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

• Published in: Toxicon (Oxford) 1996-11, Vol.34 (11), p.1243-1256
• Main Authors: Chiappinelli, Vincent A., Weaver, William R., McLane, Katya E., Conti-Fine, Bianca M., Fiordalisi, James J., Grant, Gregory A.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Molecular Sequence Data; Mutagenesis, Site-Directed; Neurotoxins - classification; Neurotoxins - genetics; Neurotoxins - metabolism; Protein Binding - genetics; Receptors, Nicotinic - classification; Receptors, Nicotinic - metabolism
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/S0041-0101(96)00110-9

The κ-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 α3- or α4-subunits ( IC 50 ≤ 100 nM). Four of these highly homologous, 66 amino acid peptides have been purified from the venom of Bungarus multicinctus [κ-bungarotoxin (κ-Bgt), κ2-Bgt, κ3-Bgt] and Bungarus flaviceps [κ-flavitoxin (κ-Fvt)]. Two approaches were taken to examine the binding of these toxins to nicotinic receptors. First, venom-derived κ-Fvt and κ-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 α3-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 κ-Bgt for binding to α3-containing receptors in autonomic ganglia and α1-containing muscle receptors. This was done using site-directed mutants of κ-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 α-neurotoxins) increased affinity for the muscle receptor by 8-fold. The results confirm that κ-neurotoxins bind potently to the α3-subunit and bind with considerably reduced affinity ( K d ≈ 10 μM) to muscle receptors. Site-directed mutagenesis of recombinant κ-Bgt is thus an important approach for the study of structure-function relationships between κ-Bgt and nicotinic receptors.