title: Binding of native kappa-neurotoxins and site-directed mutants to nicotinic acetylcholine receptors.
abstract: 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 (IC50 < or = 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-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 position 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 Gln-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 (Kd approximately 10 microM) 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.
authors.affiliation: Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, MO 63104, USA.
authors.forename: V A
authors.initials: VA
authors.lastname: Chiappinelli
authors.forename: W R
authors.initials: WR
authors.lastname: Weaver
authors.forename: K E
authors.initials: KE
authors.lastname: McLane
authors.forename: B M
authors.initials: BM
authors.lastname: Conti-Fine
authors.forename: J J
authors.initials: JJ
authors.lastname: Fiordalisi
authors.forename: G A
authors.initials: GA
authors.lastname: Grant
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011978:Receptors, Nicotinic / Q000145:classification / Q000378:metabolism*
chemical_list: D009498:Neurotoxins; D011978:Receptors, Nicotinic