title: 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(3A)Rs containing a C-terminal extension that confers high-affinity binding of alpha-bungarotoxin (alphaBgTx) was established (alphaBgTx-5-HT(3A)Rs) and used to purify alphaBgTx-5-HT(3A)Rs in a lipid environment for use in structural studies using photoaffinity labeling. alphaBgTx-5-HT(3A)Rs were expressed robustly (60 pmol of [(3)H]BRL-43694 binding sites (approximately 3 microg of receptor) per milligram of protein) and displayed the same functional properties as wild-type receptors (serotonin EC(50) = 5.3 +/- 0.04 microM). While [(125)I]alphaBgTx bound to the alphaBgTx-5-HT(3A)Rs with high affinity (K(d) = 11 nM), application of nonradioactive alphaBgTx (up to 300 microM) had no effect on serotonin-induced current responses. alphaBgTx-5-HT(3A)Rs were purified on an alphaBgTx-derivatized affinity column from detergent extracts in milligram quantities and at approximately 25% purity. The hydrophobic photolabel 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) was used to identify the amino acids at the lipid-protein interface of purified and lipid-reconstituted alphaBgTx-5-HT(3A)Rs. [(125)I]TID photoincorporation into the alphaBgTx-5-HT(3A)R subunit was initially mapped to subunit proteolytic fragments of 8 kDa, containing the M4 transmembrane segment and approximately 60% of incorporated (125)I, and 17 kDa, containing the M1-M3 transmembrane segments. Within the M4 segment, [(125)I]TID labeled Ser(451), equivalent to the [(125)I]TID-labeled residue Thr(422) at the lipid-exposed face of the Torpedo nicotinic acetylcholine receptor (nAChR) alpha1M4 alpha-helix. These results provide a first definition of the surface of the 5-HT(3A)R M4 helix that is exposed to lipid and establish that this surface is equivalent to the surface exposed to lipid in the Torpedo nAChR.
authors.affiliation: Department of Pharmacology and Neuroscience and Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
authors.forename: Mitesh
authors.initials: M
authors.lastname: Sanghvi
authors.forename: Ayman K
authors.initials: AK
authors.lastname: Hamouda
authors.forename: Margaret I
authors.initials: MI
authors.lastname: Davis
authors.forename: Russell A
authors.initials: RA
authors.lastname: Morton
authors.forename: Shouryadeep
authors.initials: S
authors.lastname: Srivastava
authors.forename: Akash
authors.initials: A
authors.lastname: Pandhare
authors.forename: Phaneendra K
authors.initials: PK
authors.lastname: Duddempudi
authors.forename: Tina K
authors.initials: TK
authors.lastname: Machu
authors.forename: David M
authors.initials: DM
authors.lastname: Lovinger
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cohen
authors.forename: Michael P
authors.initials: MP
authors.lastname: Blanton
mesh_terms: D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008074:Lipoproteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D019920:Photoaffinity Labels / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry* / Q000378:metabolism; D014101:Torpedo / Q000378:metabolism
chemical_list: D002038:Bungarotoxins; C511230:Htr3a protein, mouse; D008074:Lipoproteins; D019920:Photoaffinity Labels; D011978:Receptors, Nicotinic; D044406:Receptors, Serotonin, 5-HT3