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We characterized the differential accessibility of the nicotinic acetylcholine receptor α1 subunit in the open, closed, and desensitized states by using electrophysiology-coordinated photolabeling by several lipophilic probes followed by mass spectrometric analysis. Voltage-clamped oocytes expressing receptors were preincubated with one of the lipophilic probes and were continually exposed to acetylcholine; UV irradiation was applied during 500-ms pulses to + 40 or to -140 mV (which produced closed or ≈50% open receptors, respectively). In the open state, there was specific probe incorporation within the N-terminal domain at residues that align with the β8–β9 loop of the acetylcholine-binding protein. In the closed state, probe incorporation was identified at several sites of the N-terminal domain within the conserved cysteine loop (residues 128–142), the cytoplasmic loop (M3–M4), and M4. The labeling pattern in the M4 region is consistent with previous results, further defining the lipid-exposed face of this transmembrane α-helix. These results show regions within the N-terminal domain that are involved in gating-dependent conformational shifts, confirm that the cysteine loop resides at or near the protein-membrane interface, and show that segments of the M3–M4 loop are near to the lipid bilayer

Members of the heteropentameric ligand-gated ion channel superfamily rapidly mediate signaling across the synaptic cleft. Sequence analysis and limited experimental studies have yielded a topological model containing four transmembrane ␣-helices, labeled M1 to M4, and a large soluble, extracellular N-terminal domain. This model persists to date despite some recent structural studies that suggest it may be inappropriate. In this study, the topology of the glycine receptor was probed by limited proteolysis coupled to mass spectrometry. Of particular note, accessible cleavage sites within the putative M1 and M3 transmembrane helices were identified. Membrane-associated fragments within the postulated globular extracellular N-terminal domain were also observed. This report presents several key details incorporated in a new topological model and is the first direct experimental evidence that a subset of the transmembrane regions are too short to be membrane-spanning ␣-helices; rather, these regions are proposed to be a mix of ␣-helices and ␤-sheets. This report is also the first to exploit the capability of mass spectrometry to probe critically the topology of a class of membrane proteins of unknown structure.The glycine receptor (GlyR) 1 as well as the ␥-aminobutyric acid receptor, serotonin receptor, and nicotinic acetylcholine receptor (nAChR) comprise, in part, the ligand-gated ion channel superfamily (LGICS) (1). The GlyR is the major inhibitory neurotransmitter receptor in the post-synaptic membrane of spinal cord and lower brain. Upon binding the agonist glycine, the channel transiently opens, allowing passive flux of chloride, further hyperpolarizing the neuronal cell and thus reducing the probability of reaching an action potential. Like other members of the LGICS, the GlyR is a heteropentamer. Although the receptor is comprised of ␣ (48 kDa) and ␤ (58 kDa) subunits in vivo (2), recombinant GlyR ␣ subunits can assemble as homopentamers and retain native-like pharmacological activity (3)(4)(5). This simplified experimental system offers substantial benefits for structural investigations; we have developed a baculovirus overexpression system for the production of functional ␣1 GlyR which may be purified (5) and reconstituted 2 for subsequent characterization. The current topological model for all members of the LGICS, derived primarily from hydropathy predictions, is characterized by a large extracellular, globular N-terminal domain (residues 1-218 in ␣1 GlyR) containing the agonist-and antagonist-binding sites, four TM ␣-helices, designated M1 to M4, and a large cytoplasmic loop between M3 and M4 (6, 7). This model persists to date despite some recent structural studies that suggest it may be inappropriate. Fourier-transform infrared spectroscopy studies of nAChR suggest approximately equal proportions of ␣-helix and ␤-structure in the membrane (8), and electron diffraction studies of nAchR arrays suggested a limited helical content within the membrane (9). Recent secondary structure predictions of nAChR pl...