Probing the structure of the nicotinic acetylcholine receptor with the hydrophobic photoreactive probes [125I]TID-BE and [125I]TIDPC/16

The hydrophobic photoreactive compound 3-trifluoromethyl-3-(m-[I-125]iodophenyl) diazirine ([I-125]TID) has revealed important structural information about the pore of the ion channel and lipid-protein interface of the nicotinic acetylcholine receptor (AChR). To further characterize the structure of the AChR, we have mapped the sites of photoincorporation of a benzoic acid eater analogue of TID ([I-125]TID-BE),d a phospholipid analogue ([T-125]TIDPC/16). For each photoreactive probe, labeled sites were identified by amino-terminal sequencing of purified tryptic fragments of individual receptor subunits. [I-125]TID-BE reacted with alpha Cys-412, alpha Met-415, and alpha Cys-418 in the M4 segment 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 labeled alpha Phe-227, alpha Leu-228, and beta Leu-234, beta Ala-235, respectively. The labeling pattern in the M1 and M4 segments indicate that TID and TID-BE interact with the AChR lipid-protein interface in a similar fashion, revealing the same lipid-exposed face of each transmembrane segment. In contrast to TID, there was, however, no detectable incorporation of [I-125]TLD-BE into the channel lining beta M2 segment when the AChR was labeled in the resting state conformation. In the presence of agonist (desensitized state), [I-125]TID-BE reacted with beta Leu-257, beta Val-261, and beta-Leu-264 in beta M2; a labeling pattern 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 transmembrane segments alpha M4, alpha M1, and gamma M4, validating previous results found with small lipophilic probes.