Novel Neonicotinoid‐Agarose Affinity Column for Drosophila and Musca Nicotinic Acetylcholine Receptors

Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high‐affinity binding site for [ 3 H]IMI with K D values of 1–2 n M and B max values of 560–850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an α‐bungarotoxin (α‐BGT)‐agarose affinity column are known to be α‐subunit homooligomers. This study uses 1 ‐ [ N ‐ (6 ‐ chloro ‐ 3 ‐ pyridylmethyl) ‐ N ‐ ethyl]amino ‐ 1 ‐ amino‐2‐nitroethene (which inhibits [ 3 H]IMI binding to Drosophila and Musca head membranes at 2–3 n M ) to develop a neonicotinoid‐agarose affinity column. The procedure—introduction of Triton‐solubilized Drosophila or Musca head membranes into this neonicotinoid‐based column, elution with IMI, and analysis by lithium dodecyl sulfate‐polyacrylamide gel electrophoresis—gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the α‐BGT‐agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with 125 I‐α‐BGT‐4‐azidosalicylic acid gives a labeled derivative of 66–69 kDa. The yield is 2–5 µg of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.