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

Abstract

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.

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