Affinity of Native k-Bungarotoxin and Site-Directed Mutants for the Muscle Nicotinic Acetylcholine Receptor

k-Bungarotoxin (k-bgt) is a 66-residue peptide originally purified from snake venom that acts as an antagonist at certain acetylcholine receptors. It is one of four homologous k-neurotoxins that are distinguished from the structurally related α-neurotoxins by their ability to block the α₃-subunit-containing neuronal nicotinic acetylcholine receptor (nAChR). It has been reported that venom-purified k-bgt also displays some affinity for the α₁-subunit-containing muscle nAChR to which the α-neurotoxins bind with high affinity. Here we report the effects of particular mutations on the ability of recombinant k-bgt to block the binding of ¹²⁵I-α-bgt to nAChRs found in fetal mouse muscle and chick skeletal muscle. While the replacement of a proline residue found in all k-neurotoxins with an alanine (P-42-A) has relatively little effect, the introduction of a lysine, which is found in 90% of active α-neurotoxins at the same position (P-42-K), eliminates muscle receptor affinity at the concentrations tested. In contrast, the replacement of a glutamine in k-bgt with a tryptophan found in all active α-neurotoxins (Q-32-W) increases the affinity of k-bgt for the muscle receptor. When the arginine residue found in all active α- and k-neurotoxins is replaced by an alanine (R-40-A), the ability of k-bgt to block the muscle receptor is reduced to undetectable levels. The affinity of recombinant k-bgt for the muscle receptor is also shown to be 1–2 orders of magnitude lower than that of venom-purified k-bgt (IC₅₀ = 10 μ and 150 nM, respectively). We conclude that commercially available venom-purified k-bgt contains pharmacologically significant amounts of an α-neurotoxin, probably α-bgt, that is found in the same venom.