Probing the Structure of the Nicotinic Acetylcholine Receptor with the Hydrophobic Photoreactive Probes [125I]TID-BE and [125I]TIDPC/16

• Published in: Biochemistry (Easton) Vol. 37; no. 41; pp. 14545 - 14555
• Main Authors: Blanton, Michael P, McCardy, Elizabeth A, Huggins, Angela, Parikh, Dipen
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 13.10.1998
• Subjects: Amino Acid Sequence; Animals; Azirines - chemistry; Azirines - metabolism; Cross-Linking Reagents; Iodine Radioisotopes; Models, Molecular; Peptide Fragments - isolation & purification; Peptide Fragments - metabolism; Peptide Mapping; Phosphatidylcholines - chemistry; Phosphatidylcholines - metabolism; Phospholipids - chemistry; Phospholipids - metabolism; Receptor, Muscarinic M2; Receptor, Muscarinic M4; Receptors, Muscarinic - metabolism; Receptors, Nicotinic - chemistry; Receptors, Nicotinic - metabolism; Torpedo
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi981435q

The hydrophobic photoreactive compound 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ([125I]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 ester analogue of TID ([125I]TID-BE) and a phospholipid analogue ([125I]TIDPC/16). For each photoreactive probe, labeled sites were identified by amino-terminal sequencing of purified tryptic fragments of individual receptor subunits. [125I]TID-BE reacted with αCys-412, αMet-415, and αCys-418 in the M4 segment of the α-subunit and γCys-451 and γSer-460 in γM4. In the M1 segment of the α- and β-subunits, [125I]TID-BE labeled αPhe-227, αLeu-228, and βLeu-234, βAla-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 [125I]TID-BE into the channel lining βM2 segment when the AChR was labeled in the resting state conformation. In the presence of agonist (desensitized state), [125I]TID-BE reacted with βLeu-257, βVal-261, and β-Leu-264 in βM2; 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 [125I]TIDPC/16, receptor labeling was insensitive to the presence of agonist and the sites of incorporation mapped to the confines of the transmembrane segments αM4, αM1, and γM4, validating previous results found with small lipophilic probes.