Va. Chiappinelli et al., BINDING OF NATIVE KAPPA-NEUROTOXINS AND SITE-DIRECTED MUTANTS TO NICOTINIC ACETYLCHOLINE-RECEPTORS, Toxicon, 34(11-12), 1996, pp. 1243-1256
The kappa-neurotoxins are useful ligands for the pharmacological chara
cterization of nicotinic acetylcholine receptors because they are pote
nt 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 puri
fied from the venom of Bungarus multicinctus [kappa-bungarotoxin (kapp
a-Bgt), kappa 2-Bgt, kappa 3-Bgt] and Bungarus flaviceps [kappa-flavit
oxin (kappa-Fvt)]. Two approaches were taken to examine the binding of
these toxins to nicotinic receptors. First, venom-derived kappa-Fvt a
nd 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 recep
tor 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 sequenc
e positions in kappa-Bgt for binding to alpha 3-containing receptors i
n autonomic ganglia and alpha 1-containing muscle receptors. This was
done using site-directed mutants of kappa-Bgt produced by an Escherich
ia coli expression system. Arg-34 and position 36 were important for b
inding to both receptor subtypes, while replacing Gin-26 with Trp-26 (
an invariant in alpha-neurotoxins) increased affinity for the muscle r
eceptor by 8-fold. The results confirm that kappa-neurotoxins bind pot
ently to the alpha 3-subunit and bind with considerably reduced affini
ty (K-d approximate to 10 mu M) to muscle receptors. Site-directed mut
agenesis of recombinant kappa-Bgt is thus an important approach for th
e study of structure-function relationships between kappa-Bgt and nico
tinic receptors.