The hydrophobic photoreagent 3-(trifluoromethyl)-3-m-([125I] iodophenyl) diazirine is a novel noncompetitive antagonist of the nicotinic acetylcholine receptor.
We have shown previously that the lipophilic photoreagent 3-(trifluoromethyl)3-m-([125I]iodophenyl)-diazirine ([125I]TID) photolabels all four subunits of the Torpedo nicotinic acetylcholine receptor (AChR) and that greater than 70% of this photoincorporation is inhibited by cholinergic agonists and some noncompetitive antagonists, including histrionicotoxin (HTX), but not phencyclidine (PCP; White, B.H., and Cohen, J.B. (1988) Biochemistry 27, 8741-8751). We have now examined the effects of nonradioactive TID on (a) AChR photoincorporation of [125I]TID, (b) AChR-mediated ion transport, and (c) AChR binding of several cholinergic ligands. We find that TID inhibits [125I]TID photoincorporation into the AChR to the same extent as carbamylcholine. The saturable component of [125I]TID photolabeling is half-maximal at 4 microM [125I]TID with 0.5 mol specifically incorporated per mol of AChR after 30 min photolysis with 60 microM [125I]TID. Repeated labeling of membranes at a fixed [125I]TID concentration gave results consistent with a maximal incorporation of one [125I]TID molecule per AChR. Nonradioactive TID also noncompetitively inhibits agonist-stimulated 22Na+ efflux from Torpedo vesicles with an IC50 of 1 microM. Furthermore, TID inhibits allosterically the binding of [3H]HTX, decreasing its affinity for the AChR 5-fold both in the presence and absence of agonist. In contrast, TID has little effect on [3H]PCP binding in the absence of agonist but completely inhibits it in the presence of agonist. TID enhances the cooperativity of [3H]nicotine binding. [125I]TID is thus a photoaffinity label for a novel noncompetitive antagonist binding site on the AChR that is linked allosterically to the binding sites of both agonists and other noncompetitive antagonists. The [125I]TID site is presumably located within the central pore of the AChR.
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110.
B H
BH
White
S
S
Howard
S G
SG
Cohen
J B
JB
Cohen
D000345:Affinity Labels; D000664:Amphibian Venoms / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001389:Azirines / Q000378:metabolism / Q000494:pharmacology*; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007700:Kinetics; D046911:Macromolecular Substances; D018733:Nicotinic Antagonists; D010622:Phencyclidine / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D014101:Torpedo
D000345:Affinity Labels; D000664:Amphibian Venoms; D001389:Azirines; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D002217:Carbachol; D012964:Sodium; D010622:Phencyclidine; C007948:histrionicotoxin