Hydrophobic photolabeling studies identify the lipid-protein interface of the 5-HT3A receptor

Mitesh Sanghvi, Ayman K. Hamouda, Margaret I. Davis, Russell A. Morton, Shouryadeep Srivastava, Akash Pandhare, Phaneendra K. Duddempudi, Tina K. Machu, David M. Lovinger, Jonathan B. Cohen, Michael P. Blanton

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

AHEK-293 cell line that stably expresses mouse 5-HT3ARs 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-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 displayed 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-labeled residue Thr422 at the lipid-exposed face of the Torpedo nicotinic acetylcholine receptor (nAChR) α1M4α-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.

Original languageEnglish (US)
Pages (from-to)9278-9286
Number of pages9
JournalBiochemistry
Volume48
Issue number39
DOIs
StatePublished - Oct 6 2009
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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