Three-Dimensional Localization of the α and β Subunits and of the II-III Loop in the Skeletal Muscle L-type Ca2+ Channel |
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Authors: | John Szpyt Nancy Lorenzon Claudio F Perez Ethan Norris Paul D Allen Kurt G Beam Montserrat Samsó |
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Institution: | From the ‡Department of Anesthesia, Brigham and Women''s Hospital and Harvard Medical School, Boston, Massachusetts 02115.;the §Department of Biological Sciences, University of Denver, Denver, Colorado 80208.;the ¶Department of Physiology and Biophysics, University of Colorado-Denver, Aurora, Colorado 80045, and ;the ‖Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, Virginia 23298 |
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Abstract: | The L-type Ca2+ channel (dihydropyridine receptor (DHPR) in skeletal muscle acts as the voltage sensor for excitation-contraction coupling. To better resolve the spatial organization of the DHPR subunits (α1s or CaV1.1, α2, β1a, δ1, and γ), we created transgenic mice expressing a recombinant β1a subunit with YFP and a biotin acceptor domain attached to its N- and C- termini, respectively. DHPR complexes were purified from skeletal muscle, negatively stained, imaged by electron microscopy, and subjected to single-particle image analysis. The resulting 19.1-Å resolution, three-dimensional reconstruction shows a main body of 17 × 11 × 8 nm with five corners along its perimeter. Two protrusions emerge from either face of the main body: the larger one attributed to the α2-δ1 subunit that forms a flexible hook-shaped feature and a smaller protrusion on the opposite side that corresponds to the II-III loop of CaV1.1 as revealed by antibody labeling. Novel features discernible in the electron density accommodate the atomic coordinates of a voltage-gated sodium channel and of the β subunit in a single docking possibility that defines the α1-β interaction. The β subunit appears more closely associated to the membrane than expected, which may better account for both its role in localizing the α1s subunit to the membrane and its suggested role in excitation-contraction coupling. |
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Keywords: | Calcium Channels Cell Signaling Electron Microscopy (EM) Excitation-Contraction Coupling Protein Expression 3D Reconstruction Cav1 1 Dihydropyridine Receptor L-type Channel Protein Structure |
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