TY - JOUR
T1 - Analysis of the dihydropyridine receptor site of L-type calcium channels by alanine-scanning mutagenesis
AU - Peterson, Blaise Z.
AU - Johnson, Barry D.
AU - Hockerman, Gregory H.
AU - Acheson, Matthew
AU - Scheuer, Todd
AU - Catterall, William A.
PY - 1997
Y1 - 1997
N2 - The dihydrepyridine Ca2+ antagonist drugs used in the therapy of cardiovascular 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 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.
AB - The dihydrepyridine Ca2+ antagonist drugs used in the therapy of cardiovascular 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 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.
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U2 - 10.1074/jbc.272.30.18752
DO - 10.1074/jbc.272.30.18752
M3 - Article
C2 - 9228048
AN - SCOPUS:0030855109
SN - 0021-9258
VL - 272
SP - 18752
EP - 18758
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 30
ER -