5-HTThe 5-HT 3 1 receptor is a member of the Cys loop family of ligand-gated ion channels, which includes nicotinic acetylcholine (nACh), GABA A and glycine receptors. These receptors are pentamers, usually formed by the co-assembly of one to four different subunits each with a large extracellular N-terminal region and four putative transmembrane domains (M1-M4). Two 5-HT 3 receptor subunits, 5-HT 3A (1) and 5-HT 3B (2), have been identified so far, and receptors can function as either homo-oligomeric (A only) or hetero-oligomeric receptors (2). Evidence suggests that the Cys loop family of receptors is modular in design, with the extracellular N-terminal domain containing the ligand binding site and the transmembrane regions containing the pore (3). There is good evidence from a variety of studies that the second transmembrane segment, M2, lines the channel (4). Studies on acetylcholine receptors, for example, have identified rings of residues that alter conductance (5) or the selectivity among monovalent (5, 6) or divalent (7) cations or channel gating (8). The high resolution structure of a protein homologous to the extracellular domain of the acetylcholine receptor was recently determined (9); however, so far details of the complete structure of any of this family of receptors are lacking.The substituted cysteine accessibility method (SCAM) has been used to identify systematically the residues that line an ion channel. Here residues in a membrane-spanning segment are individually mutated to cysteine and each mutant receptor expressed in Xenopus oocytes. If the mutant receptors have similar properties to wild type, it can be assumed that their structure is similar to that of wild type. The accessibility of each residue can then be determined by examining the ability of small sulfydryl-specific reagents to react with the cysteine. The information gained is able to provide information on the secondary structure of channel-lining segments and the location of ion channel gates and selectivity filters and to map binding sites within the channel (10). SCAM has been used to identify pore-lining residues in a variety of ion channels, including the nACh and GABA A receptors. The M2 regions in these receptors gave a similar but not identical pattern of labeling and supported previous studies suggesting that this region is largely ␣-helical. There are, however, some discrepancies, particularly in the region surrounding the conserved central leucine residue.The amino acid sequence of the 5-HT 3A receptor subunit displays strong sequence similarity with nACh receptor subunits, especially in the M2 region (e.g. the ␣ 1 nACh receptor subunit as illustrated in Fig. 1). We therefore wanted to confirm that the water-accessible residues in this receptor are similar to those in the nACh and GABA A receptors and also explore whether the use of homomeric receptors could provide additional information about the structure and function of M2. The data revealed a similar but not identical pattern of wateraccessible residues in the por...

GABA A receptors are ligand-gated ion channels formed by the pseudosymmetrical assembly of five homologous subunits around the central channel axis. The five M2 membrane-spanning segments largely line the channel. In the present work we probed the water surface accessibility of the ␤ 1 subunit M2 segment using the substituted cysteine accessibility method. We assayed the reaction of the negatively charged sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS ؊ ), by its effect on subsequent currents elicited by EC 50 and saturating GABA concentrations. pCMBS ؊ , applied with GABA, reacted with 14 of the 19 residues tested. At the M2 cytoplasmic end from 2 to 6 only ␤ 1 A252C (2) and ␤ 1 T256C (6) were pCMBS ؊ -reactive in the presence of GABA. We infer that the M2 segments are tightly packed in this region. Toward the extracellular half of M2 all residues from ␤ 1 T262C (12) through ␤ 1 E270C (20) reacted with pCMBS ؊ applied with GABA. We infer that this region is highly mobile and loosely packed against the rest of the protein. Based on differences in pCMBS ؊ reaction rates two domains can be distinguished on the putative channel-lining side of M2. A faster reacting domain includes the 2, 9, 12, 13, and 16 residues. The slower reacting face contains the 6, 10, and 14 residues. We hypothesize that these may represent the channel-lining faces in the closed and open states and that gating involves an 80 -100°rotation of the M2 segments. These results are consistent with the loose packing of the M2 segments inferred from the structure of the homologous Torpedo nicotinic acetylcholine receptor.