The calcium release channel of sarcoplasmic reticulum is modulated by FK-506 binding protein: Effect of FKBP-12 on single channel activity of the skeletal muscle ryanodine receptor

Our results show that FKBP12.6 binds to RyR1 and RyR2 in the same orientation and suggest new insights into the discrete structural domains responsible for channel binding and inhibition. FRET mapping of RyR-bound FKBP12.6 is consistent with the predictions of a previous cryoelectron microscopy study and strongly supports the proposed structural model. The 2.3-MDa ryanodine receptor (RyR)2 /Ca 2ϩ release channel isoforms expressed in skeletal muscle (RyR1) and cardiac muscle (RyR2) function in complex with smaller regulatory proteins, which include FK506-binding proteins (FKBP12 and FKBP12.6) and calmodulin (CaM) (1, 2). The FKBPs tightly bind to RyR channels and may function to stabilize channels in a fully closed conformational state while minimizing the occurrence of subconductance states (3, 4).Disruption of FKBP binding to RyRs has been proposed to underlie increased channel openings in response to ␤-adrenergic stimulation (5), oxidation/nitrosylation (6 -8), or diseasecausing channel mutations (9 -11), and the FKBP binding interface is now under investigation as a therapeutic target for disordered Ca 2ϩ regulation in cardiac and skeletal muscle. However, the fundamental cellular and molecular mechanisms that govern FKBP binding remain poorly defined, and the significance of reduced FKBP binding in RyR channelopathies is controversial (12-16). Because no atomic structure of an FKBP⅐RyR complex is currently available, uncertainty regarding the specific structural domains that comprise the binding interface remains a significant gap in understanding.Molecular determinants of FKBP binding have been investigated previously through mutagenesis of FKBP12 and FKBP12.6 (17,18). However, the key determinants thus far identified are broadly distributed throughout the FKBP threedimensional structure and do not provide a clear indication of a major RyR binding interface. Cryoelectron microscopy (cryo-EM) and single-particle three-dimensional reconstruction of RyRs in the absence and presence of FKBP12/12.6 have demonstrated that the FKBPs bind within a pocket formed by the intersection of domains 3 and 9 of the RyR1 and RyR2 cytoplasmic assemblies (19,20). More recently, Samsó et al. (21) advanced this approach to higher resolution and were able to dock the atomic structure of FKBP12 into the three-dimensional difference map of FKBP12 in a unique orientation. These results suggested a distinct mode of binding, in which the surface formed by the ␤-sheet and adjacent loops of FKBP12 forms a major binding interface with domain 9 of the RyR1 channel, and the hydrophobic drug-binding pocket of FKBP12 faces RyR1 domain 3.Here, we extend an approach (22) based on site-directed labeling of channel regulatory proteins and fluorescence resonance energy transfer (FRET) to further investigate the structural basis of FKBP binding to RyR channels. We identify structural determinants of both high affinity binding and RyR inhibition. We define the orientation of FKBP12.6 when bound to both the RyR1 and the RyR2 channel isoforms. Ou...

“…The FKBPs tightly bind to RyR channels and may function to stabilize channels in a fully closed conformational state while minimizing the occurrence of subconductance states (3,4).…”

Section: The 23-mda Ryanodine Receptor (Ryr)mentioning

confidence: 99%

Mapping the Ryanodine Receptor FK506-binding Protein Subunit Using Fluorescence Resonance Energy Transfer

Cornea

Nitu

Samsó

et al. 2010

“…In lipid bilayers, RyR1 is profoundly activated by the depletion of FKBP12 by using FK506 or rapamycin, as evidenced by a greater mean open probability due to longer mean open time and dominant sub-conductance states (13,(27)(28)(29)(30). These drugs also alter the sensitivity of RyR1 to effectors and affect full gating properties (13,16,25,27,28).…”

mentioning

confidence: 99%

N-terminal Region of FKBP12 Is Essential for Binding to the Skeletal Ryanodine Receptor

Lee

Rho

Kwon

et al. 2004

It is known that the two types of FK506-binding proteins FKBP12 and FKBP12.6 are tightly associated with the skeletal (RyR1) and cardiac ryanodine receptors (RyR2), respectively, and their interactions are important for channel functions of the RyR. In the case of cardiac muscle, three amino acid residues (Gln-31, Asn-32, and Phe-59) of FKBP12.6 could be essential for the selective binding to RyR2 (Xin, H. B., Rogers, K., Qi, Y., Kanematsu, T., and Fleischer, S. (1999) J. Biol. Chem. 274, 15315-15319). In this study to identify amino acid residues of FKBP12 that are important for the selective binding to RyR1, we mutated 9 amino acid residues of FKBP12 that differ from the counterparts of FKBP12.6 (Q3E, R18A, E31Q, D32N, M49R, R57A, W59F, H94A, and K105A), and we examined binding properties of these mutants to RyR1 by in vitro binding assay by using glutathione S-transferase-fused proteins of the mutants and Triton X-100-solubilized, FKBP12-depleted rabbit skeletal sarcoplasmic reticulum vesicles. Among the nine mutants tested, only Q3E and R18A lost their selective binding ability to RyR1. Furthermore, co-immunoprecipitation of RyR1 with 33 various mutants for the 9 positions produced by introducing different size, charge, and hydrophobicity revealed that an integration of the hydrogen bonds by the irreplaceable Gln-3 and the hydrophobic interactions by the residues Arg-18 and Met-49 could be a possible mechanism for the binding of FKBP12 to RyR1. Therefore, these results suggest that the N-terminal regions of FKBP12 (Gln-3 and Arg-18) and Met-49 are essential and unique for binding of FKBP12 to RyR1 in skeletal muscle.

“…Furthermore it has been recently suggested that FKBP12 may be responsible for cooperative gating between neighboring channels (14). The RyR1 complex deficient of FKBP12 appears to be more sensitive to activation by caffeine and Ca 2ϩ (10,15) and enhances the sensitivity of fibers to depolarization and caffeine (16).…”

mentioning

confidence: 99%

Bastadin 10 Stabilizes the Open Conformation of the Ryanodine-sensitive Ca2+ Channel in an FKBP12-dependent Manner

Chen

Molinski

Pessah

1999