Functional characterization of a mutated chicken α7 nicotinic acetylcholine receptor subunit with a leucine residue inserted in transmembrane domain 2

1. Site-directed mutagenesis was used to create an altered form of the chicken α7 nicotinic acetylcholine (ACh) receptor subunit (α7x61) in which a leucine residue was inserted between residues Leu9' and Ser10' in transmembrane domain 2. The properties of α7x61 receptors are distinct from those of the wild-type receptor. 2. Oocytes expressing wild-type α7 receptors responded to 10 μM nicotine with rapid inward currents that desensitized with a time-constant of 710 ± 409 ms (mean ± s.e.mean, n = 5). However in α7x61 receptors 10 μM 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-β-erythroidine (DHβE) acted as an antagonist on both receptors. 3. Molecular modelling of the α7x61 receptor channel pore formed by a bundle of M2 α-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 α7x61 receptors. However, block was more dependent on membrane potential for the α7x61 receptors. 5. We conclude that the leucine insertion in transmembrane domain 2 has the unusual effect of slowing desensitization without altering apparent agonist affinity.