Tomizawa_1996_J.Neurochem_67_1669

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 [3H]IMI with KD values of 1-2 nM and Bmax values of 560-850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an alpha-bungarotoxin (alpha-BGT)-agarose affinity column are known to be alpha-subunit homooligomers. This study uses 1-[N-(6-chloro-3-pyridylmethyl)-N-ethyl]amino-1-amino-2-nitroethene++ + (which inhibits [3H]IMI binding to Drosophila and Musca head membranes at 2-3 nM) 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-polyacrylamicle 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 alpha-BGT-agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with 125I-alpha-BGT-4-azidosalicylic acid gives a labeled derivative of 66-69 kDa. The yield is 2-5 micrograms 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.