Identifying the Lipid−Protein Interface of the α4β2 Neuronal Nicotinic Acetylcholine Receptor: Hydrophobic Photolabeling Studies with 3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

Abstract

Using an acetylcholine-derivatized affinity column, we have purified human α4β2 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 α4β2 nAChR. In this first study, the lipid−protein interface of purified and lipid-reconstituted α4β2 nAChRs was directly examined using photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[¹²⁵I]iodophenyl)diazirine ([¹²⁵I]TID). [¹²⁵I]TID photoincorporated into both α4 and β2 subunits, and for each subunit the labeling was initially mapped to fragments containing the M4 and M1−M3 transmembrane segments. For both the α4 and β2 subunits, ∼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, [¹²⁵I]TID labeled homologous amino acids α4-Cys⁵⁸²/β2-Cys⁴⁴⁵, which are also homologous to the [¹²⁵I]TID-labeled residues α1-Cys⁴¹⁸ and β1-Cys⁴⁴⁷ in the lipid-exposed face of Torpedo nAChR α1M4 and β1M4, respectively. Within the α4M1 segment, [¹²⁵I]TID labeled residues Cys²²⁶ and Cys²³¹, which correspond to the [¹²⁵I]TID-labeled residues Cys²²² and Phe²²⁷ at the lipid-exposed face of the Torpedo α1M1 segment. In β2M1, [¹²⁵I]TID labeled β2-Cys²²⁰, which is homologous to α4-Cys²²⁶. We conclude from these studies that the α4β2 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 α4β2 nAChR and the Torpedo nAChR display a high degree of structural homology.