Identification of the Sites of Incorporation of [3H]Ethidium Diazide within the Torpedo Nicotinic Acetylcholine Receptor Ion Channel

The binding sites of ethidium, a noncompetitive antagonist of the nicotinic acetylcholine receptor (nAChR), have been localized in the Torpedo nAChR in the desensitized state by use of a photoactivatible derivative, [3H]ethidium diazide. At 10 μM [3H]ethidium diazide, incorporation into the α-, β-, and δ-subunits was inhibited by the presence of phencyclidine (PCP). Within the α-subunit, the incorporation was mapped to a 20-kDa fragment beginning at αSer-173 and containing the first three transmembrane segments, αM1, αM2, and αM3. Further digestion of this fragment generated two fragments with PCP-inhibitable incorporation, one containing αM1 and one containing both αM2 and αM3. Within αM2, specific incorporation was present in αLeu-251 and αSer-252, residues that have been previously shown to line the lumen of the ion channel. Digestion of the δ-subunit with S. aureus V8 protease generated a 14-kDa and a 20-kDa fragment, both of which began at Ile-192 and contained PCP-inhibitable labeling. The 14-kDa fragment, containing δM1 and δM2, was further digested to generate a 3-kDa fragment, containing δM2 alone, with PCP-inhibitable incorporation. Digestion of the 20-kDa fragment, which contained δM1, δM2, and δM3, generated two fragments with incorporation, one containing the δM1 segment and the other containing δM2 and δM3. These results establish that in the desensitized state of the nAChR, the high-affinity binding site of ethidium is within the lumen of the ion channel and that the bound drug is in contact with amino acids from both the M1 and M2 hydrophobic segments.