Mutagenesis of non-conventional toxin WTX from Naja kaouthia is performed.•
Activity of WTX and its mutants on muscle-type and α7-type of nAChRs was studied.•
R31 and R32 from the loop II is important for binding to both types of receptors.•
Modeling revealed the loop II entering into the orthosteric site of the α7 nAChRs.
‘Three-finger’ toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with 125I-α-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human α7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at α7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs.