Vol. 59, Issue 6, 1514-1522, June 2001

Interactions between 3-(Trifluoromethyl)-3-(m-[¹²⁵I]iodophenyl)diazirine and Tetracaine, Phencyclidine, or Histrionicotoxin in the Torpedo Species Nicotinic Acetylcholine Receptor Ion Channel

Martin J. Gallagher,¹ David C. Chiara, and Jonathan B. Cohen

Department of Neurobiology, Harvard Medical School, Boston, Massachusetts

3-(Trifluoromethyl)-3-(m-[¹²⁵I]iodophenyl)diazirine ([¹²⁵I]TID) and [³H]tetracaine, an aromatic amine, are noncompetitive antagonists (NCAs) of the Torpedo species nicotinic acetylcholine receptor (nAChR), which have been shown by photoaffinity labeling to bind to a common site in the ion channel in the closed state. Although tetracaine and TID bind to the same site, the amine NCAs phencyclidine (PCP) and histrionicotoxin (HTX), which are also believed to bind within the ion channel, interact competitively with tetracaine but allosterically with TID. To better characterize drug interactions within the nAChR ion channel in the closed state, we identified the amino acids photoaffinity labeled by [¹²⁵I]TID in the presence of tetracaine, PCP, or HTX. In the absence of other drugs, [¹²⁵I]TID reacts with Leu-251 (M2-9) and Val-255 (M2-13) and the homologous residues in each of the other subunits. None of the NCAs shifted the sites of [¹²⁵I]TID labeling to other residues within the ion channel. Tetracaine inhibited [¹²⁵I]TID labeling of M2-9 and M2-13 without changing the relative¹²⁵I incorporation at these positions, whereas PCP and HTX each altered the pattern of [¹²⁵I]TID incorporation at M2-9 and M2-13. These results indicate that tetracaine and TID bind in a mutually exclusive manner to a common site in the closed channel that is spatially separated from the binding sites for PCP and HTX.