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, [H-3]ethidium diazide. At 10 mu M [H-3]ethidium diazide, incorporation into the alpha-, beta-, and delta-subunits was inhibited by the presence of phencyclidine (PCP). Within the alpha-subunit, the incorporation was mapped to a 20-kDa fragment beginning at alpha Ser-173 and containing the first three transmembrane segments, alpha M1, alpha M2, and alpha M3. Further digestion of this fragment generated two fragments with PCP-inhibitable incorporation, one containing alpha M1 and one containing both alpha M2 and alpha M3. Within alpha M2, specific incorporation was present in alpha Leu-251 and alpha Ser-252, residues that have been previously shown to line the lumen of the ion channel. Digestion of the g-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 delta M1 and delta M2, was further digested to generate a 3-kDa fragment, containing delta M2 alone, with PCP-inhibitable incorporation. Digestion of the 20-kDa fragment, which contained delta M1, dM2, and delta M3, generated two fragments with incorporation, one containing the delta M1 segment and the other containing dM2 and delta M3. 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.