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

The 5-HT3 1 receptor is a member of the Cys-loop superfamily of ligand-gated ion channels (LGICs) and mediates excitatory fast synaptic transmission in the central and peripheral nervous systems (1). Presynaptic localized 5-HT3 receptors modulate the release of several neurotransmitters, including acetylcholine, dopamine, and γ-aminobutyric acid (2, 3) . In common with all members of the Cys-loop family of LGICs, 5-HT3 receptors are assembled as a pentamer of subunits that surround, in a pseudosymetric manner, a cation-selective ion channel (4–6). Of the five subunits (A–E) cloned to date, only the 5-HT3A and 5-HT3B subunits have been demonstrated to have functional significance in the central or peripheral nervous system (1, 7–9). The 5-HT3A subunit is able to assemble as a homomeric pentamer and is the predominant form of the 5-HT3 receptors present in the rodent brain (10). In contrast, the other 5-HT3R subunits (B–E) must coassemble with the 5-HT3A subunit to form functional receptors (9). The 5-HT3A subunit, along with all Cys-loop LGIC subunits, share a common structural architecture that is comprised of a large extracellular N-terminal domain, four transmembrane domains/α-helices (M1–M4), connected by extracellular (M2–M3) and intracellular (M1–M2 and M3–M4) loops, and an extracellular C-terminus (11–12). The five M2 helices are arranged about a central axis orthogonal to the membrane forming the channel lumen, and the M1, M3, and M4 helices form an outer ring that shields M2 from the lipid bilayer. Due to the abundance and purity of muscle-type nicotinic acetylcholine receptors (nAChRs) in preparations from the electric organ of the Torpedo electric ray, it is the most studied and understood member of the Cys-loop LGIC superfamily, and many of the structural/functional features of the Torpedo nAChR may be generalized to other member of the Cys-loop superfamily. The 5-HT3A subunit shares about 22–30% sequence identity/homology with Torpedo AChR subunits (1, 13) and a variety of studies utilizing different methodologies, including the construction of functional α7AChR/5-HT3AR chimeras, have contributed to the consensus view that the Torpedo AChR, the 5-HT3AR and other Cys-loop LGIC members display a common three-dimensional architecture (11–14). Nevertheless, direct structural information regarding the 5-HT3AR is currently lacking. This is in large part due to the low expression level of 5-HT3ARs (<0.3 µg receptor/mg of tissue) in the central and peripheral nervous system as well in most established mammalian cell lines (14–17). The establishment of a rich-source of 5-HT3AR protein and a means of purifying the receptor to or near homogeneity are two important steps in the effort to directly study the structure of the 5-HT3AR. We report here: 1) the establishment of an HEK-293 cell line (αBgTx-5-HT3AR) that stably and robustly (3–8 µg receptor/mg of membrane protein) expresses mouse 5-HT3ARs containing a C-terminal extension (pharmatope tag) that confers high affinity binding of the AChR competitive antagonist alpha bungarotoxin (αBgTx; 18–19) that does not bind to native mouse 5-HT3AR; and 2) purification on an αBgTx-derivatized affinity column of αBgTx-5-HT3ARs in mg quantities and at ~25% purity, reconstituted into lipid vesicles. We used these purified receptors to identify the amino acids photolabeled by 3-trifluoromethyl-3-(m-[125I] iodophenyl) diazirine ([125I]TID, the hydrophobic, photoreactive probe that has been used to identify amino acids at the lipid interface of Torpedo and α4β2 nAChRs (20–22).