Binding of native kappa-neurotoxins and site-directed mutants to nicotinic acetylcholine receptors

The kappa-neurotoxins are useful ligands for the pharmacological characterization of nicotinic acetylcholine receptors because they are potent antagonists at only a subgroup of these receptors containing either alpha 3- or alpha 4-subunits (IC (50) less than or equal to 100 nM). Four of these highly homologous, 66 amino acid peptides have been purified from the venom of Bungarus multicinctus [kappa-bungarotoxin (kappa-Bgt), kappa 2-Bgt, kappa 3-Bgt] and Bungarus flaviceps [kappa-flavitoxin (kappa-Fvt)]. Two approaches were taken to examine the binding of these toxins to nicotinic receptors. First, venom-derived kappa-Fvt and kappa-Bgt were radioiodinated and the specific binding was measured of these toxins to overlapping synthetic peptides (16-20 amino acids in length) prepared based on the known sequence of the nicotinic receptor alpha 3-subunit. At least two main regions of interaction between the toxins and the receptor subunit were identified, both lying in the N-terminal region of the subunit that is exposed to the extracellular space. The second approach examined the importance of several sequence positions in kappa-Bgt for binding to alpha 3-containing receptors in autonomic ganglia and alpha 1-containing muscle receptors. This was done using site-directed mutants of kappa-Bgt produced by an Escherichia coli expression system. Arg-34 and position 36 were important for binding to both receptor subtypes, while replacing Gin-26 with Trp-26 (an invariant in alpha-neurotoxins) increased affinity for the muscle receptor by 8-fold. The results confirm that kappa-neurotoxins bind potently to the alpha 3-subunit and bind with considerably reduced affinity (K-d approximate to 10 mu M) to muscle receptors. Site-directed mutagenesis of recombinant kappa-Bgt is thus an important approach for the study of structure-function relationships between kappa-Bgt and nicotinic receptors.