The binding site for alpha-bungarotoxin resides in the sequence 188-201 of the alpha-subunit of acetylcholine receptor: structure, conformation and binding characteristics of peptide [Lys] 188-201.
In order to study where the binding site of cholinergic agents is in the sequence of the alpha-subunit of nicotinic acetylcholine receptor (AChR), we have synthetized 3 peptides with an amino acid sequence corresponding to the following sequences of the alpha-subunit of Torpedo californica AChR: 125-143, 158-167, [Lys] 188-201. For binding studies the peptides were immobilized on Sepharose 4B. Only the peptide [Lys] 188-201 binds 125I-alpha-bungarotoxin (alpha-Bgtx) with Kd of 1.03 microM. The binding of 125I-alpha-Bgtx to the peptide is reduced by 85% after reduction of the S-S bridge present between 192-193 cysteines indicating that an intact disulfide bond is important for toxin binding. The 125I-alpha-Bgtx binding is inhibited by curare, decamethonium, hexamethonium but not by carbamylcholine and Naja naja siamensis alpha-toxin and P15 toxin. All these data provide direct evidence that the sequence 188-201 of the alpha-subunit of AChR binds alpha-Bgtx and that this binding has a pharmacological profile similar to that of nicotinic acetylcholine receptor.
Gotti
C
C
Department of Pharmacology, University of Milano, Italy.
Mazzola
G
G
Longhi
R
R
Fornasari
D
D
Clementi
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism; D007700:Kinetics; D008968:Molecular Conformation; D010084:Oxidation-Reduction; D010277:Parasympathomimetics / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000032:analysis*; D011978:Receptors, Nicotinic; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014101:Torpedo; D064569:alpha7 Nicotinic Acetylcholine Receptor
D002038:Bungarotoxins; D010277:Parasympathomimetics; D010446:Peptide Fragments; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; D013438:Sulfhydryl Compounds; D064569:alpha7 Nicotinic Acetylcholine Receptor