GABA A receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABA A receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their concentration, occupy two possible sites in ELIC. An intrasubunit site is adjacent to the GABA-recognition site but faces the channel vestibule. A second intersubunit site partially overlaps with the GABA site and likely corresponds to a low-affinity benzodiazepine-binding site in GABA A receptors that mediates inhibitory effects of the benzodiazepine flurazepam. Our study offers a structural view how GABA and benzodiazepines are recognized at a GABA-activated ion channel.
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...
Background: Pentameric ligand-gated ion channels are modulated by general anesthetics.Results: The crystal structure of ELIC in complex with bromoform reveals anesthetic binding in the channel pore and in novel sites in the transmembrane and extracellular domain.Conclusion: General anesthetics allosterically modulate channel function via multisite binding.Significance: Our data reveal detailed insight into multisite recognition of general anesthetics at the structural level.
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.
The mechanism by which agonist binding triggers pore opening in ligand-gated ion channels is poorly understood. Here, we used unnatural amino acid mutagenesis to introduce subtle changes to the side chains of tyrosine residues (Tyr141, Tyr143, Tyr153, and Tyr234), which dominate the 5-HT 3 receptor binding site. Heterologous expression in oocytes, combined with radioligand binding data and a model of 5-HT (serotonin) computationally docked into the binding site, has allowed us to determine which of these residues are responsible for binding and/or gating. We have shown that Tyr 143 forms a hydrogen bond that is essential for receptor gating but does not affect binding, whereas a hydrogen bond formed by Tyr153 is involved in both binding and gating of the receptor. The aromatic group of Tyr234 is essential for binding and gating, whereas its hydroxyl does not affect binding but plays a steric role in receptor gating. Tyr141 is not involved in agonist binding or receptor gating but is important for antagonist interactions. These data, combined with a new model of the nonliganded 5-HT 3 receptor, lead to a mechanistic explanation of the interactions that initiate the conformational change leading to channel opening. Thus, we suggest that agonist entry into the binding pocket may displace Tyr143 and Tyr153 and results in their forming new hydrogen bonds. These bonds may form part of the network of bond rearrangements that trigger the conformational change leading to channel opening. Similar rearrangements may initiate gating in all Cys-loop receptors.
RDL receptors are GABA-activated inhibitory Cys-loop receptors found throughout the insect CNS. They are a key target for insecticides. Here, we characterize the GABA binding site in RDL receptors using computational and electrophysiological techniques. A homology model of the extracellular domain of RDL was generated and GABA docked into the binding site. Molecular dynamics simulations predicted critical GABA binding interactions with aromatic residues F206, Y254, and Y109 and hydrophilic residues E204, S176, R111, R166, S176, and T251. These residues were mutated, expressed in Xenopus oocytes, and their functions assessed using electrophysiology. The data support the binding mechanism provided by the simulations, which predict that GABA forms many interactions with binding site residues, the most significant of which are cation-π interactions with F206 and Y254, H-bonds with E204, S205, R111, S176, T251, and ionic interactions with R111 and E204. These findings clarify the roles of a range of residues in binding GABA in the RDL receptor, and also show that molecular dynamics simulations are a useful tool to identify specific interactions in Cys-loop receptors.
Recent studies on the pathogenic mechanisms of recessive hyperekplexia indicate disturbances in glycine receptor (GlyR) ␣1 biogenesis. Here, we examine the properties of a range of novel glycine receptor mutants identified in human hyperekplexia patients using expression in transfected cell lines and primary neurons. All of the novel mutants localized in the large extracellular domain of the GlyR ␣1 have reduced cell surface expression with a high proportion of receptors being retained in the ER, although there is forward trafficking of glycosylated subpopulations into the ER-Golgi intermediate compartment and cis-Golgi compartment. CD spectroscopy revealed that the mutant receptors have proportions of secondary structural elements similar to wild-type receptors. Two mutants in loop B (G160R, T162M) were functional, but none of those in loop D/2-3 were. One nonfunctional truncated mutant (R316X) could be rescued by coexpression with the lacking C-terminal domain. We conclude that a proportion of GlyR ␣1 mutants can be transported to the plasma membrane but do not necessarily form functional ion channels. We suggest that loop D/2-3 is an important determinant for GlyR trafficking and functionality, whereas alterations to loop B alter agonist potencies, indicating that residues here are critical elements in ligand binding.
Non-technical summaryNerve signals are transmitted across cell membranes by receptors that can consist of multiple different subunits. The 5-HT3 receptor is a pentamer which can function with A subunits alone, or with a mixture of A and B subunits. As 5-HT activates the receptor by binding at the interface of adjacent subunits, it is important to know which subunits are adjacent. Here we show that in both A-only and A+B receptors there is at least one A–A interface, without which the receptor cannot function. This knowledge is important for understanding the receptor mechanism, and also will allow the design of more specific drugs that act at the 5-HT binding site.
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