Molecular mechanisms of α-conotoxin [S9K]TxID selectively targeting the rat α3β4 nicotinic acetylcholine receptor

The α3β4 nicotinic acetylcholine receptor (nAChR) is a potential therapeutic target for the treatment of drug dependence and addiction, lung cancer, overeating and obesity and other disorders. α-Conotoxin analogue [S9K]TxID, obtained based on the modification of TxID, exhibited an IC₅₀ (half-maximal inhibitory concentration) of 9.0 nM on rat α3β4 nAChRs and > 950-fold higher selectivity than other subtypes, which could be one of the most potent and selective ligands for this receptor. In the hope to identify key residues responsible for the receptor selectivity of [S9K]TxID, in total 131 mutants of the α3β4 nAChR were generated with single amino acid substitution switching α3 and β4 to α6/α3 (chimera of α6 and α3 subunits) and β2 subunits respectively, against which the potencies of [S9K]TxID were evaluated using an electrophysiological approach. Fourteen amino acid residues in the α3β4 nAChR were identified as determinants responsible for the sensitivity to [S9K]TxID, among which the mutation at α3-K152, α3-Q195, β4-I108 and β4-Q116 decreased the [S9K]TxID activity to the greatest extent (7.4, 11, 18 and 14-fold, respectively). Molecular modeling was utilized to provide a possible explanation for the [S9K]TxID selectivity against α3β4 nAChRs. These results will contribute to the application of [S9K]TxID as a novel marine drug lead for α3β4 nAChR-related diseases.