The Conus peptides α-conotoxin ImI (α-ImI) and ImII (α-Imll) differ by only three of 11 residues in their primary sequences and yet are shown to inhibit the human α7 nicotinic acetylcholine receptor (nAChR) by targeting different sites. Mutations at both faces of the classical ligand binding site of the α7 nAChR strongly affect antagonism by α-ImI but not α-ImII. The effects of the mutations on α-ImI binding and functional antagonism are explained by computational docking of the NMR structure of α-ImI to a homology model of the ligand binding domain of the α7 nAChR. A distinct binding site for α-ImII is further demonstrated by its weakened antagonism for a chimeric receptor in which the membrane-spanning domains and intervening linkers of the α7 nAChR are replaced with the corresponding sequence from the serotonin type-3 receptor (5HT3). The two toxins also discriminate between different subtypes of human nicotinic receptors; α-ImII most strongly blocks the human α7 and α1β1δε receptor subtypes, while α-ImI most potently blocks the human α3β2 subtype. Collectively, the data show that while α-ImI targets the classical competitive ligand binding site in a subtype selective manner, α-ImII is a probe of a novel inhibitory site in homomeric α7 nAChRs.
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