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 cardi ovacular 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 bind ing, The amino acid residues that are required for high affinity bindi ng 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 b lock of Ba2+ currents through expressed Ca2+ channels, Eleven amino ac id residues in transmembrane segments IIIS6 and IVS6 were identified w hose mutation reduced the affinity for the Ca2+ antagonist PN200-110 b y 2-25-fold. Both amino acid residues conserved among Ca2+ channels an d those specific to L-type Ca2+ channels were found to be required for high affinity dihydropyridine binding, In addition, mutation F1462A i ncreased the affinity for the dihydropyridine Ca2+ antagonist PN200-11 0 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 fac es of these two alpha helices, supporting a ''domain interface model'' of dihydropyridine binding and action in which the IIIS6 and IVS6 int eract to form a high affinity dihydropyridine receptor site on L-type Ca2+ channels.