FRET-based mapping of calmodulin bound to the RyR1 Ca
            <sup>2+</sup>
            release channel

Cornea, Rǎzvan L.; Nitu, Florentin R.; Gruber, Simon; Kohler, Katherine; Satzer, Michael; Thomas, David D.; Fruen, Bradley R.

doi:10.1073/pnas.0813010106

Cited by 56 publications

(91 citation statements)

References 27 publications

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“…This assumption is well supported by the disposition of FKBP and CaM subunits in cryo-EM structures (19), by our own previous analysis (22), and by the donor-acceptor distances derived in this study (Table 1). A schematic is included to illustrate the predicted distance relationships among multiple donors and acceptors bound within an ordered array of RyR channels, in the context of the limited range of distances over which FRET may occur (supplemental Fig.…”

Section: Methodssupporting

confidence: 66%

“…The FKBP12 isoform is more widely and abundantly expressed and copurifies with the RyR1 channel from mammalian skeletal muscle, where FKBP12.6 is absent. However, in vitro FKBP12.6 binds with higher affinity and selectivity to both the RyR1 and RyR2 channels (17) and thus provides an ideal means of targeting fluorescent probes to each of the two channel isoforms (22).…”

Section: Resultsmentioning

confidence: 99%

“…Bound Alexa 555-labeled FKBP12 was determined from the integrated fluorescence intensity from 563 to 591 nm, acquired using a Gemini EM microplate fluorometer (Molecular Devices, Sunnyvale, CA) with excitation at 542 nm and a 550-nm emission long pass filter (no contribution of Alexa 488-labeled FKBPs to the fluorescence signal). FRET Measurements and Calculation of Donor-Acceptor Distances-FRET between RyR-bound F-FKBPs and F-CaM was measured as described previously (22) in KCl/PIPES buffers containing 30 M Ca 2ϩ . SR membranes (0.4 mg/ml) were preincubated with 50 nM Alexa 488-labeled FKBP12.6 (donor) for 90 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

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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: Methodssupporting

confidence: 66%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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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“…Difference mapping of threedimensional reconstructions of RyR with and without FKBP12 or 12.6 places the FKBPs binding site between subdomains 3, 5, and 9 (Wagenknecht et al 1996;Wagenknecht et al 1997;Samsó et al 2006;Sharma et al 2006). In agreement with this localization, FRET studies have localized the FKBP12 (Cornea et al 2009) and the FKBP12.6 (Cornea et al 2010) binding site to the same area as the model from Samsó et al 2006. Furthermore, both FKBP12 and 12.6 bind RyR1 and RyR2 in the same orientation (Cornea et al 2010).…”

Section: Calsequestrinsupporting

confidence: 64%

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

Lanner¹,

Georgiou²,

Joshi³

et al. 2010

Cold Spring Harbor Perspectives in Biology

656

572

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Ryanodine receptors (RyRs) are located in the sarcoplasmic/endoplasmic reticulum membrane and are responsible for the release of Ca 2þ from intracellular stores during excitation-contraction coupling in both cardiac and skeletal muscle. RyRs are the largest known ion channels (.2MDa) and exist as three mammalian isoforms (RyR 1 -3), all of which are homotetrameric proteins that interact with and are regulated by phosphorylation, redox modifications, and a variety of small proteins and ions. Most RyR channel modulators interact with the large cytoplasmic domain whereas the carboxy-terminal portion of the protein forms the ion-conducting pore. Mutations in RyR2 are associated with human disorders such as catecholaminergic polymorphic ventricular tachycardia whereas mutations in RyR1 underlie diseases such as central core disease and malignant hyperthermia. This chapter examines the current concepts of the structure, function and regulation of RyRs and assesses the current state of understanding of their roles in associated disorders.

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“…A single‐cysteine mutant of the human small immunophillin FK‐506–binding FKBP12.6 isoform (T14C‐FKBP12.6) was labeled by the thiol‐specific maleimide derivative of Alexa Fluor ® 568 (Invitrogen, Carlsbad, CA), as previously described 20. This F‐FKBP binds to and regulates RyRs in SR vesicles and permeabilized myocytes such as wild‐type FKBP12.6 9…”

Section: Methodsmentioning

confidence: 99%

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

Galice

Xie

Yang

et al. 2018

JAHA

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BackgroundRyanodine receptors (RyR) mediate sarcoplasmic reticulum calcium (Ca2+) release and influence myocyte Ca2+ homeostasis and arrhythmias. In cardiac myocytes, RyRs are found in clusters of various sizes and shapes, and RyR cluster size may critically influence normal and arrhythmogenic Ca2+ spark and wave formation. However, the actual RyR cluster sizes at specific Ca2+ spark sites have never been measured in the physiological setting.Methods and ResultsHere we measured RyR cluster size and Ca2+ sparks simultaneously to assess how RyR cluster size influences Ca2+ sparks and sarcoplasmic reticulum Ca2+ leak. For small RyR cluster sizes (<50), Ca2+ spark frequency is very low but then increases dramatically at larger cluster sizes. In contrast, Ca2+ spark amplitude is nearly maximal even at relatively small RyR cluster size (≈10) and changes little at larger cluster size. These properties agreed with computational simulations of RyR gating within clusters.ConclusionsOur study explains how this combination of properties may limit arrhythmogenic Ca2+ sparks and wave propagation (at many junctions) while preserving the efficacy and spatial synchronization of Ca2+‐induced Ca2+‐release during normal excitation‐contraction coupling. However, variations in RyR cluster size among individual junctions and RyR sensitivity could exacerbate heterogeneity of local sarcoplasmic reticulum Ca2+ release and arrhythmogenesis under pathological conditions.

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FRET-based mapping of calmodulin bound to the RyR1 Ca ²⁺ release channel

Cited by 56 publications

References 27 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

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

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

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FRET-based mapping of calmodulin bound to the RyR1 Ca 2+ release channel

Cited by 56 publications

References 27 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

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

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

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FRET-based mapping of calmodulin bound to the RyR1 Ca ²⁺ release channel