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Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes.

Bhattacharya D, Mehle A, Kamp TJ, Balijepalli RC - PLoS ONE (2015)

Bottom Line: The dihydropyridine receptor (DHPR) β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1) complex is still debatable.Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP.The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Arrhythmia Research Program, Department of Medicine, University of Wisconsin-Madison, Wisconsin, United States of America.

ABSTRACT
The dihydropyridine receptor (DHPR) β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1) complex is still debatable. We used fluorescence resonance energy transfer (FRET) to probe proximity relationships within the β1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP) was used as the FRET donor. Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1) myotubes revealed that in only one construct (H458 CCPGCC β1a -CFP) FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

No MeSH data available.


Related in: MedlinePlus

FRET signals from ß1a subunits incorporating the CFP-CCPGCC concatamer in different domains or doubly transfected CFP and CCPGCC tags expressed in WT myotubes.(A) Myotubes co-transfected with CFP- ß1a (construct 6) and CCPGCC- ß1a (construct 7), constructs demonstrate unchanged FRET efficiency after FlAsH clearance with 7.5 mM EDT indicating that ß1a intermolecular interactions do not contribute to FRET in the present study. Myotubes individually transfected with the CFP-CCPGCC concatemers (B) M1 CFP-CCPGCC ß1a (construct 8), (C) Q57 CFP-CCPGCC ß1a (construct 9), (D) P249 CFP-CCPGCC ß1a (construct 10), (E) H458 CFP-CCPGCC ß1a (construct 11) or (F) M524 CFP-CCPGCC ß1a (construct 12) show ~2 fold FRET efficiency after FlAsH clearance with 7.5mM EDT. The numbers indicated represents the fluorescence intensity of the region in the myotubes before and after FlAsH clearance with EDT for FRET measurements. (G) Schematic diagrams of tagged ß1a constructs (constructs 6 to 12) that were used to assess the accessibility of FlAsH to tertracycteine tags incorporated in different domain of ß1a. (H) Comparison of the FRET efficiencies for different ß1a constructs (constructs 6 to 12) expressed in WT myotubes that shows in panel A to G with number of tested cells for each group in parentheses. Mean FRET efficiencies ± standard errors are shown.
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pone.0131399.g002: FRET signals from ß1a subunits incorporating the CFP-CCPGCC concatamer in different domains or doubly transfected CFP and CCPGCC tags expressed in WT myotubes.(A) Myotubes co-transfected with CFP- ß1a (construct 6) and CCPGCC- ß1a (construct 7), constructs demonstrate unchanged FRET efficiency after FlAsH clearance with 7.5 mM EDT indicating that ß1a intermolecular interactions do not contribute to FRET in the present study. Myotubes individually transfected with the CFP-CCPGCC concatemers (B) M1 CFP-CCPGCC ß1a (construct 8), (C) Q57 CFP-CCPGCC ß1a (construct 9), (D) P249 CFP-CCPGCC ß1a (construct 10), (E) H458 CFP-CCPGCC ß1a (construct 11) or (F) M524 CFP-CCPGCC ß1a (construct 12) show ~2 fold FRET efficiency after FlAsH clearance with 7.5mM EDT. The numbers indicated represents the fluorescence intensity of the region in the myotubes before and after FlAsH clearance with EDT for FRET measurements. (G) Schematic diagrams of tagged ß1a constructs (constructs 6 to 12) that were used to assess the accessibility of FlAsH to tertracycteine tags incorporated in different domain of ß1a. (H) Comparison of the FRET efficiencies for different ß1a constructs (constructs 6 to 12) expressed in WT myotubes that shows in panel A to G with number of tested cells for each group in parentheses. Mean FRET efficiencies ± standard errors are shown.

Mentions: After validating the FlAsH-based ex vivo FRET system using the CFP-CCPGCC concatemers in myotubes, we sought to evaluate FRET in tagged ß1a subunits. Control experiments revealed that FRET was not detected in the CFP- ß1a fusion that lacked the tetracysteine tag (construct 6) (Fig 2G and 2H), demonstrating that nonspecific binding of FlAsH does not yield any spurious FRET. We also tested for intermolecular FRET between ß1a subunits, given that ß subunits can exhibit intermolecular interaction under certain conditions [48–50]. Co-expressing N-terminal CFP- ß1a and CCPGCC- ß1a in wild type myotubes (Fig 2A) did not result in detectable FlAsH-mediated FRET (Fig 2G and 2H; co-transfected with constructs 6 and 7). Therefore, expression of constructs containing both FRET donor and acceptor in the same ß1a molecule will report intramolecular proximity relationships without the complication of intermolecular FRET between different ß1a subunits.


Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes.

Bhattacharya D, Mehle A, Kamp TJ, Balijepalli RC - PLoS ONE (2015)

FRET signals from ß1a subunits incorporating the CFP-CCPGCC concatamer in different domains or doubly transfected CFP and CCPGCC tags expressed in WT myotubes.(A) Myotubes co-transfected with CFP- ß1a (construct 6) and CCPGCC- ß1a (construct 7), constructs demonstrate unchanged FRET efficiency after FlAsH clearance with 7.5 mM EDT indicating that ß1a intermolecular interactions do not contribute to FRET in the present study. Myotubes individually transfected with the CFP-CCPGCC concatemers (B) M1 CFP-CCPGCC ß1a (construct 8), (C) Q57 CFP-CCPGCC ß1a (construct 9), (D) P249 CFP-CCPGCC ß1a (construct 10), (E) H458 CFP-CCPGCC ß1a (construct 11) or (F) M524 CFP-CCPGCC ß1a (construct 12) show ~2 fold FRET efficiency after FlAsH clearance with 7.5mM EDT. The numbers indicated represents the fluorescence intensity of the region in the myotubes before and after FlAsH clearance with EDT for FRET measurements. (G) Schematic diagrams of tagged ß1a constructs (constructs 6 to 12) that were used to assess the accessibility of FlAsH to tertracycteine tags incorporated in different domain of ß1a. (H) Comparison of the FRET efficiencies for different ß1a constructs (constructs 6 to 12) expressed in WT myotubes that shows in panel A to G with number of tested cells for each group in parentheses. Mean FRET efficiencies ± standard errors are shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482598&req=5

pone.0131399.g002: FRET signals from ß1a subunits incorporating the CFP-CCPGCC concatamer in different domains or doubly transfected CFP and CCPGCC tags expressed in WT myotubes.(A) Myotubes co-transfected with CFP- ß1a (construct 6) and CCPGCC- ß1a (construct 7), constructs demonstrate unchanged FRET efficiency after FlAsH clearance with 7.5 mM EDT indicating that ß1a intermolecular interactions do not contribute to FRET in the present study. Myotubes individually transfected with the CFP-CCPGCC concatemers (B) M1 CFP-CCPGCC ß1a (construct 8), (C) Q57 CFP-CCPGCC ß1a (construct 9), (D) P249 CFP-CCPGCC ß1a (construct 10), (E) H458 CFP-CCPGCC ß1a (construct 11) or (F) M524 CFP-CCPGCC ß1a (construct 12) show ~2 fold FRET efficiency after FlAsH clearance with 7.5mM EDT. The numbers indicated represents the fluorescence intensity of the region in the myotubes before and after FlAsH clearance with EDT for FRET measurements. (G) Schematic diagrams of tagged ß1a constructs (constructs 6 to 12) that were used to assess the accessibility of FlAsH to tertracycteine tags incorporated in different domain of ß1a. (H) Comparison of the FRET efficiencies for different ß1a constructs (constructs 6 to 12) expressed in WT myotubes that shows in panel A to G with number of tested cells for each group in parentheses. Mean FRET efficiencies ± standard errors are shown.
Mentions: After validating the FlAsH-based ex vivo FRET system using the CFP-CCPGCC concatemers in myotubes, we sought to evaluate FRET in tagged ß1a subunits. Control experiments revealed that FRET was not detected in the CFP- ß1a fusion that lacked the tetracysteine tag (construct 6) (Fig 2G and 2H), demonstrating that nonspecific binding of FlAsH does not yield any spurious FRET. We also tested for intermolecular FRET between ß1a subunits, given that ß subunits can exhibit intermolecular interaction under certain conditions [48–50]. Co-expressing N-terminal CFP- ß1a and CCPGCC- ß1a in wild type myotubes (Fig 2A) did not result in detectable FlAsH-mediated FRET (Fig 2G and 2H; co-transfected with constructs 6 and 7). Therefore, expression of constructs containing both FRET donor and acceptor in the same ß1a molecule will report intramolecular proximity relationships without the complication of intermolecular FRET between different ß1a subunits.

Bottom Line: The dihydropyridine receptor (DHPR) β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1) complex is still debatable.Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP.The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Arrhythmia Research Program, Department of Medicine, University of Wisconsin-Madison, Wisconsin, United States of America.

ABSTRACT
The dihydropyridine receptor (DHPR) β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1) complex is still debatable. We used fluorescence resonance energy transfer (FRET) to probe proximity relationships within the β1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP) was used as the FRET donor. Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1) myotubes revealed that in only one construct (H458 CCPGCC β1a -CFP) FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

No MeSH data available.


Related in: MedlinePlus