Scorpion alpha-toxins LqhalphaIT, Lqh-2, and Lqh-3 are representatives of three groups of alpha-toxins that differ in their preference for insects and mammals. These alpha-insect, antimammalian, and alpha-like toxins bind to voltage-gated sodium channels and slow down channel inactivation. Sodium channel mutagenesis studies using various alpha-toxins have shown that they interact with receptor site 3, which is composed mainly of a short stretch of amino-acid residues between S3 and S4 of domain 4. Variation in this region results in marked differences between various subtypes of sodium channels with respect to their sensitivity to the three Lqh toxins. We incorporated the S3-S4 linker of domain 4 from hNaV1.2/hNaV1.1, hNaV1.3, hNaV1.6, and hNaV1.7 channels as well as individual point mutations into the rNaV1.4 skeletal muscle sodium channel. Our data show that the affinity of Lqh-3 and LqhalphaIT to sodium channels is markedly determined by an aspartate residue (Asp1428 in rNaV1.4); when mutated to glutamate, as is present in NaV1.1-1.3 channels, Lqh-3-channel interactions are abolished. The interaction of Lqh-2 and LqhalphaIT, however, is strongly reduced when a lysine residue (Lys1432 in rNaV1.4) is replaced by threonine (as in hNaV1.7), whereas this substitution is without effect for Lqh-3. The influence of Lys1432 on Lqh-2 and LqhalphaIT strongly depends on the context of the Asp/Glu site at position 1428, giving rise to a wide variety of toxicological phenotypes by means of a combinatorial mixing and matching of only a few residues in receptor site 3.