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 125I-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 alpha-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.(ABSTRACT TRUNCATED AT 250 WORDS)

[1]  J. Merlie,et al.  ACh receptor-rich membrane domains organized in fibroblasts by recombinant 43-kildalton protein , 1991, Science.

[2]  David A. Agard,et al.  α-Bungaratoxin structure revealed by a rapid method for averaging electron density of non-crystallographically translationally related molecules , 1980 .

[3]  P. Corfield,et al.  Erabutoxin b. Structure/function relationships following initial protein refinement at 0.140-nm resolution. , 1986, European journal of biochemistry.

[4]  G. A. Grant,et al.  Purification of an active species of recombinant kappa-bungarotoxin. , 1992, Protein expression and purification.

[5]  A. Drake,et al.  Molecular conformation of α-cobratoxin as studied by nuclear magnetic resonance and circular dichroism , 1982 .

[6]  R E Oswald,et al.  Solution structure of neuronal bungarotoxin determined by two-dimensional NMR spectroscopy: calculation of tertiary structure using systematic homologous model building, dynamical simulated annealing, and restrained molecular dynamics. , 1992, Biochemistry.

[7]  F. Inagaki,et al.  Proton-Nuclear-Magnetic-Resonance Study on Molecular Conformations of Long Neurotoxins , 1981 .

[8]  P. Clarke.  The fall and rise of neuronal α-bungarotoxin binding proteins , 1992 .

[9]  V. Basus,et al.  Structural studies of alpha-bungarotoxin. 2. 1H NMR assignments via an improved relayed coherence transfer nuclear overhauser enhancement experiment. , 1988, Biochemistry.

[10]  G. A. Grant,et al.  Synthesis and expression in Escherichia coli of a gene for kappa-bungarotoxin. , 1991, Biochemistry.

[11]  A. Drake,et al.  Molecular conformation of α-bungarotoxin as studied by nuclear magnetic resonance and circular dichroism , 1985 .

[12]  J. Richardson,et al.  Three dimensional structure of erabutoxin b neurotoxic protein: inhibitor of acetylcholine receptor. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[13]  D. Colquhoun,et al.  Effects of inhibitors of the binding of iodinated alpha-bungarotoxin to acetylcholine receptors in rat muscle. , 1976, Molecular pharmacology.

[14]  J. B. Cohen,et al.  Characterization of a snake venom neurotoxin which blocks nicotinic transmission in the avian ciliary ganglion , 1984, Neuroscience.

[15]  V. Chiappinelli,et al.  Kappa-neurotoxins: heterodimer formation between different neuronal nicotinic receptor antagonists. , 1989, Biochemistry.

[16]  A. Maelicke,et al.  Three-dimensional structure of the "long" neurotoxin from cobra venom. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[17]  V. Chiappinelli,et al.  κ-Bungarotoxin: binding of a neuronal nicotinic receptor antagonist to chick optic lobe and skeletal muscle , 1988, Brain Research.

[18]  G. A. Grant,et al.  κ2-Bungarotoxin and κ3-bungarotoxin: two new neuronal nicotinic receptor antagonists isolated from the venom of Bungarus multicinctus , 1990, Brain Research.

[19]  A. Ménez,et al.  Direct expression in E. coli of a functionally active protein A--snake toxin fusion protein. , 1989, Protein engineering.

[20]  C. Dobson,et al.  Solution structure of neuronal bungarotoxin determined by two-dimensional NMR spectroscopy: sequence-specific assignments, secondary structure, and dimer formation. , 1991, Biochemistry.

[21]  P. Corfield,et al.  The crystal structure of erabutoxin a at 2.0-A resolution. , 1990, The Journal of biological chemistry.

[22]  S. Zinn-Justin,et al.  Genetic engineering of snake toxins. Role of invariant residues in the structural and functional properties of a curaremimetic toxin, as probed by site-directed mutagenesis. , 1993, The Journal of biological chemistry.

[23]  C. Yu,et al.  Two-dimensional NMR studies and secondary structure of cobrotoxin in aqueous solution. , 1990, European journal of biochemistry.

[24]  C. Betzel,et al.  The refined crystal structure of alpha-cobratoxin from Naja naja siamensis at 2.4-A resolution. , 1993, The Journal of biological chemistry.

[25]  R. Loring,et al.  Amino acid sequence of toxin F, a snake venom toxin that blocks neuronal nicotinic receptors , 1986, Brain Research.

[26]  G. Scapin,et al.  Crystallization of κ-bungarotoxin: Preliminary X-ray data obtained from the venom-derived protein , 1992 .

[27]  I. Kuntz,et al.  Structural studies of alpha-bungarotoxin. 1. Sequence-specific 1H NMR resonance assignments. , 1988, Biochemistry.

[28]  R M Stroud,et al.  The crystal structure of alpha-bungarotoxin at 2.5 A resolution: relation to solution structure and binding to acetylcholine receptor. , 1986, Protein engineering.

[29]  L. Pillet,et al.  A recombinant snake neurotoxin generated by chemical cleavage of a hybrid protein recovers full biological properties , 1990, FEBS letters.

[30]  E. Hawrot,et al.  Functional expression and site-directed mutagenesis of a synthetic gene for alpha-bungarotoxin. , 1994, The Journal of biological chemistry.

[31]  J. Lee,et al.  kappa-Bungarotoxin. Self-association of a neuronal nicotinic receptor probe. , 1985, The Journal of biological chemistry.

[32]  C. Roumestand,et al.  Three-dimensional solution structure of a curaremimetic toxin from Naja nigricollis venom: a proton NMR and molecular modeling study. , 1992, Biochemistry.

[33]  Zhong-Wei Zhang,et al.  Nicotinic receptors that bind α-bungarotoxin on neurons raise intracellular free ca2+ , 1992, Neuron.

[34]  G. A. Grant,et al.  Site-directed mutagenesis of kappa-bungarotoxin: implications for neuronal receptor specificity. , 1994, Biochemistry.

[35]  J. B. Cohen,et al.  The effects of α- and β-neurotoxins from the venoms of various snakes on transmission in autonomic ganglia , 1981, Brain Research.

[36]  V. Chiappinelli,et al.  alpha-Bungarotoxin blocks nicotinic transmission in the avian ciliary ganglion. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[37]  V. Chiappinelli.  Kappa-Bungarotoxin: a probe for the neuronal nicotinic receptor in the avian ciliary ganglion , 1983, Brain Research.