Functional characterization of a mutated chicken alpha7 nicotinic acetylcholine receptor subunit with a leucine residue inserted in transmembrane domain 2

Br J Pharmacol. 1998 Jun;124(4):747-55. doi: 10.1038/sj.bjp.0701866.

Abstract

1. Site-directed mutagenesis was used to create an altered form of the chicken alpha7 nicotinic acetylcholine (ACh) receptor subunit (alpha7x61) in which a leucine residue was inserted between residues Leu9' and Ser10' in transmembrane domain 2. The properties of alpha7x61 receptors are distinct from those of the wild-type receptor. 2. Oocytes expressing wild-type alpha7 receptors responded to 10 microM nicotine with rapid inward currents that desensitized with a time-constant of 710+/-409 ms (mean+/-s.e.mean, n=5). However in alpha7x61 receptors 10 microM nicotine resulted in slower onset inward currents that desensitized with a time-constant of 5684+/-3403 ms (mean+/-s.e.mean, n = 4). No significant difference in the apparent affinity of nicotine or acetylcholine between mutant and wild-type receptors was observed. Dihydro-beta-erythroidine (DHbetaE) acted as an antagonist on both receptors. 3. Molecular modelling of the alpha7x61 receptor channel pore formed by a bundle of M2 alpha-helices suggested that three of the channel lining residues would be altered by the leucine insertion i.e.; Ser10 would be replaced by the leucine insertion, Val13' and Phe14' would be replaced, by Thr and Val, respectively. 4 When present in the LEV-1 nicotinic ACh receptor subunit from Caenorhabditis elegans the same alteration conferred resistance to levamisole anthelmintic drug. Levamisole blocked responses to nicotine of wild-type and alpha7x61 receptors. However, block was more dependent on membrane potential for the alpha7x61 receptors. 5. We conclude that the leucine insertion in transmembrane domain 2 has the unusual effect of slowing desensitization without altering apparent agonist affinity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholine / pharmacology
  • Amino Acid Sequence
  • Animals
  • Anthelmintics / pharmacology
  • Base Sequence
  • Chickens
  • Dihydro-beta-Erythroidine / pharmacology
  • Female
  • In Vitro Techniques
  • Leucine / chemistry
  • Leucine / genetics*
  • Levamisole / pharmacology
  • Membrane Potentials
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation*
  • Nicotine / pharmacology
  • Nicotinic Agonists / pharmacology
  • Oocytes
  • Protein Conformation
  • Receptors, Nicotinic / chemistry
  • Receptors, Nicotinic / drug effects
  • Receptors, Nicotinic / genetics
  • Receptors, Nicotinic / physiology*
  • Xenopus laevis

Substances

  • Anthelmintics
  • Nicotinic Agonists
  • Receptors, Nicotinic
  • Dihydro-beta-Erythroidine
  • Levamisole
  • Nicotine
  • Leucine
  • Acetylcholine