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

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Abstract
The hydrophobic photoreactive compound 3-trifluoromethyl-3-(m-( 125 I)iodophenyl) diazirine (( 125 I)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 (( 125 I)TID-BE) and a phospholipid analogue (( 125 I)TIDPC/16). For each photoreactive probe, labeled sites were identified by amino-terminal sequencing of purified tryptic fragments of individual receptor subunits. ( 125 I)TID-BE reacted with RCys-412, RMet-415, and RCys-418 in the M4 segment of the R-subunit and ÁCys-451 and ÁSer-460 in ÁM4. In the M1 segment of the R- and ‚-subunits, ( 125 I)TID- BE labeled RPhe-227, RLeu-228, and ‚Leu-234, ‚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 ( 125 I)TID-BE into the channel lining ‚M2 segment when the AChR was labeled in the resting state conformation. In the presence of agonist (desensitized state), ( 125 I)TID-BE reacted with ‚Leu-257, ‚Val-261, and ‚-Leu-264 in ‚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 ( 125 I)TIDPC/16, receptor labeling was insensitive to the presence of agonist and the sites of incorporation mapped to the confines of the transmembrane segments RM4, RM1, and ÁM4, validating previous results found with small lipophilic probes. Abbreviations: AChR, nicotinic acetylcholine receptor; TTD-BE, 4'-(3-trifluoromethyl-3H-diazirin-3-yl)-2'-tributylstannylbenzyl ben- zoate; TTDPC/16, 1-O-hexadecanoyl-2-O-(9-(((2-tributylstannyl)-4- (trifluoromethyl-3H-diazirin-3-yl)benzyl)oxy)carbonyl)nonanoyl)- sn- glycero-3-phosphocholine; TLC, thin-layer chromatography; ( 125 I)TID, 3-trifluoromethyl-3-(m-(