PROBING THE STRUCTURE OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR WITH THE HYDROPHOBIC PHOTOREACTIVE PROBES [I-125] TID-BE AND [I-125] TIDPC 16/

The hydrophobic photoreactive compound 3-trifluoromethyl-3-(m-[I-125]i odophenyl) diazirine ([I-125]TID) has revealed important structural in formation about the pore of the ion channel and lipid-protein interfac e of the nicotinic acetylcholine receptor (AChR). To further character ize the structure of the AChR, we have mapped the sites of photoincorp oration of a benzoic acid eater analogue of TID ([I-125]TID-BE),d a ph ospholipid analogue ([T-125]TIDPC/16). For each photoreactive probe, l abeled sites were identified by amino-terminal sequencing of purified tryptic fragments of individual receptor subunits. [I-125]TID-BE react ed with alpha Cys-412, alpha Met-415, and alpha Cys-418 in the M4 segm ent of the alpha-subunit and gamma Cys-451 and gamma Ser-460 in gamma M4. In the M1 segment of the alpha- and beta-subunits, [I-125]TID-BE l abeled alpha Phe-227, alpha Leu-228, and beta Leu-234, beta Ala-235, r espectively. The labeling pattern in the M1 and M4 segments indicate t hat TID and TID-BE interact with the AChR lipid-protein interface in a similar fashion, revealing the same lipid-exposed face of each transm embrane segment. In contrast to TID, there was, however, no detectable incorporation of [I-125]TLD-BE into the channel lining beta M2 segmen t when the AChR was labeled in the resting state conformation. In the presence of agonist (desensitized state), [I-125]TID-BE reacted with b eta Leu-257, beta Val-261, and beta-Leu-264 in beta M2; a labeling pat tern which indicates that, in comparison to TID, the binding loci for TID-BE is located closer to the extracellular end of the channel. For [I-125]TIDPC/16, receptor labeling was insensitive to the presence of agonist and the sites of incorporation mapped to the confines of the t ransmembrane segments alpha M4, alpha M1, and gamma M4, validating pre vious results found with small lipophilic probes.