Binding sites for α-bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the α-subunit of the nicotinic acetylcholine receptor

The binding of the competitive antagonist α-bungarotoxin (α-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the α-subunit of the Torpedo acetylcholine receptor has been characterized. 125I-α-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by α-Btx d-tubocurarine and NaCl. In the presence of 0.02% sodium dodecyl sulfate, 125I-α-Btx bound to the 56-residue peptide with a KD of 3.5 nM, as determined by equilibrium saturation binding studies. Because αBtx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptide comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, [3H]PCP was bound to the 172-227 peptide. [3H]PCP binding was inhibited by chlorpromazine, tetracaine, and dibucaine. It is concluded that a high-affinity binding site for PCP is located between residues 205 and 227, which includes the first 18 residues of transmembrane segment M1, and that a low-affinity site is located in the competitive antagonist binding site between residues 173 and 204. These results show that a synthetic peptide comprising residues 172-227 of the α subunit contains three binding sites, one for α-Btx and two for PCP. Previous studies on the intact receptor indicate high-affinity PCP binding occurs in the receptor channel