[Sensitive nonradioactive screening method for compounds interacting with alpha7-cholinoreceptor].
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).
Korotina
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AS
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, GSP Moscow, 117997 Russia.
Kriukova
E V
EV
Azeeva
E A
EA
Sheval'e
A F
AF
Utkin
Iu N
IuN
Tsetlin
V I
VI
D000691:Anabasine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D001921:Brain; D002217:Carbachol / Q000378:metabolism; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005109:Extracellular Matrix / Q000378:metabolism; D008024:Ligands; D009474:Neurons / Q000737:chemistry*; D009538:Nicotine / Q000378:metabolism; D051381:Rats; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D014403:Tubocurarine / Q000378:metabolism; D064569:alpha7 Nicotinic Acetylcholine Receptor
C579165:Chrna7 protein, rat; D003039:Cobra Neurotoxin Proteins; D008024:Ligands; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; C025154:alpha-cobratoxin; D009538:Nicotine; D001710:Biotin; D002217:Carbachol; C091713:anabaseine; D000691:Anabasine; D014403:Tubocurarine