title: Molecular determinants of high affinity dihydropyridine binding in L-type calcium channels.
abstract: The pore-forming alpha1 subunit of L-type voltage-gated Ca2+ channels is pharmacologically modulated by dihydropyridine (DHP) Ca2+ antagonists and agonists. Site-directed mutation of amino acids within transmembrane segments IIIS6 and IVS6 to those characteristic of DHP-insensitive channels revealed 2 mutations in IIIS6 (I1049F and I1052F) and 4 mutations in IVS6 (Y1365I, M1366F, I1372M, and I1373L) with increased KD values for (+)-[3H]PN200-110 binding. A tyrosine residue (Y1048) in IIIS6 that is conserved between DHP-sensitive and -insensitive Ca2+ channels was also altered by mutagenesis. Y1048F had a KD for (+)-[3H]PN200-110 binding that was increased 12-fold, and Y1048A had a KD at least 1000-fold higher than that of wild-type. These results support the hypothesis that transmembrane segments IIIS6 and IVS6 both contribute critical amino acid residues to the DHP receptor site and that Tyr-1048 within transmembrane segment IIIS6 is required for high affinity DHP binding, even though it is conserved between DHP-sensitive and -insensitive Ca2+ channels.
authors.affiliation: Department of Pharmacology, University of Washington, Seattle, 98195-7280, USA.
authors.forename: B Z
authors.initials: BZ
authors.lastname: Peterson
authors.forename: T N
authors.initials: TN
authors.lastname: Tanada
authors.forename: W A
authors.initials: WA
authors.lastname: Catterall
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002121:Calcium Channel Blockers / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D003001:Cloning, Molecular; D004095:Dihydropyridines / Q000378:metabolism*; D017275:Isradipine / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011817:Rabbits; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014316:Tritium
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D004095:Dihydropyridines; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014316:Tritium; C038806:1,4-dihydropyridine; D017275:Isradipine