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

Abstract

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 α_1C 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.