Hydrophobic Photolabeling Studies Identify the Lipid−Protein Interface of the 5-HT3A Receptor

Abstract

A HEK-293 cell line that stably expresses mouse 5-HT_3ARs containing a C-terminal extension that confers high-affinity binding of α-bungarotoxin (αBgTx) was established (αBgTx-5-HT_3ARs) and used to purify αBgTx-5-HT_3ARs in a lipid environment for use in structural studies using photoaffinity labeling. αBgTx-5-HT_3ARs were expressed robustly (60 pmol of [³H]BRL-43694 binding sites (∼3 μg of receptor) per milligram of protein) and displayed the same functional properties as wild-type receptors (serotonin EC₅₀ = 5.3 ± 0.04 μM). While [¹²⁵I]αBgTx bound to the αBgTx-5-HT_3ARs with high affinity (K_d = 11 nM), application of nonradioactive αBgTx (up to 300 μM) had no effect on serotonin-induced current responses. αBgTx-5-HT_3ARs 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-HT_3ARs. [¹²⁵I]TID photoincorporation into the αBgTx-5-HT_3AR 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-labeled 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-HT_3AR M4 helix that is exposed to lipid and establish that this surface is equivalent to the surface exposed to lipid in the Torpedo nAChR.