The amino terminal domain of the human α7 nicotinic acetylcholine receptor subunit leads to the functional expression of human/insect receptors.

BACKGROUND AND PURPOSE: Human α7 nicotinic acetylcholine receptor subunit forms a cluster with insect orthologs, suggesting they can form homomeric receptors in heterologous systems, such as Xenopus oocytes. However, it is difficult to express insect α7 homomeric receptors to study their properties. We investigated whether the N-terminal domain of each subunit could lead to functional receptors activated by acetylcholine.

EXPERIMENTAL APPROACH: Human, honeybee and cockroach α7 subunits were used to synthetise chimeric receptors. Mutagenesis analysis on the N-terminal domain of each α7 subunit, with confocal imaging, and electrophysiological studies, were used to build human/insect and insect/human homomeric constructs in Xenopus oocytes. Effects of the positive allosteric modulator, PNU-120596 and inhibitor, α-bungarotoxin, were evaluated on each chimera. Homology modelling investigation provided a preliminary and qualitative description of the environment of the acetylcholine in the receptor's surroundings.

KEY

RESULTS

Human/insect chimeric constructs α7H/α7A and α7H/α7P, which include the N-terminal domain of the human α7 subunit, make up the functional receptors which are activated by acetylcholine, and inhibited by α-bungarotoxin. In comparison, the insect/human chimeric constructs α7A/α7H and α7P/α7H, which contain the N-terminal domains of insect α7 subunits, are not able to form functional receptors. However, insertion of the cys-loop from the human α7 subunit into the cockroach α7 subunit leads to functional receptors activated by acetylcholine, and modulated by PNU.

CONCLUSION AND IMPLICATIONS: This study identifies chimeric insect receptors which can be used to study the pharmacological properties of insect α7 homomeric receptors, to aid the understanding of insecticide compounds.