AFFINITY OF NATIVE KAPPA-BUNGAROTOXIN AND SITE-DIRECTED MUTANTS FOR THE MUSCLE NICOTINIC ACETYLCHOLINE-RECEPTOR

kappa-Bungarotoxin (kappa-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 kappa-neurotoxins that are distinguished from the structurally related alpha-neurotoxins by their ability to block the alpha(3)-subunit-containing neuronal nicotinic acetylcholine receptor (nAChR). It has been reported that venom-purified kappa-bgt also displays some affinity for the alpha(1)-subunit-containing muscle nAChR to which the alpha-neurotoxins bind with high affinity. Here we report the effects of particular mutations on the ability of recombinant kappa-bgt to block the binding. of I-125-alpha-bgt to nAChRs found in fetal mouse muscle and chick skeletal muscle. While the replacement of a proline residue found in all kappa-neurotoxins with an alanine (P-42-A) has relatively little effect, the introduction of a lysine, which is found in 90% of active a-neurotoxins at the same position (P-42-K), eliminates muscle receptor affinity at the concentrations tested. In contrast, the replacement of a glutamine in kappa-bgt with a tryptophan found in all active alpha-neurotoxins (Q-32-W) increases the affinity of kappa-bgt for the muscle receptor. When the arginine residue found in all active alpha- and kappa-neurotoxins is replaced by an alanine (R-40-A), the ability of kappa-bgt to block the muscle receptor is reduced to undetectable levels. The affinity of recombinant kappa-bgt for the muscle receptor is also shown to be 1-2 orders of magnitude lower than that of venom-purified kappa-bgt (IC50 = 10 mu M and 150 nM, respectively). We conclude that commercially available venom-purified kappa-bgt contains pharmacologically significant amounts of an alpha-neurotoxin, probably alpha-bgt, that is found in the same venom.