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

To identify residues in the neuronal alpha(7) acetylcholine subunit that confer high affinity for the neuronal-specific toxin conotoxin ImI (CTx ImI), we constructed alpha(7)-alpha(1) chimeras containing segments of the muscle alpha(1) subunit inserted into equivalent positions of the neuronal alpha(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) 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 alpha(7)/5HT-3 over alpha(1)/5HT-3 homooligomers. Two of these pairs, alpha(7)Trp(55)/alpha(1)Arg(55) and alpha Ser(59)/alpha(1)Gln(59), 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, alpha(7)Thr(77)/alpha(1)Lys(77), 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 alpha(1) and alpha(7) subunits exchanges the affinities of the binding sites for CTx ImI, suggesting that the alpha(7) and alpha(1) subunits, despite sequence identity of only 38%, share similar protein scaffolds.