title: Labelling of the 3D structure of the cardiac L-type voltage-gated calcium channel.
abstract: Voltage-gated calcium channels (VGCCs) regulate calcium influx into all excitable cells. In the heart, the main calcium channels are the L-type VGCCs (LTCCs). These are localised to the sarcolemmal membrane, and are hetero-oligomeric complexes comprised of three non-covalently associated polypeptides; alpha1 (CaV1.2), alpha2delta and beta. We recently reported the 3D structure for a monomeric form of the cardiac LTCC1 using electron microscopy and single particle analysis. We also determined the first medium/low resolution structure of a T-type voltage gated calcium channel (CaV3.1) polypeptide. We identified the transmembrane and cytoplasmic domains of the T-type channel using labelling studies to determine the position of the C-terminus. By modelling of the CaV3.1 structure (comparable at these resolutions to CaV1.2) into the cardiac LTCC volume, we were able to delineate the subunit boundaries of the cardiac LTCC, leading to a proposal for a putative orientation of the LTCC with respect to the membrane bilayer. We have now extended these studies to include labelling of the extracellular alpha2 polypeptide using affinity purified antibodies raised against the Von Willebrand Factor A (VWA) domain and calmodulin-gold labelling of the C-terminus of CaV1.2. These data provide further support for the proposed orientation of the 3D structure of the cardiac LTCC.
authors.affiliation: Cardiovascular Medicine, School of Clinical & Laboratory Sciences, Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK.
authors.forename: Conor P
authors.initials: CP
authors.lastname: Walsh
authors.forename: Anthony
authors.initials: A
authors.lastname: Davies
authors.forename: Manuela
authors.initials: M
authors.lastname: Nieto-Rostro
authors.forename: Annette C
authors.initials: AC
authors.lastname: Dolphin
authors.forename: Ashraf
authors.initials: A
authors.lastname: Kitmitto
mesh_terms: D000906:Antibodies; D001665:Binding Sites; D020746:Calcium Channels, L-Type / Q000737:chemistry*; D002147:Calmodulin / Q000378:metabolism; D002462:Cell Membrane; D003600:Cytosol; D006321:Heart / Q000502:physiology*; D006801:Humans; D015640:Ion Channel Gating; D008854:Microscopy, Electron; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D014841:von Willebrand Factor / Q000276:immunology
chemical_list: D000906:Antibodies; D020746:Calcium Channels, L-Type; D002147:Calmodulin; D014841:von Willebrand Factor