To identify residues in the neuronal ␣ 7 acetylcholine subunit that confer high affinity for the neuronal-specific toxin conotoxin ImI (CTx ImI), we constructed ␣ 7 -␣ 1 chimeras containing segments of the muscle ␣ 1 subunit inserted into equivalent positions of the neuronal ␣ 7 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)

more »

... . 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 ␣ 7 /5HT-3 over ␣ 1 /5HT-3 homooligomers. Two of these pairs, ␣ 7 Trp 55 /␣ 1 Arg 55 and ␣ 7 Ser 59 /␣ 1 Gln 59 , are within one of the four loops that contribute to the traditional non-␣ subunit face of the muscle receptor binding site. The third pair, ␣ 7 Thr 77 / ␣ 1 Lys 77 , is not within previously described loops of either the ␣ or non-␣ faces and may represent a new loop or an allosterically coupled loop. Exchanging these residues between ␣ 1 and ␣ 7 subunits exchanges the affinities of the binding sites for CTx ImI, suggesting that the ␣ 7 and ␣ 1 subunits, despite sequence identity of only 38%, share similar protein scaffolds.