The ligand-gated ion channel superfamily of neurotransmitter receptors are proteins responsible for rapid transmission of nerve impulses at the synapse and have, therefore, been the subject of intensive research for many years. The cys-loop family, of which the 5-HT3 receptor is a member, includes the nicotinic acetylcholine receptor, the GABAA receptor and the glycine receptor. A diverse range of endogenous and artificial ligands activate these receptors, but, nevertheless, the family shares many similarities of structure and function. Several important questions, however, still remain to be determined, including the mechanism of agonist recognition at the binding site, the nature of the connection between the agonist binding and channel domains, the structure of the transmembrane regions and the mechanism of ion permeation and selectivity. This article reviews recent advances in the characterization of the molecular properties of the 5-HT3 receptor and their role in its function, and assesses its suitability as a model system for the study of the above questions.

We have used a homology model of the extracellular domain of the 5-HT 3 receptor to dock granisetron, a 5-HT 3 receptor antagonist, into the binding site using AUTODOCK. This yielded 13 alternative energetically favorable models. The models fell into 3 groups. In model type A the aromatic rings of granisetron were between Trp-90 and Phe-226 and its azabicyclic ring was between Trp-183 and Tyr-234, in model type B this orientation was reversed, and in model type C the aromatic rings were between Asp-229 and Ser-200 and the azabicyclic ring was between Phe-226 and Asn-128. Residues located no more than 5 Å from the docked granisetron were identified for each model; of 26 residues identified, 8 were found to be common to all models, with 18 others being represented in only a subset of the models. To identify which of the docking models best represents the ligand-receptor complex, we substituted each of these 26 residues with alanine and a residue with similar chemical properties. The mutant receptors were expressed in human embryonic kidney (HEK)293 cells and the affinity of granisetron determined using radioligand binding. Mutation of 2 residues (Trp-183 and Glu-129) ablated binding, whereas mutation of 14 other residues caused changes in the [ 3 H]granisetron binding affinity in one or both mutant receptors. The data showed that residues both in and close to the binding pocket can affect antagonist binding and overall were found to best support model B.The 5-HT 3 receptor is the only member of the 5-HT (serotonin) receptor family that is a ligand-gated ion channel. It is a member of the Cys-loop ligand-gated ion channel family, which includes nicotinic acetylcholine (nACh), 1 glycine and GABA A receptors. The receptors function as a pentameric arrangement of subunits. Each subunit has a large extracellular N-terminal region and four transmembrane domains (M1-M4). Five 5-HT 3 receptor subunits (A-E) have been identified although to date only homomeric (A only) or heteromeric (A and B) subunit complexes have been functionally characterized (1, 2). 5-HT 3 receptors may be evolutionarily the oldest members of the Cys-loop family (3), and this, combined with the ability of the A subunit to yield functional homomeric proteins, has meant that 5-HT 3 receptors provide a useful model system for understanding critical features of all Cys loop receptors (4). Most work on this family of proteins has been performed using nACh receptors, but despite many years of study structural details of the receptor-ligand interactions at the atomic level remain unknown. The determination of the structure of the acetylcholinebinding protein (AChBP), which is homologous to the extracellular domain of the nACh receptor, and indeed all Cys loop receptors, has significantly improved our knowledge of the ligand binding domain (5). However, some differences have emerged between the AChBP structure and cryoelectron microscopy data from the nACh receptor. For example, the extracellular domains of the nACh receptor ␣ subunits in the closed state di...

5-HT(3) receptors demonstrate significant structural and functional homology to other members of the Cys-loop ligand-gated ion channel superfamily. The extracellular domains of these receptors share similar sequence homology (approximately 20%) with Limnaea acetylcholine binding protein, for which an x-ray crystal structure is available. We used this structure as a template for computer-based homology modeling of the 5-HT(3) receptor extracellular domain. AutoDock software was used to dock 5-HT into the putative 5-HT(3) receptor ligand-binding site, resulting in seven alternative energetically favorable models. Residues located no more than 5 A from the docked 5-HT were identified for each model; of these, 12 were found to be common to all seven models with five others present in only certain models. Some docking models reflected the cation-pi interaction previously demonstrated for W183, and data from these and other studies were used to define our preferred models.

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...

Summary Circadian rhythms offer an excellent opportunity to dissect the neural circuits underlying innate behavior because the genes and neurons involved are relatively well-understood. We first sought to understand how Drosophila clock neurons interact in the simple circuit that generates circadian rhythms in larval light avoidance. We used genetics to manipulate two groups of clock neurons, increasing or reducing excitability, stopping their molecular clocks and blocking neurotransmitter release and reception. Our results revealed that Lateral Neuron (LNv) clock neurons promote and Dorsal Neurons (DN1s) inhibit light avoidance, that these neurons probably signal at different times of day, and that both signals are required for rhythmic behavior. We found similar principles apply in the more complex adult circadian circuit that generates locomotor rhythms. Thus the changing balance in activity between clock neurons with opposing behavioral effects generates robust circadian behavior and likely helps organisms transition between discrete behavioral states such as sleep and wakefulness.

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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.

5-HT3A receptors select among permeant ions based on size and charge. The membrane-associated (MA) helix lines the portals into the channel’s cytoplasmic vestibule in the 4-Å resolution structure of the homologous acetylcholine receptor. 5-HT3A MA helix residues are important determinants of single-channel conductance. It is unknown whether the portals into the cytoplasmic vestibule also determine the size selectivity of permeant ions. We sought to determine whether the portals form the size selectivity filter. Recently, we showed that channels functioned when the entire 5-HT3A M3–M4 loop was replaced by the heptapeptide M3–M4 loop sequence from GLIC, a bacterial Cys-loop neurotransmitter gated ion channel homologue from Gloebacter violaceus. We used homomeric 5-HT3A receptors with either a wild-type (WT) M3–M4 loop or the chimeric heptapeptide (5-HT3A–glvM3M4) loop, i.e., with or without portals. In Na+-containing buffer, the WT receptor current–voltage relationship was inwardly rectifying. In contrast, the 5-HT3A–glvM3M4 construct had a negative slope conductance region at voltages less than −80 mV. Glutamine substitution for the heptapeptide M3–M4 loop arginine eliminated the negative slope conductance region. We measured the relative permeabilities and conductances of a series of inorganic and organic cations ranging from 0.9 to 4.5 Å in radius (Li+, Na+, ammonium, methylammonium, ethanolammonium, 2-methylethanolammonium, dimethylammonium, diethanolammonium, tetramethylammonium, choline, tris [hydroxymethyl] aminomethane, and N-methyl-d-glucamine). Both constructs had measurable conductances with Li+, ammonium, and methylammonium (size range of 0.9–1.8-Å radius). Many of the organic cations >2.4 Å acted as competitive antagonists complicating measurement of conductance ratios. Analysis of the permeability ratios by excluded volume theory indicates that the minimal pore radius for 5-HT3A and 5-HT3–glvM3M4 receptors was similar, ∼5 Å. We infer that the 5-HT3A size selectivity filter is located in the transmembrane channel and not in the portals into the cytoplasmic vestibule. Thus, the determinants of size selectivity and conductance are located in physically distinct regions of the channel protein.