Identifying the lipid-protein interface of the α4β2 neuronal nicotinic acetylcholine receptor:: Hydrophobic photolabeling studies with 3-(Trifluorornethyl)-3-(m-[125I]iodophenyl)diazirine

Using an acetylcholine-derivatized affinity column, we have purified human alpha 4 beta 2 neuronal nicotinic acetylcholine receptors (nAChRs) from a stably transfected HEK-293 cell line. Both the quantity and the quality of the purified receptor are suitable for applying biochemical methods to directly study the structure of the alpha 4 beta 2 nAChR. In this first study, the lipid-protein interface of purified and lipid-reconstituted alpha 4 beta 2 nAChRs was directly examined using photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[I-125]iodophenyl)diazirine ([I-125]TID). [I-125]TID photoincorporated into both alpha 4 and beta 2 subunits, and for each subunit the labeling was initially mapped to fragments containing the M4 and M1-M3 transmembrane segments. For both the alpha 4 and beta 2 subunits, similar to 60% of the total labeling was localized within fragments that contain the M4 segment, which suggests that the M4 segment has the greatest exposure to lipid. Within M4 segments, [I-125]TID labeled homologous amino acids alpha 4-CyS582/beta 2-CyS445, which are also homologous to the [I-125]TID-labeled residues alpha 1-CyS418 and beta 1-Cys 441 in the lipid-exposed face of Torpedo nAChR alpha 1M4 and beta 1M4, respectively. Within the alpha 4M1 segment, [I-125]TID labeled residues Cys(226) and Cys(231), which correspond to the [I-125]TID-labeled residues CyS222 and Phe(227) at the lipid-exposed face of the Torpedo alpha 1M1 segment. In beta 2M1, [I-125]TID labeled beta 2-CyS220, which is homologous to alpha 4-CyS226. We conclude from these studies that the alpha 4 beta 2 nAChR can be purified from stably transfected HEK-293 cells in sufficient quantity and purity for structural studies and that the lipid-protein interfaces of the neuronal alpha 4 beta 2 nAChR and the Torpedo nAChR display a high degree of structural homology.