title: Roles of amino acids and subunits in determining the inhibition of nicotinic acetylcholine receptors by competitive antagonists.
abstract: BACKGROUND
  Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the alpha-delta and alpha-epsilon subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the alpha-epsilon interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors.
  
  
  METHODS
  HEK293 cells were transfected with wild-type or mutant (alphaY198F, epsilonD59A, epsilonD59N, epsilonD173A, epsilonD173N, deltaD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcholine in the absence and presence of antagonist. Concentration-response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates.
  
  
  RESULTS
  Effects of mutations were antagonist specific. alphaY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to epsilonD173 mutations. epsilonD59 mutations had large effects on metocurine and cisatracurium. deltaD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two delta subunits but no epsilon subunit. Differences in IC50 arose from differences in both dissociation and association rates.
  
  
  CONCLUSIONS
  Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the alpha-epsilon interface. Pancuronium, vecuronium, and cisatracurium were selective for the alpha-delta interface. This suggests the possibility of synergistic inhibition by pairs of antagonists.
authors.affiliation: Department of Anesthesiology, Stony Brook University, NY 11794-8480, USA, and Department of Anesthesiology, Surugadai Nihon University Hospital, Kanda, Tokyo, Japan. james.dilger@stonybrook.edu
authors.forename: James P
authors.initials: JP
authors.lastname: Dilger
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Vidal
authors.forename: Man
authors.initials: M
authors.lastname: Liu
authors.forename: Claire
authors.initials: C
authors.lastname: Mettewie
authors.forename: Takahiro
authors.initials: T
authors.lastname: Suzuki
authors.forename: Anh
authors.initials: A
authors.lastname: Pham
authors.forename: Deeptankar
authors.initials: D
authors.lastname: Demazumder
mesh_terms: D000596:Amino Acids / Q000502:physiology*; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects / Q000235:genetics*; D002478:Cells, Cultured; D051379:Mice; D016296:Mutagenesis; D003473:Neuromuscular Nondepolarizing Agents / Q000494:pharmacology*; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D010197:Pancuronium / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D014403:Tubocurarine / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D003473:Neuromuscular Nondepolarizing Agents; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D010197:Pancuronium; D014403:Tubocurarine