Functional characterization of a mutated chicken alpha7 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 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.
The Babraham Institute Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, UK.
S D
SD
Buckingham
C
C
Adcock
M S
MS
Sansom
D B
DB
Sattelle
H A
HA
Baylis
D000109:Acetylcholine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000871:Anthelmintics / Q000494:pharmacology; D001483:Base Sequence; D002645:Chickens; D004079:Dihydro-beta-Erythroidine / Q000494:pharmacology; D005260:Female; D066298:In Vitro Techniques; D007930:Leucine / Q000737:chemistry / Q000235:genetics*; D007978:Levamisole / Q000494:pharmacology; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D009865:Oocytes; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D014982:Xenopus laevis
D000871:Anthelmintics; D018722:Nicotinic Agonists; D011978:Receptors, Nicotinic; D004079:Dihydro-beta-Erythroidine; D007978:Levamisole; D009538:Nicotine; D007930:Leucine; D000109:Acetylcholine