title: Fluorescence Resonance Energy Transfer-based Structural Analysis of the Dihydropyridine Receptor α1S Subunit Reveals Conformational Differences Induced by Binding of the β1a Subunit.
abstract: The skeletal muscle dihydropyridine receptor α1S subunit plays a key role in skeletal muscle excitation-contraction coupling by sensing membrane voltage changes and then triggering intracellular calcium release. The cytoplasmic loops connecting four homologous α1S structural domains have diverse functions, but their structural arrangement is poorly understood. Here, we used a novel FRET-based method to characterize the relative proximity of these intracellular loops in α1S subunits expressed in intact cells. In dysgenic myotubes, energy transfer was observed from an N-terminal-fused YFP to a FRET acceptor, ReAsH (resorufin arsenical hairpin binder), targeted to each α1S intracellular loop, with the highest FRET efficiencies measured to the α1S II-III loop and C-terminal tail. However, in HEK-293T cells, FRET efficiencies from the α1S N terminus to the II-III and III-IV loops and the C-terminal tail were significantly lower, thus suggesting that these loop structures are influenced by the cellular microenvironment. The addition of the β1a dihydropyridine receptor subunit enhanced FRET to the II-III loop, thus indicating that β1a binding directly affects II-III loop conformation. This specific structural change required the C-terminal 36 amino acids of β1a, which are essential to support EC coupling. Direct FRET measurements between α1S and β1a confirmed that both wild type and truncated β1a bind similarly to α1S These results provide new insights into the role of muscle-specific proteins on the structural arrangement of α1S intracellular loops and point to a new conformational effect of the β1a subunit in supporting skeletal muscle excitation-contraction coupling.
authors.affiliation: From the Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115.
authors.forename: Mohana
authors.initials: M
authors.lastname: Mahalingam
authors.affiliation: From the Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115 cperez@zeus.bwh.harvard.edu.
authors.forename: Claudio F
authors.initials: CF
authors.lastname: Perez
authors.affiliation: From the Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115 jfessenden@zeus.bwh.harvard.edu.
authors.forename: James D
authors.initials: JD
authors.lastname: Fessenden
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D009119:Muscle Contraction / Q000502:physiology*; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits
chemical_list: C412223:CACNA1S protein, human; C529218:CACNA1S protein, mouse; C492923:Cacnb1 protein, mouse; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D009124:Muscle Proteins; D021122:Protein Subunits
keywords: calcium channel; dihydropyridine receptor (DHPR); excitation-contraction coupling (E-C coupling); fluorescence resonance energy transfer (FRET); skeletal muscle