Ligand-dependent Conformational Changes in the Clamp Region of the Cardiac Ryanodine Receptor

Tian, Xixi; Liu, Yingjie; Liu, Ying; Wang, Ruiwu; Wagenknecht, Terence; Liu, Zheng; Chen, S. R. Wayne

doi:10.1074/jbc.m112.427864

Cited by 14 publications

(13 citation statements)

References 53 publications

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“…For example, fluorescent protein (FP) fusions have been used to target both donor and acceptor fluorophores to the ryanodine receptor [6], [7], [8]. While these proteins can be inserted with essentially 100% labeling efficiency, their bulky size can disrupt the native protein structure or function, which in turn limits the number of potential insertion sites.…”

Section: Resultsmentioning

confidence: 99%

“…For example, the position and orientation of key regulatory proteins on the RyR such as FK506 binding protein 12 kDa (FKBP12) and calmodulin (CaM) have been elucidated using FRET measurements [3], [4], [5]. In addition, small changes in FRET efficiencies between fluorescent proteins fused into the RyR have been observed that may reflect agonist-induced conformational changes within the RyR [6], [7], [8].…”

Section: Introductionmentioning

confidence: 99%

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Site-Specific Labeling of the Type 1 Ryanodine Receptor Using Biarsenical Fluorophores Targeted to Engineered Tetracysteine Motifs

Fessenden

Mahalingam

2013

PLoS ONE

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The type 1 ryanodine receptor (RyR1) is an intracellular Ca2+ release channel that mediates skeletal muscle excitation contraction coupling. While the overall shape of RyR1 has been elucidated using cryo electron microscopic reconstructions, fine structural details remain elusive. To better understand the structure of RyR1, we have previously used a cell-based fluorescence resonance energy transfer (FRET) method using a fused green fluorescent protein (GFP) donor and a fluorescent acceptor, Cy3NTA that binds specifically to short poly-histidine ‘tags’ engineered into RyR1. However, the need to permeabilize cells to allow Cy3NTA entry as well as the noncovalent binding of Cy3NTA to the His tag limits future applications of this technique for studying conformational changes of the RyR. To overcome these problems, we used a dodecapeptide sequence containing a tetracysteine (Tc) motif to target the biarsenical fluorophores, FlAsH and ReAsH to RyR1. These compounds freely cross intact cell membranes where they then bind covalently to the tetracysteine motif. First, we used this system to conduct FRET measurements in intact cells by fusing a yellow fluorescent protein (YFP) FRET donor to the N-terminus of RyR1 and then targeting the FRET acceptor, ReAsH to an adjacent Tc tag. Moderate energy transfer (∼33%) was observed whereas ReAsH incubation of a YFPRyR1 fusion protein lacking the Tc tag resulted in no detectable FRET. We also developed a FRET-based system that did not require RyR fluorescent protein fusions by labeling N-terminal Tc-tagged RyR1 with FlAsH, a FRET donor and then targeting the FRET acceptor Cy3NTA to an adjacent decahistidine (His10) tag. A high degree of energy transfer (∼66%) indicated proper binding of both compounds to these unique recognition sequences in RyR1. Thus, these two systems should provide unprecedented flexibility in future FRET-based structural determinations of RyR1.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Site-Specific Labeling of the Type 1 Ryanodine Receptor Using Biarsenical Fluorophores Targeted to Engineered Tetracysteine Motifs

Fessenden

Mahalingam

2013

PLoS ONE

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“…RyR activation is induced either by direct interaction with DHPR (for RyR1), by a small amount of Ca 2+ influx from DHPR (for RyR2) or by Ca 2+ released from SR stores through nearby RyRs (RyR1 and RyR2). Global conformational changes underlie RyR activation, including changes in the clamp region (Tian et al, 2013), in the transmembrane region that result in ion channel activation (Samsó et al, 2009), and, as we show here for the first time, in the CaM-binding region of RyR1. Upon RyR activation the two fluorophore-labeled sequences move apart, a structural change that is likely to be influenced by CaM.…”

Section: Conformational Changes In the Cam-binding Region Of Ryr And mentioning

confidence: 85%

Two potential calmodulin-binding sequences in the ryanodine receptor contribute to a mobile, intra-subunit calmodulin-binding domain

Huang

Liu

Wang

et al. 2013

Journal of Cell Science

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SummaryCalmodulin (CaM), a 16 kDa ubiquitous calcium-sensing protein, is known to bind tightly to the calcium release channel/ryanodine receptor (RyR), and modulate RyR function. CaM binding studies using RyR fragments or synthetic peptides have revealed the presence of multiple, potential CaM-binding regions in the primary sequence of RyR. In the present study, we inserted GFP into two of these proposed CaM-binding sequences and mapped them onto the three-dimensional structure of intact cardiac RyR2 by cryo-electron microscopy. Interestingly, we found that the two potential CaM-binding regions encompassing, Arg3595 and Lys4269, respectively, are in close proximity and are adjacent to the previously mapped CaM-binding sites. To monitor the conformational dynamics of these CaM-binding regions, we generated a fluorescence resonance energy transfer (FRET) pair, a dual CFP-and YFP-labeled RyR2 (RyR2 R3595-CFP/K4269-YFP ) with CFP inserted after Arg3595 and YFP inserted after Lys4269. We transfected HEK293 cells with the RyR2 R3595-CFP/K4269-YFP cDNA, and examined their FRET signal in live cells. We detected significant FRET signals in transfected cells that are sensitive to the channel activator caffeine, suggesting that caffeine is able to induce conformational changes in these CaMbinding regions. Importantly, no significant FRET signals were detected in cells co-transfected with cDNAs encoding the single CFP (RyR2 R3595-CFP ) and single YFP (RyR2 K4269-YFP ) insertions, indicating that the FRET signal stemmed from the interaction between R3595-CFP and K4269-YFP that are in the same RyR subunit. These observations suggest that multiple regions in the RyR2 sequence may contribute to an intra-subunit CaM-binding pocket that undergoes conformational changes during channel gating.

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“…Caffeine and chlorophenol derivatives, structurally different RyR agonists, are commonly used as pharmacological research tools and diagnostic drugs for MH (Figure A) (DiJulio et al ., ; Westerblad et al ., ; Baur et al ., ; Varadi and Rutter, ; Gerbershagen et al ., ). These two classes of structure may have different binding sites to RyRs (Tian et al ., ). The chlorophenol derivatives 4‐chloro‐3‐ethylphenol (4‐CEP) and 4‐chloro‐ m ‐cresol (4‐C m C) can induce a robust release of intracellular Ca 2+ by stimulating RyRs at concentrations about 10‐fold lower than caffeine (Islam et al ., ; Jacobson et al ., ), and have advantages over caffeine and ryanodine as diagnostic drugs for MH (Gerbershagen et al ., ).…”

Section: Introductionmentioning

confidence: 97%

The ryanodine receptor agonist 4‐chloro‐3‐ethylphenol blocks ORAI store‐operated channels

Zeng

Chen

Daskoulidou

et al. 2014

British J Pharmacology

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BACKGROUNDDepletion of the Ca 2+ store by ryanodine receptor (RyR) agonists induces store-operated Ca 2+ entry (SOCE). 4-Chloro-3-ethylphenol (4-CEP) and 4-chloro-m-cresol (4-CmC) are RyR agonists commonly used as research tools and diagnostic reagents for malignant hyperthermia. Here, we investigated the effects of 4-CEP and its analogues on SOCE. EXPERIMENTAL APPROACHSOCE and ORAI1-3 currents were recorded by Ca 2+ imaging and whole-cell patch recordings in rat L6 myoblasts and in HEK293 cells overexpressing STIM1/ORAI1-3. KEY RESULTS4-CEP induced a significant release of Ca 2+ in rat L6 myoblasts, but inhibited SOCE. The inhibitory effect was concentration-dependent and more potent than its analogues 4-CmC and 4-chlorophenol (4-ClP). In the HEK293 T-REx cells overexpressing STIM1/ORAI1-3, 4-CEP inhibited the ORAI1, ORAI2 and ORAI3 currents evoked by thapsigargin. The 2-APB-induced ORAI3 current was also blocked by 4-CEP. This inhibitory effect was reversible and independent of the Ca 2+ release. The two analogues, 4-CmC and 4-ClP, also inhibited the ORAI1-3 channels. Excised patch and intracellular application of 4-CEP demonstrated that the action site was located extracellularly. Moreover, 4-CEP evoked STIM1 translocation and subplasmalemmal clustering through its Ca 2+ store-depleting effect via the activation of RyR, but no effect on STIM1 redistribution was observed in cells co-expressing STIM1/ORAI1-3. CONCLUSION AND IMPLICATIONS4-CEP not only acts as a RyR agonist to deplete the Ca 2+ store and trigger STIM1 subplasmalemmal translocation and clustering, but also directly inhibits ORAI1-3 channels. These findings demonstrate a novel pharmacological property for the chlorophenol derivatives that act as RyR agonists. Abbreviations

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Ligand-dependent Conformational Changes in the Clamp Region of the Cardiac Ryanodine Receptor

Cited by 14 publications

References 53 publications

Site-Specific Labeling of the Type 1 Ryanodine Receptor Using Biarsenical Fluorophores Targeted to Engineered Tetracysteine Motifs

Site-Specific Labeling of the Type 1 Ryanodine Receptor Using Biarsenical Fluorophores Targeted to Engineered Tetracysteine Motifs

Two potential calmodulin-binding sequences in the ryanodine receptor contribute to a mobile, intra-subunit calmodulin-binding domain

The ryanodine receptor agonist 4‐chloro‐3‐ethylphenol blocks ORAI store‐operated channels

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