Loop2 Size Modification Reveals Significant Impacts on the Potency of a-Conotoxin TxID

alpha 4/6-conotoxin TxID, which was identified from Conus textile, simultaneously blocks rat (r) alpha 3 beta 4 and r alpha 6/alpha 3 beta 4 nicotinic acetylcholine receptors (nAChRs) with IC50 values of 3.6 nM and 33.9 nM, respectively. In order to identify the effects of loop2 size on the potency of TxID, alanine (Ala) insertion and truncation mutants were designed and synthesized in this study. An electrophysiological assay was used to evaluate the activity of TxID and its loop2-modified mutants. The results showed that the inhibition of 4/7-subfamily mutants [+9A]TxID, [+10A]TxID, [+14A]TxID, and all the 4/5-subfamily mutants against r alpha 3 beta 4 and r f alpha 6/ alpha 3 beta 4 nAChRs decreased. Overall, alainsertion or truncation of the 9th, 10th, and 11th amino acid results in a loss of inhibition and the truncation of loop2 has more obvious impacts on its functions. Our findings have strengthened the understanding of ff-conotoxin, provided guidance for further modifications, and offered a perspective for future studies on the molecular mechanism of the interaction between ff-conotoxins and nAChRs.

Automated summary

In this study, the activity of TxID and its loop2-modified mutants were evaluated using an electrophysiological assay. The results showed a decrease in inhibition against r alpha 3 beta 4 and r f alpha 6/ alpha 3 beta 4 nAChRs for the 4/7-subfamily mutants [+9A]TxID, [+10A]TxID, [+14A]TxID, and all the 4/5-subfamily mutants, indicating that the insertion or truncation of the 9th, 10th, and 11th amino acid results in a loss of inhibition, and the truncation of loop2 has more pronounced impacts on its functions. These findings enhance the understanding of ff-conotoxin, provide guidance for further modifications, and offer a perspective for future studies on the molecular mechanism of the interaction between ff-conotoxins and nAChRs.