FUNCTIONAL-CHARACTERIZATION OF A MUTATED CHICKEN ALPHA(7) 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 a, nicotinic acetylcholine (ACh) receptor subunit (alpha(7)x61 ) in which a leucine residue was inserted between residues Leu9' and S er10' in transmembrane domain . The properties of a(7)x61 receptors ar e distinct from those of the wild-type receptor. 2 Oocytes expressing wild-type alpha(7) receptors responded to 10 mu M nicotine with rapid inward currents that desensitized with a time-constant of 710 +/- 409 ms (mean +/- s.e.mean, n = 5). However in alpha(7)x61 receptors 10 mu M nicotine resulted in slower onset inward currents that desensitized with a time-constant of 5684 +/- 3403 ms (mean +/- s.e.mean, n = 4). N o significant difference in the apparent affinity of nicotine or acety lcholine between mutant and wild-type receptors was observed. Dihydro- beta-erythroidine (DK beta E) acted as an antagonist on both receptors . 3 Molecular modelling of the alpha(7)x61 receptor channel pore forme d 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 b e replaced, by Thr and Val, respectively. 4 When present in the LEV-1 nicotinic ACh receptor subunit from Caenorhabditis elegans the same al teration conferred resistance to levamisole anthelmintic drug. Levamis ole blocked responses to nicotine of wild-type and alpha(7)x61 recepto rs. However, block was more dependent on membrane potential for the al pha(7)x61 receptors. 5 We conclude that the leucine insertion in trans membrane domain 2 has the unusual effect of slowing desensitization wi thout altering apparent agonist affinity.