Analysis of the Dihydropyridine Receptor Site ofl-type Calcium Channels by Alanine-scanning Mutagenesis

• Published in: The Journal of biological chemistry Vol. 272; no. 30; pp. 18752 - 18758
• Main Authors: Peterson, Blaise Z., Johnson, Barry D., Hockerman, Gregory H., Acheson, Matthew, Scheuer, Todd, Catterall, William A.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 25.07.1997; American Society for Biochemistry and Molecular Biology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.30.18752

The dihydropyridine Ca2+antagonist drugs used in the therapy of cardiovacular disorders inhibitl-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 α1C 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 α 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.