Effect of Methionine Oxidation and Substitution of α-Conotoxin TxID on α3β4 Nicotinic Acetylcholine Receptor

alpha-Conotoxin TxID was discovered from Conus textile by gene cloning, which has 4/6 inter-cysteine loop spacing and selectively inhibits alpha 3 beta 4 nicotinic acetylcholine receptor (nAChR) subtype. However, TxID is susceptible to modification due to it containing a methionine (Met) residue that easily forms methionine sulfoxide (MetO) in oxidative environment. In this study, we investigated how Met-11 and its derivatives affect the activity of TxID using a combination of electrophysiological recordings and molecular modelling. The results showed most TxID analogues had substantially decreased activities on alpha 3 beta 4 nAChR with more than 10-fold potency loss and 5 of them demonstrated no inhibition on alpha 3 beta 4 nAChR. However, one mutant, [M11I]TxID, displayed potent inhibition at alpha 3 beta 4 nAChR with an IC50 of 69 nM, which only exhibited 3.8-fold less compared with TxID. Molecular dynamics simulations were performed to expound the decrease in the affinity for alpha 3 beta 4 nAChR. The results indicate replacement of Met with a hydrophobic moderate-sized Ile in TxID is an alternative strategy to reduce the impact of Met oxidation, which may help to redesign conotoxins containing methionine residue.