Overexpression of FK-506–Binding Protein 12.0 Modulates Excitation–Contraction Coupling in Adult Rabbit Ventricular Cardiomyocytes

Seidler, Tim; Loughrey, Christopher M.; Zibrova, Darya; Kettlewell, Sarah; Teucher, Nils; Kögler, Harald; Hasenfuß, Gerd; Smith, Godfrey L.

doi:10.1161/circresaha.107.154609

Cited by 29 publications

(25 citation statements)

References 20 publications

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“…Previous studies have documented important differences in the affinity and functional activity of FKBP12 and FKBP12.6 (17,35,36). These differences are probably a function of both the FKBP isoform and the RyR isoform, and understanding these differences remains an important goal.…”

Section: Discussionmentioning

confidence: 99%

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

Cornea

Nitu

Samsó

et al. 2010

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 99%

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

Cornea

Nitu

Samsó

et al. 2010

Journal of Biological Chemistry

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“…Recently, FKBP12.0 was acutely overexpressed in isolated adult rabbit cardiac myocytes by adenoviral gene transfer (809). A threefold FKBP12.0 overexpression increased SR Ca 2ϩ content similar to FKBP12.6 (809). This acute gene transfer model also uncovered several functional differences between FKBP isoforms.…”

Section: Fkbp126/calstabin2mentioning

confidence: 93%

“…This mouse model implicates a key role for FKBP12.0 in the early functioning myocardium, but the physiological function of FKBP12.0 versus FKBP12.6 in the adult heart remains unclear. Recently, FKBP12.0 was acutely overexpressed in isolated adult rabbit cardiac myocytes by adenoviral gene transfer (809). A threefold FKBP12.0 overexpression increased SR Ca 2ϩ content similar to FKBP12.6 (809).…”

Section: Fkbp126/calstabin2mentioning

confidence: 99%

Designing Heart Performance by Gene Transfer

Davis¹,

Westfall²,

Townsend³

et al. 2008

Physiological Reviews

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The birth of molecular cardiology can be traced to the development and implementation of high-fidelity genetic approaches for manipulating the heart. Recombinant viral vector-based technology offers a highly effective approach to genetically engineer cardiac muscle in vitro and in vivo. This review highlights discoveries made in cardiac muscle physiology through the use of targeted viral-mediated genetic modification. Here the history of cardiac gene transfer technology and the strengths and limitations of viral and nonviral vectors for gene delivery are reviewed. A comprehensive account is given of the application of gene transfer technology for studying key cardiac muscle targets including Ca2+handling, the sarcomere, the cytoskeleton, and signaling molecules and their posttranslational modifications. The primary objective of this review is to provide a thorough analysis of gene transfer studies for understanding cardiac physiology in health and disease. By comparing results obtained from gene transfer with those obtained from transgenesis and biophysical and biochemical methodologies, this review provides a global view of cardiac structure-function with an eye towards future areas of research. The data presented here serve as a basis for discovery of new therapeutic targets for remediation of acquired and inherited cardiac diseases.

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“…However, isolated RyR2 channels from FKBP12-knockout mice display sub-conductance behaviour (Shou et al, 1998). Furthermore, adenovirus-mediated FKBP12 overexpression in isolated cardiomyocytes reduces excitation-contraction coupling gain and decreases Ca 2+ spark frequency (Seidler et al, 2007). Assuming that a cardiac-specific FKBP12 post-translational modification is required for RyR2 interaction would explain the latter two studies.…”

Section: Ryr-fkbp Association In Heart 1765mentioning

confidence: 98%

“…FKBP12-deficient mice have severe cardiac defects and die in utero or shortly after birth, whereas RyR2 channels isolated from these mice display sub-conductance behaviour (Shou et al, 1998). Adenovirusmediated FKBP12 overexpression in isolated cardiomyocytes increases SR Ca 2+ content and decreases Ca 2+ spark frequency (Seidler et al, 2007). However, FKBP12 fails to modulate recombinant RyR2 in heterologous expression systems (George et al, 2003;Goonasekera et al, 2005) or in permeabilised cardiomyocytes (Guo et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Disparate Ryanodine Receptor Association with the FK506-binding Proteins in Mammalian Heart

Zissimopoulos¹,

Seifan²,

Maxwell³

et al. 2012

Journal of Cell Science

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SummaryThe FK506-binding proteins (FKBP12 and FKBP12.6; also known as FKBP1A and FKBP1B, respectively) are accessory subunits of the ryanodine receptor (RyR) Ca 2+ release channel. Aberrant RyR2-FKBP12.6 interactions have been proposed to be the underlying cause of channel dysfunction in acquired and inherited cardiac disease. However, the stoichiometry of the RyR2 association with FKBP12 or FKBP12.6 in mammalian heart is currently unknown. Here, we describe detailed quantitative analysis of cardiac stoichiometry between RyR2 and FKBP12 or FKBP12.6 using immunoblotting and [3 H]ryanodine-binding assays, revealing striking disparities between four mammalian species. In mouse and pig heart, RyR2 is found complexed with both FKBP12 and FKBP12.6, although the former is the most abundant isoform. In rat heart, RyR2 is predominantly associated with FKBP12.6, whereas in rabbit it is associated with FKBP12 only. Co-immunoprecipitation experiments demonstrate RyR2-specific interaction with both FKBP isoforms in native cardiac tissue. Assuming four FKBP-binding sites per RyR2 tetramer, only a small proportion of available sites are occupied by endogenous FKBP12.6. FKBP interactions with RyR2 are very strong and resistant to drug (FK506, rapamycin and cyclic ADPribose) and redox (H 2 O 2 and diamide) treatment. By contrast, the RyR1-FKBP12 association in skeletal muscle is readily disrupted under oxidative conditions. This is the first study to directly assess association of endogenous FKBP12 and FKBP12.6 with RyR2 in native cardiac tissue. Our results challenge the widespread perception that RyR2 associates exclusively with FKBP12.6 to near saturation, with important implications for the role of the FK506-binding proteins in RyR2 pathophysiology and cardiac disease.

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Overexpression of FK-506–Binding Protein 12.0 Modulates Excitation–Contraction Coupling in Adult Rabbit Ventricular Cardiomyocytes

Cited by 29 publications

References 20 publications

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

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

Designing Heart Performance by Gene Transfer

Disparate Ryanodine Receptor Association with the FK506-binding Proteins in Mammalian Heart

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