IDENTIFICATION OF RESIDUES IN THE NEURONAL ALPHA(7) ACETYLCHOLINE-RECEPTOR THAT CONFER SELECTIVITY FOR CONOTOXIN IMI

To identify residues in the neuronal alpha(7) acetylcholine subunit th at confer high affinity for the neuronal-specific toxin conotoxin ImI (CTx ImI), we constructed alpha(7)-alpha(1) chimeras containing segmen ts of the muscle alpha(1) subunit inserted into equivalent positions o f the neuronal alpha(7) subunit. To achieve high expression in 293 hum an embryonic kidney cells and formation of homo-oligomers, we joined t he extracellular domains of each chimera to the M1 junction of the 5-h ydroxytryptamine-3 (5HT-3) subunit, Measurements of CTx ImI binding to the chimeric receptors reveal three pairs of residues in equivalent p ositions 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 pai rs, 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 n on-alpha subunit face of the muscle receptor binding site. The third p air, alpha(7)Thr(77)/alpha(1)Lys(77), is not within previously describ ed loops of either the alpha or non-alpha faces and may represent a ne w loop or an allosterically coupled loop. Exchanging these residues be tween alpha(1) and alpha(7) subunits exchanges the affinities of the b inding sites for CTx ImI, suggesting that the alpha(7) and alpha(1) su bunits, despite sequence identity of only 38%, share similar protein s caffolds.