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Keywords:

  • Nicotinic acetylcholine receptor;
  • Insect;
  • Subunit composition;
  • Xenopus oocyte;
  • Functional expression;
  • Immunoprecipitation;
  • α-Bungarotoxin

Abstract: Although neuronal nicotinic acetylcholine receptors from insects have been reconstituted in vitro more than a decade ago, our knowledge about the subunit composition of native receptors as well as their functional properties still remains limited. Immunohistochemical evidence has suggested that two α subunits, α-like subunit (ALS) and Drosophilaα2 subunit (Dα2), are colocalized in the synaptic neuropil of the Drosophila CNS and therefore may be subunits of the same receptor complex. To gain further understanding of the composition of these nicotinic receptors, we have examined the possibility that a receptor may imbed more than one α subunit using immunoprecipitations and electrophysiological investigations. Immunoprecipitation experiments of fly head extracts revealed that ALS-specific antibodies coprecipitate Dα2, and vice versa, and thereby suggest that these two α subunits must be contained within the same receptor complex, a result that is supported by investigations of reconstituted receptors in Xenopus oocytes. Discrimination between binary (ALS/β2 or Dα2/β2) and ternary (ALS/Dα2/β2) receptor complexes was made on the basis of their dose-response curve to acetylcholine as well as their sensitivity to α-bungarotoxin or dihydro-β-erythroidine. These data demonstrate that the presence of the two α subunits within a single receptor complex confers new receptor properties that cannot be predicted from knowledge of the binary receptor’s properties.