Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes
نویسندگان
چکیده
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.
منابع مشابه
RyR1 reorganizes the cytoplasmic interface of α1S 1 Fluorescence Resonance Energy Transfer (FRET) Indicates that Association with the Type I Ryanodine Receptor (RyR1) Causes Reorientation of Multiple Cytoplasmic Domains of the Dihydropyridine Receptor (DHPR) α1S Subunit*
متن کامل
A novel interaction between the DHPR Ca 2 + channel β subunit and ryanodine receptor type 1 strengthens excitation - contraction coupling
Previous studies have shown that the skeletal dihydropydidine receptor (DHPR) pore subunit CaV1.1 (α1S) physically interacts with ryanodine receptor type 1 (RyR1) and a molecular signal is transmitted from α1S to RyR1 to trigger excitation-contraction (EC) coupling. We show that the β subunit of the skeletal DHPR also binds RyR1 and participates in this signaling process. A novel binding site f...
متن کاملThe Skeletal L - type Ca 2 + Current Is a Major Contributor to Excitation - coupled Ca 2 + entry Roger
The skeletal muscle L-type Ca 2+ channel (1,4-dihydropyridine receptor [DHPR]) serves as the voltage sensor for excitation – contraction (EC) coupling ( Tanabe et al., 1988 ). Conformational changes in the DHPR in response to membrane depolarization are coupled to gating of the type 1 RYR (RYR1) of the SR ( Beam and Horowicz, 2004 ). The resultant Ca 2+ effl ux via RYR1 into the myoplasm initia...
متن کاملFunctional analysis of the R1086H malignant hyperthermia mutation in the DHPR reveals an unexpected influence of the III-IV loop on skeletal muscle EC coupling.
Malignant hyperthermia (MH) is an inherited pharmacogenetic disorder caused by mutations in the skeletal muscle ryanodine receptor (RyR1) and the dihydropyridine receptor (DHPR) alpha(1S)-subunit. We characterized the effects of an MH mutation in the DHPR cytoplasmic III-IV loop of alpha(1S) (R1086H) on DHPR-RyR1 coupling after reconstitution in dysgenic (alpha(1S) null) myotubes. Compared with...
متن کاملThe beta 1a subunit is essential for the assembly of dihydropyridine-receptor arrays in skeletal muscle.
Homozygous zebrafish of the mutant relaxed (red(ts25)) are paralyzed and die within days after hatching. A significant reduction of intramembrane charge movements and the lack of depolarization-induced but not caffeine-induced Ca(2+) transients suggested a defect in the skeletal muscle dihydropyridine receptor (DHPR). Sequencing of DHPR cDNAs indicated that the alpha(1S) subunit is normal, wher...
متن کامل