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

Blaise Z. Peterson, Barry D. Johnson, Gregory H. Hockerman, Matthew Acheson, Todd Scheuer, William A. Catterall

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)18752-18758
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number30
DOIs
Publication statusPublished - Aug 12 1997

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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