Identification of residues in the neuronal alpha7 acetylcholine receptor that confer selectivity for conotoxin ImI.
To identify residues in the neuronal alpha7 acetylcholine subunit that confer high affinity for the neuronal-specific toxin conotoxin ImI (CTx ImI), we constructed alpha7-alpha1 chimeras containing segments of the muscle alpha1 subunit inserted into equivalent positions of the neuronal alpha7 subunit. To achieve high expression in 293 human embryonic kidney cells and formation of homo-oligomers, we joined the extracellular domains of each chimera to the M1 junction of the 5-hydroxytryptamine-3 (5HT-3) subunit. Measurements of CTx ImI binding to the chimeric receptors reveal three pairs of residues in equivalent positions of the primary sequence that confer high affinity of CTx ImI for alpha7/5HT-3 over alpha1/5HT-3 homo-oligomers. Two of these pairs, alpha7Trp55/alpha1Arg55 and alpha7Ser59/alpha1Gln59, are within one of the four loops that contribute to the traditional non-alpha subunit face of the muscle receptor binding site. The third pair, alpha7Thr77/alpha1Lys77, is not within previously described loops of either the alpha or non-alpha faces and may represent a new loop or an allosterically coupled loop. Exchanging these residues between alpha1 and alpha7 subunits exchanges the affinities of the binding sites for CTx ImI, suggesting that the alpha7 and alpha1 subunits, despite sequence identity of only 38%, share similar protein scaffolds.
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Receptor Biology Laboratory, Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905, USA.
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D020916:Conotoxins; D006801:Humans; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D017354:Point Mutation; D011950:Receptors, Cholinergic / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012701:Serotonin / Q000378:metabolism
D020916:Conotoxins; D009842:Oligopeptides; D011950:Receptors, Cholinergic; C087686:alpha-conotoxin ImI; D012701:Serotonin