Dataset Information

Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) ?1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes.

ABSTRACT: The dihydropyridine receptor (DHPR) ?1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of ?1a 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 ?1a 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 ?1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of ?1a 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 ?1a 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 ?1a -CFP) FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the ?1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of ?1a and RyR1 in resting myotubes.

SUBMITTER: Bhattacharya D

PROVIDER: S-EPMC4482598 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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 Dipankar D Mehle Andrew A Kamp Timothy J TJ Balijepalli Ravi C RC

PloS one 20150626 6

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 exhi ...[more]

PMID: 26114725

Similar Datasets

Project description:Molecular determinants essential for skeletal-type excitation-contraction (EC) coupling have been described in the cytosolic loops of the dihydropyridine receptor (DHPR) alpha1S pore subunit and in the carboxyl terminus of the skeletal-specific DHPR beta1a-subunit. It is unknown whether EC coupling domains present in the beta-subunit influence those present in the pore subunit or if they act independent of each other. To address this question, we investigated the EC coupling signal that is generated when the endogenous DHPR pore subunit alpha1S is paired with the heterologous heart/brain DHPR beta2a-subunit. Studies were conducted in primary cultured myotubes from beta1 knockout (KO), ryanodine receptor type 1 (RyR1) KO, ryanodine receptor type 3 (RyR3) KO, and double RyR1/RyR3 KO mice under voltage clamp with simultaneous monitoring of confocal fluo-4 fluorescence. The beta2a-mediated Ca2+ current recovered in beta1 KO myotubes lacking the endogenous DHPR beta1a-subunit verified formation of the alpha1S/beta1a pair. In myotube genotypes which express no or low-density L-type Ca2+ currents, namely beta1 KO and RyR1 KO, beta2a overexpression recovered a wild-type density of nifedipine-sensitive Ca2+ currents with a slow activation kinetics typical of skeletal myotubes. Concurrent with Ca2+ current recovery, there was a drastic reduction of voltage-dependent, skeletal-type EC coupling and emergence of Ca2+ transients triggered by the Ca2+ current. A comparison of beta2a overexpression in RyR3 KO, RyR1 KO, and double RyR1/RyR3 KO myotubes concluded that both RyR1 and RyR3 isoforms participated in Ca2+-dependent Ca2+ release triggered by the beta2a-subunit. In beta1 KO and RyR1 KO myotubes, the Ca2+-dependent EC coupling promoted by beta2a overexpression had the following characteristics: 1), L-type Ca2+ currents had a wild-type density; 2), Ca2+ transients activated much slower than controls overexpressing beta1a, and the rate of fluorescence increase was consistent with the activation kinetics of the Ca2+ current; 3), the voltage dependence of the Ca2+ transient was bell-shaped and the maximum was centered at approximately +30 mV, consistent with the voltage dependence of the Ca2+ current; and 4), Ca2+ currents and Ca2+ transients were fully blocked by nifedipine. The loss in voltage-dependent EC coupling promoted by beta2a was inferred by the drastic reduction in maximal Ca2+ fluorescence at large positive potentials (DeltaF/Fmax) in double dysgenic/beta1 KO myotubes overexpressing the pore mutant alpha1S (E1014K) and beta2a. The data indicate that beta2a, upon interaction with the skeletal pore subunit alpha1S, overrides critical EC coupling determinants present in alpha1S. We propose that the alpha1S/beta pair, and not the alpha1S-subunit alone, controls the EC coupling signal in skeletal muscle.

Dataset Information

Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) ?1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes.

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets