BINDING-SITES FOR ALPHA-BUNGAROTOXIN AND THE NONCOMPETITIVE INHIBITOR PHENCYCLIDINE ON A SYNTHETIC PEPTIDE COMPRISING RESIDUES 172-227 OF THE ALPHA-SUBUNIT OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR

The binding of the competitive antagonist alpha-bungarotoxin (alpha-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the alpha-subunit of the Torpedo acetylcholine receptor has been characterized. I-125-alpha-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by alpha-Btx (IC50 = 5.0 X 10(-8) M), d-tubocurarine (IC50 = 5.9 X 10(-5) M), and NaCl (IC50 = 7.9 X 10(-2) M). In the presence of 0.02% sodium dodecyl sulfate, I-125-alpha-Btx bound to the 56-residue peptide with a K(D) of 3.5 nM, as determined by equilibrium saturation binding studies. Because alpha-Btx 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, [H-3]PCP was bound to the 172-227 peptide. [H-3]PCP binding was inhibited by chlorpromazine (IC50 = 6.3 X 10(-5) M), tetracaine (IC50 = 4.2 X 10(-6) M), and dibucaine (IC50 = 2.7 X 10(-4) M). Equilibrium saturation binding studies in the presence of 0.02% sodium dodecyl sulfate showed that [H-3]PCP bound at two sites, a major site of high affinity with an apparent K(D) of 0.4-mu-M and a minor low-affinity site with an apparent K(D) of 4.6-mu-M. High-affinity binding occurred at a single site on peptide 205-227 (K(D) = 0.27-mu-M) and was competed by chlorpromazine but not by alpha-Btx. alpha-Btx and chlorpromazine competed the minor binding component on the 172-227 and 173-204 peptides. 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 alpha-subunit contains three binding sites, one for alpha-Btx and two for PCP. Previous studies on the intact receptor indicate high-affinity PCP binding occurs in the receptor channel. Thus, the high-affinity site within residues 205-227 may be exposed or closely approximated to the ion channel of the receptor. The proximity of the agonist binding site to a noncompetitive inhibitor binding site on the linear sequence of the alpha-subunit suggests that these sites could be tightly coupled and that this region could be involved in gating of the channel.