Article date: June 1998
By: Steven D Buckingham, Charlotte Adcock, Mark S P Sansom, David B Sattelle, Howard A Baylis, in Volume 124, Issue 4, pages 747-755
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.
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.
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.
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.
We conclude that the leucine insertion in transmembrane domain 2 has the unusual effect of slowing desensitization without altering apparent agonist affinity.
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.
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.
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.
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.
We conclude that the leucine insertion in transmembrane domain 2 has the unusual effect of slowing desensitization without altering apparent agonist affinity.
DOI: 10.1038/sj.bjp.0701866
View this article