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The lipid requirements of the Torpedo californica nicotinic acetylcholine receptor (nAChR) were assessed by reconstituting purified receptors into lipid vesicles of defined composition to determine functionality. The results are consistent with a functional requirement of a single shell of lipids surrounding the nAChR and specific anionic lipid- and sterol (CH)-protein interactions.

A HEK-293 cell line expressing mouse 5-H[T.sub.3A]Rs containing a C-terminal extension that confers high-affinity binding of [alpha]-bungarotoxin ([alpha]BgTx) is established ([alpha]BgTx-5-H[T.sub.3A]Rs) and is used for purifying [alpha]BgTx-5-H[T.sub.3A]Rs in a lipid environment for use in structural studies by using photoaffinity labeling. The results have described the surface of the 5-H[T.sub.3A]RM4 helix that is exposed to lipid and has established that the surface is equivalent to the surface exposed to lipid in the Torpedo nicotinic acetylcholine receptor.

The structural studies of the lipid-protein interface of purified and lipid-reconstituted human [alpha]4[beta]2 neuronal nicotinic acetylcholine receptors (nAChRs) is reported. A high degree of structural homology is obtained by using stably transfected HEK-293 cell line.

A HEK-293 cell line that stably expresses mouse 5-HT3ARs containing a C-terminal extension that confers high-affinity binding of ct-bungarotoxin (αBgTx) was established (αBgTx-5-HT3ARs) and used to purify αBgTx-5-HT3ARs in a lipid environment for use in structural studies using photoaffinity labeling. αBgTx-5-HT3ARs were expressed robustly (60 pmol of [³H]BRL-43694 binding sites (∼3 μg of receptor) per milligram of protein) and dipIayed the same functional properties as wild-type receptors (serotonin EC50 = 5.3 ± 0.04 μM). While [125I]αBgTx bound to the αBgTx-5-HT3ARs with high affinity (Kd = 11 nM), application of nonradioactive αBgTx (up to 300 μM) had no effect on serotonin-induced current responses. αBgTx-5-HT3ARs were purified on an αBgTx-derivatized affinity column from detergent extracts in milligram quantities and at ∼25% purity. The hydrophobic photolabel 3-trifluoromethyl- 3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to identify the amino acids at the lipid-protein interface of purified and lipid-reconstituted αBgTx-5-HT3ARs. [125I]TID photoincorporation into the αBgTx-5-HT3AR subunit was initially mapped to subunit proteolytic fragments of 8 kDa, containing the M4 transmembrane segment and ∼60% of incorporated 125I and 17 kDa, containing the M1 - M3 transmembrane segments. Within the M4 segment, [125I]TID labeled Ser451, equivalent to the (125I]TID-Iabeled residue Thr422 at the lipid-exposed face of the Torpedo nicotinic acetylcholine receptor (nAChR) α1M4 α-helix. These results provide a first definition of the surface of the 5-HT3AR M4 helix that is exposed to lipid and establish that this surface is equivalent to the surface exposed to lipid in the Torpedo nAChR. [ABSTRACT FROM AUTHOR]

Using an acetylcholine-derivatized affinity column, we have purified human alpha4beta2 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 alpha4beta2 nAChR. In this first study, the lipid-protein interface of purified and lipid-reconstituted alpha4beta2 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 alpha4 and beta2 subunits, and for each subunit the labeling was initially mapped to fragments containing the M4 and M1-M3 transmembrane segments. For both the alpha4 and beta2 subunits, approximately 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 alpha4-Cys582/beta2-Cys445, which are also homologous to the [125I]TID-labeled residues alpha1-Cys418 and beta1-Cys447 in the lipid-exposed face of Torpedo nAChR alpha1M4 and beta1M4, respectively. Within the alpha4M1 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 alpha1M1 segment. In beta2M1, [125I]TID labeled beta2-Cys220, which is homologous to alpha4-Cys226. We conclude from these studies that the alpha4beta2 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 alpha4beta2 nAChR and the Torpedo nAChR display a high degree of structural homology.