Analysis of the dihydropyridine receptor site of L-type calcium channels by alanine-scanning mutagenesis

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 alpha(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 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.