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PubMed · 12947760

[Sensitive nonradioactive screening method for compounds interacting with alpha7-cholinoreceptor].

A S Korotina·E V Kriukova·E A Azeeva·A F Sheval'e·Iu N Utkin·V I Tsetlin
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Abstract

A sensitive nonradioactive method for detection of substances interacting with the neuronal nicotinic acetylcholine alpha 7-type receptor (AChR) was proposed. The method uses biotinylated alpha-cobratoxin (Bt-CTX) and is based on the ability of the N-terminal ligand-binding extracellular domain (LBED) of AChR to interact with alpha-cobratoxin (CTX) as does the whole receptor. LBED was produced by heterologic expression of a gene fragment of the alpha 7 subunit of AChR from the rat brain in Escherichia coli cells sorbed on wells of a 96-well plate and incubated with Bt-CTX. The specifically bound Bt-CTX was determined by staining with streptavidin-peroxidase complex. The ability of other compounds to interact with alpha 7-AChR was checked according to the degree with which they inhibit Bt-CTX binding to LBED. Nicotine, carbamylcholine, d-tubocurarin, anabaseine, conotoxin ImI, and neurotoxin II were used as model compounds. The sensitivity of this method was comparable with that of the radioligand method (up to 10 pmol).

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AnabasineAnimalsBinding SitesBiotinBrainCarbacholCobra Neurotoxin ProteinsDrug Evaluation, PreclinicalEscherichia coliExtracellular MatrixLigandsNeuronsNicotineRatsReceptors, NicotinicSensitivity and SpecificityTubocurarinealpha7 Nicotinic Acetylcholine ReceptorJournal Article

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A S Korotina, E V Kriukova, E A Azeeva, A F Sheval'e, Iu N Utkin, V I Tsetlin. [Sensitive nonradioactive screening method for compounds interacting with alpha7-cholinoreceptor].. https://doi.org/10.1023/a%3A1024949101261

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