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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

Intramolecular FRET in the ß1a subunit depends upon donor-acceptor position and RyR1 expression.(A) FRET efficiencies for ß1a subunit constructs in myotubes when CFP donor was located at the N-terminus and CCPGCC acceptor site was tested at five different positions of ß1a labeled M1 (construct 8), Q57 (construct 14), P249 (construct 15), H458 (construct 16) and M524 (construct 17), as indicated in schematic above bar graphs. (B) FRET efficiencies for same ß1a subunit constructs in myotubes except the CFP donor was located at the C-terminus (construct 12, 18, 19, 20 and 21). For both panels A and B the ß1a constructs were expressed in wild type (black) or RyR1 KO (gray) myotubes. Only the construct H458-CCGPCC ß1a-CFP (construct 21) displayed significant difference FRET efficiency (*p<0.001) in the presence (in WT 0.12 ± 0.01) versus absence (in KO 0.19 ± 0.01) of RyR1 (in Table 1 boxed). (C) Ca2+ current expression at test potentials of –30 mV and +30 mV in ß1 KO myotubes expressing H458-CCGPCC ß1a-CFP (construct 21) compared to non-transfected (NT) ß KO control which demonstrates that this construct forms functional ß subunits. (D) Comparison of FRET efficiency of H458 CCPGCC ß1a-CFP (construct 21) transfected in either WT or dyspedic myotubes. Transfection of RyR1 into dyspedic myotubes eliminated the increase in FRET observed in the absence of RyR1. The RyR1 expression marker (CD8) or the presence of endogenous ß1a subunits has no effect on the measured FRET efficiencies. The FRET efficiency of construct 21 was 0.12 ± 0.01, when it was transfected in either wild type (WT) or ß KO or RyR1 KO cells in presence of RyR1 + CD8 plasmids (triple transfected). Additionally a comparable FRET efficiency observed in RyR1 KO cells between construct 21 alone (0.19 ± 0.01) and construct 21 with CD8 plasmid co-transfection (0.18 ± 0.01), which was significantly higher than the WT (*p<0.001; see results in Table 1) (E) Expression and intramolecular FRET of H458CCPGCC ß1a- CFP alone and co-transfected with RyR1 in dyspedic myotubes with the number represents the fluorescence intensity of the region in the myotube and the arrow indicated CD8 beads.
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pone.0131399.g004: Intramolecular FRET in the ß1a subunit depends upon donor-acceptor position and RyR1 expression.(A) FRET efficiencies for ß1a subunit constructs in myotubes when CFP donor was located at the N-terminus and CCPGCC acceptor site was tested at five different positions of ß1a labeled M1 (construct 8), Q57 (construct 14), P249 (construct 15), H458 (construct 16) and M524 (construct 17), as indicated in schematic above bar graphs. (B) FRET efficiencies for same ß1a subunit constructs in myotubes except the CFP donor was located at the C-terminus (construct 12, 18, 19, 20 and 21). For both panels A and B the ß1a constructs were expressed in wild type (black) or RyR1 KO (gray) myotubes. Only the construct H458-CCGPCC ß1a-CFP (construct 21) displayed significant difference FRET efficiency (*p<0.001) in the presence (in WT 0.12 ± 0.01) versus absence (in KO 0.19 ± 0.01) of RyR1 (in Table 1 boxed). (C) Ca2+ current expression at test potentials of –30 mV and +30 mV in ß1 KO myotubes expressing H458-CCGPCC ß1a-CFP (construct 21) compared to non-transfected (NT) ß KO control which demonstrates that this construct forms functional ß subunits. (D) Comparison of FRET efficiency of H458 CCPGCC ß1a-CFP (construct 21) transfected in either WT or dyspedic myotubes. Transfection of RyR1 into dyspedic myotubes eliminated the increase in FRET observed in the absence of RyR1. The RyR1 expression marker (CD8) or the presence of endogenous ß1a subunits has no effect on the measured FRET efficiencies. The FRET efficiency of construct 21 was 0.12 ± 0.01, when it was transfected in either wild type (WT) or ß KO or RyR1 KO cells in presence of RyR1 + CD8 plasmids (triple transfected). Additionally a comparable FRET efficiency observed in RyR1 KO cells between construct 21 alone (0.19 ± 0.01) and construct 21 with CD8 plasmid co-transfection (0.18 ± 0.01), which was significantly higher than the WT (*p<0.001; see results in Table 1) (E) Expression and intramolecular FRET of H458CCPGCC ß1a- CFP alone and co-transfected with RyR1 in dyspedic myotubes with the number represents the fluorescence intensity of the region in the myotube and the arrow indicated CD8 beads.

Mentions: To map the domains of ß1a by FRET we kept the donor (CFP) position fixed at either the N-terminus or C-terminus and introduced the acceptor (CCPGCC motif) at the same five critical positions described above in ß1a molecule (constructs 8, 12 and 14 to 21). FRET efficiency measurements were conducted in wild type as well as dyspedic (lacking RyR1) myotubes. In general, comparatively weaker FRET was detected when CFP and the tetracysteine tag were separated by intervening ß1a sequence compared to inserted CFP-CCPGCC concatemers; however, for each of the inserted positions high FRET efficiency was observed (Fig 4A and 4B). The results were indistinguishable between wild type and dyspedic myotubes for all constructs except for a significant difference (p<0.001) in case of H458 CCPGCC ß1a-CFP (construct 21). The FRET efficiency at this location was 0.12 ± 0.01 in wild type myotubes and 0.19 ± 0.01 in dyspedic myotubes (Fig 4B, construct 21 and Table 1). These data indicate a conformational change in the C-terminus of ß1a induced by the presence of RyR1. They lower FRET between excitation pairs at H458 CCPGCC and the C-terminus CFP further suggests that the C-terminal region assumes a more extended conformation in the presence of RyR1.


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)

Intramolecular FRET in the ß1a subunit depends upon donor-acceptor position and RyR1 expression.(A) FRET efficiencies for ß1a subunit constructs in myotubes when CFP donor was located at the N-terminus and CCPGCC acceptor site was tested at five different positions of ß1a labeled M1 (construct 8), Q57 (construct 14), P249 (construct 15), H458 (construct 16) and M524 (construct 17), as indicated in schematic above bar graphs. (B) FRET efficiencies for same ß1a subunit constructs in myotubes except the CFP donor was located at the C-terminus (construct 12, 18, 19, 20 and 21). For both panels A and B the ß1a constructs were expressed in wild type (black) or RyR1 KO (gray) myotubes. Only the construct H458-CCGPCC ß1a-CFP (construct 21) displayed significant difference FRET efficiency (*p<0.001) in the presence (in WT 0.12 ± 0.01) versus absence (in KO 0.19 ± 0.01) of RyR1 (in Table 1 boxed). (C) Ca2+ current expression at test potentials of –30 mV and +30 mV in ß1 KO myotubes expressing H458-CCGPCC ß1a-CFP (construct 21) compared to non-transfected (NT) ß KO control which demonstrates that this construct forms functional ß subunits. (D) Comparison of FRET efficiency of H458 CCPGCC ß1a-CFP (construct 21) transfected in either WT or dyspedic myotubes. Transfection of RyR1 into dyspedic myotubes eliminated the increase in FRET observed in the absence of RyR1. The RyR1 expression marker (CD8) or the presence of endogenous ß1a subunits has no effect on the measured FRET efficiencies. The FRET efficiency of construct 21 was 0.12 ± 0.01, when it was transfected in either wild type (WT) or ß KO or RyR1 KO cells in presence of RyR1 + CD8 plasmids (triple transfected). Additionally a comparable FRET efficiency observed in RyR1 KO cells between construct 21 alone (0.19 ± 0.01) and construct 21 with CD8 plasmid co-transfection (0.18 ± 0.01), which was significantly higher than the WT (*p<0.001; see results in Table 1) (E) Expression and intramolecular FRET of H458CCPGCC ß1a- CFP alone and co-transfected with RyR1 in dyspedic myotubes with the number represents the fluorescence intensity of the region in the myotube and the arrow indicated CD8 beads.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482598&req=5

pone.0131399.g004: Intramolecular FRET in the ß1a subunit depends upon donor-acceptor position and RyR1 expression.(A) FRET efficiencies for ß1a subunit constructs in myotubes when CFP donor was located at the N-terminus and CCPGCC acceptor site was tested at five different positions of ß1a labeled M1 (construct 8), Q57 (construct 14), P249 (construct 15), H458 (construct 16) and M524 (construct 17), as indicated in schematic above bar graphs. (B) FRET efficiencies for same ß1a subunit constructs in myotubes except the CFP donor was located at the C-terminus (construct 12, 18, 19, 20 and 21). For both panels A and B the ß1a constructs were expressed in wild type (black) or RyR1 KO (gray) myotubes. Only the construct H458-CCGPCC ß1a-CFP (construct 21) displayed significant difference FRET efficiency (*p<0.001) in the presence (in WT 0.12 ± 0.01) versus absence (in KO 0.19 ± 0.01) of RyR1 (in Table 1 boxed). (C) Ca2+ current expression at test potentials of –30 mV and +30 mV in ß1 KO myotubes expressing H458-CCGPCC ß1a-CFP (construct 21) compared to non-transfected (NT) ß KO control which demonstrates that this construct forms functional ß subunits. (D) Comparison of FRET efficiency of H458 CCPGCC ß1a-CFP (construct 21) transfected in either WT or dyspedic myotubes. Transfection of RyR1 into dyspedic myotubes eliminated the increase in FRET observed in the absence of RyR1. The RyR1 expression marker (CD8) or the presence of endogenous ß1a subunits has no effect on the measured FRET efficiencies. The FRET efficiency of construct 21 was 0.12 ± 0.01, when it was transfected in either wild type (WT) or ß KO or RyR1 KO cells in presence of RyR1 + CD8 plasmids (triple transfected). Additionally a comparable FRET efficiency observed in RyR1 KO cells between construct 21 alone (0.19 ± 0.01) and construct 21 with CD8 plasmid co-transfection (0.18 ± 0.01), which was significantly higher than the WT (*p<0.001; see results in Table 1) (E) Expression and intramolecular FRET of H458CCPGCC ß1a- CFP alone and co-transfected with RyR1 in dyspedic myotubes with the number represents the fluorescence intensity of the region in the myotube and the arrow indicated CD8 beads.
Mentions: To map the domains of ß1a by FRET we kept the donor (CFP) position fixed at either the N-terminus or C-terminus and introduced the acceptor (CCPGCC motif) at the same five critical positions described above in ß1a molecule (constructs 8, 12 and 14 to 21). FRET efficiency measurements were conducted in wild type as well as dyspedic (lacking RyR1) myotubes. In general, comparatively weaker FRET was detected when CFP and the tetracysteine tag were separated by intervening ß1a sequence compared to inserted CFP-CCPGCC concatemers; however, for each of the inserted positions high FRET efficiency was observed (Fig 4A and 4B). The results were indistinguishable between wild type and dyspedic myotubes for all constructs except for a significant difference (p<0.001) in case of H458 CCPGCC ß1a-CFP (construct 21). The FRET efficiency at this location was 0.12 ± 0.01 in wild type myotubes and 0.19 ± 0.01 in dyspedic myotubes (Fig 4B, construct 21 and Table 1). These data indicate a conformational change in the C-terminus of ß1a induced by the presence of RyR1. They lower FRET between excitation pairs at H458 CCPGCC and the C-terminus CFP further suggests that the C-terminal region assumes a more extended conformation in the presence of RyR1.

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