodine Receptors in Permeabilized Cardiac Myocytes and Effects on Ca Sparks

Abstract

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 ...

Size Matters: Ryanodine Receptor Cluster Size Affects Arrhythmogenic Sarcoplasmic Reticulum Calcium Release

“…Adult Sprague‐Dawley rats were euthanitized, and their ventricular cardiac myocytes were isolated using previously described methods 9. Briefly, anesthesia (isoflurane, 5%) and heart excision were followed by 5 minutes of perfusion (37°C) with minimal essential medium (MEM, GIBCO Life Technologies) gassed with 95% O 2 /5% CO 2 before collagenase B (0.5 mg/mL, Boehringer Mannheim, Mannheim, Germany) and protease (0.02 mg/mL, Sigma, St. Louis, MO) were added to dissociate myocytes.…”

“…The 2D frame mode with 512×512‐pixel resolution was used. Due to trade‐offs of spatial versus temporal resolution, we used the same zoom factor for simultaneous Ca 2+ spark and initial F‐FKBP recordings 9. Afterward, we increased zoom factor and measured Z‐stack image of F‐FKBP fluorescence to refine analysis of RyR density and distribution.…”

“…Individual RyR cluster size can vary over a continuous distribution (from <10 up to 250 RyRs), but some smaller clusters are quite close to others (eg, at the same junction) and, because of their proximity (within 100 nm), may function as a single junctional cluster. This proximity explains how a small local Ca 2+ influx can activate multiple RyRs via Ca 2+ ‐induced Ca 2+ ‐release (CICR) during the normal action potential–induced Ca 2+ transient and also during diastolic Ca 2+ sparks 7, 8, 9, 10, 11. Ca 2+ sparks are stochastic local CICR events that are typically restricted spatially to 1 cluster/junction without propagation to nearest‐neighbor junctions spaced 1.8 to 1.9 μm longitudinally or 0.4 to 0.8 μm transversely 8, 9, 10, 11, 12.…”

“…We used our well‐characterized experimental system, which allows simultaneous confocal visualization in live myocytes of Ca 2+ sparks and the number of local RyRs 9. In this method we fluorescently label virtually all RyR monomers with FKBP12.6 that is covalently tagged with a small red fluorescent probe (F‐FKBP) in the same cell where Ca 2+ sparks are measured.…”

“…In this method we fluorescently label virtually all RyR monomers with FKBP12.6 that is covalently tagged with a small red fluorescent probe (F‐FKBP) in the same cell where Ca 2+ sparks are measured. We have previously quantified that F‐FKBP binds to RyR in situ with high affinity (K d <1 nmol/L) and ≈1:1 binding stoichiometry per RyR monomer, with only a slight influence on Ca 2+ spark properties 9. Thus, we can measure the relative number of RyRs at each site where Ca 2+ sparks occur.…”