title: Analysis of the dihydropyridine receptor site of L-type calcium channels by alanine-scanning mutagenesis.
abstract: The dihydropyridine Ca2+ antagonist drugs used in the therapy of cardiovacular disorders inhibit L-type Ca2+ channels by binding to a single high affinity site. Photoaffinity labeling and analysis of mutant Ca2+ channels implicate the IIIS6 and IVS6 segments in high affinity binding. The amino acid residues that are required for high affinity binding of dihydropyridine Ca2+ channel antagonists were probed by alanine-scanning mutagenesis of the alpha1C subunit, transient expression in mammalian cells, and analysis by measurements of ligand binding and block of Ba2+ currents through expressed Ca2+ channels. Eleven amino acid residues in transmembrane segments IIIS6 and IVS6 were identified whose mutation reduced the affinity for the Ca2+ antagonist PN200-110 by 2-25-fold. Both amino acid residues conserved among Ca2+ channels and those specific to L-type Ca2+ channels were found to be required for high affinity dihydropyridine binding. In addition, mutation F1462A increased the affinity for the dihydropyridine Ca2+ antagonist PN200-110 by 416-fold with no effect on the affinity for the Ca2+ agonist Bay K8644. The residues in transmembrane segments IIIS6 and IVS6 that are required for high affinity binding are primarily aligned on single faces of these two alpha helices, supporting a "domain interface model" of dihydropyridine binding and action in which the IIIS6 and IVS6 interact to form a high affinity dihydropyridine receptor site on L-type Ca2+ channels.
authors.affiliation: Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280, USA.
authors.forename: B Z
authors.initials: BZ
authors.lastname: Peterson
authors.forename: B D
authors.initials: BD
authors.lastname: Johnson
authors.forename: G H
authors.initials: GH
authors.lastname: Hockerman
authors.forename: M
authors.initials: M
authors.lastname: Acheson
authors.forename: T
authors.initials: T
authors.lastname: Scheuer
authors.forename: W A
authors.initials: WA
authors.lastname: Catterall
mesh_terms: D000409:Alanine / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics; D020746:Calcium Channels, L-Type; D002460:Cell Line; D006801:Humans; D017275:Isradipine / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D011817:Rabbits
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D000409:Alanine; D017275:Isradipine