Erabutoxin mutants demonstrate interface selectivity at human fetal and adult muscle-type nicotinic acetylcholine receptors

α-Neurotoxins, a prominent class of snake toxins belonging to the three-finger toxin (3FTx) superfamily, function as orthosteric antagonists of postsynaptic nicotinic acetylcholine receptors (nAChRs). Due to their high selectivity, α-neurotoxins have been widely used to characterize these physiologically important receptors. In this study, we investigated the concentration-response relationships of wild-type erabutoxin A (Ea), an α-neurotoxin, and six of its single-residue mutants by assessing their ability to inhibit acetylcholine-evoked currents mediated by α1β1γδ (fetal) and α1β1γε (adult) isoforms of human muscle-type nAChRs. These receptors were heterologously expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamp electrophysiology. Our results revealed that two mutants exhibited selectivity for the α1-δ interface: the S8T mutant displayed a selectivity pattern of α1-δ > α1-γ > α1-ε, whereas the K47E mutant showed α1-δ ≥ α1-γ > α1-ε. In contrast, the K27E and D31H mutants preferentially targeted the α1-ε interface (α1-ε > α1-γ ≥ α1-δ). The Q10A mutant showed no interface selectivity, and the R33E mutant largely lost its antagonist activity. These findings allowed us to define interface selectivity based on interactions between the mutants and six structural loops of the nAChR. This functional characterization enhances our understanding of receptor subtype selectivity and supports the future design of functionally selective protein probes targeting nAChRs and other ligand-gated ion channels.