Identification of residues in the neuronal α₇ acetylcholine receptor that confer selectivity for conotoxin ImI

To identify residues in the neuronal α₇ acetylcholine subunit that confer high affinity for the neuronal-specific toxin conotoxin ImI (CTx ImI), we constructed α₇-α₁ chimeras containing segments of the muscle α₁ subunit inserted into equivalent positions of the neuronal α₇ 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 α₇/5HT-3 over α₁/5HT-3 homo-oligomers. Two of these pairs, α₇Trp⁵⁵/α₁Arg⁵⁵ and α₇Ser⁵⁹/α₁Gln⁵⁹, are within one of the four loops that contribute to the traditional non-α subunit face of the muscle receptor binding site. The third pair, α₇Thr⁷⁷/α₁Lys⁷⁷, is not within previously described loops of either the α or non-α faces and may represent a new loop or an allosterically coupled loop. Exchanging these residues between α₁ and α₇ subunits exchanges the affinities of the binding sites for CTx ImI, suggesting that the α₇ and α₁ subunits, despite sequence identity of only 38%, share similar protein scaffolds.