Two Amino Acid Residues Contribute to a Cation-π Binding Interaction in the Binding Site of an Insect GABA Receptor

Cys-loop receptor binding sites characteristically possess an "aromatic box," where several aromatic amino acid residues surround the bound ligand. A cation-π interaction between one of these residues and the natural agonist is common, although the residue type and location are not conserved. Even in the closely related vertebrate GABA_A and GABA_C receptors, residues in distinct locations perform this role: in GABA_A receptors, a Tyr residue in loop A forms a cation-π interaction with GABA, while in GABA_C receptors it is a loop B residue. GABA-activated Cys-loop receptors also exist in invertebrates, where they have distinct pharmacologies and are the target of a range of pesticides. Here we examine the location of GABA in an insect binding site by incorporating a series of fluorinated Phe derivatives into the receptor binding pocket using unnatural amino acid mutagenesis, and evaluating the resulting receptors when expressed in Xenopus oocytes. A homology model suggests that two aromatic residues (in loops B and C) are positioned such that they could contribute to a cation-π interaction with the primary ammonium of GABA, and the data reveal a clear correlation between the GABA EC_(50) and the cation-π binding ability both at Phe206 (loop B) and Tyr254 (loop C), demonstrating for the first time the contribution of two aromatic residues to a cation-π interaction in a Cys-loop receptor

Structural Requirements in the Transmembrane Domain of GLIC Revealed by Incorporation of Noncanonical Histidine Analogs

The cyanobacterial pentameric ligand-gated ion channel GLIC, a homolog of the Cys-loop receptor superfamily, has provided useful structural and functional information about its eukaryotic counterparts. X-ray diffraction data and site-directed mutagenesis have previously implicated a transmembrane histidine residue (His234) as essential for channel function. Here, we investigated the role of His234 via synthesis and incorporation of histidine analogs and α-hydroxy acids using in vivo nonsense suppression. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell voltage-clamp electrophysiology was used to monitor channel activity. We show that an interhelix hydrogen bond involving His234 is important for stabilization of the open state, and that the shape and basicity of its side chain are highly sensitive to perturbations. In contrast, our data show that two other His residues are not involved in the acid-sensing mechanism

A Cation-π Interaction in the Binding Site of the Glycine Receptor Is Mediated by a Phenylalanine Residue

Cys-loop receptor binding sites characteristically contain many aromatic amino acids. In nicotinic ACh and 5-HT3 receptors, a Trp residue forms a cation-{pi} interaction with the agonist, whereas in GABAA receptors, a Tyr performs this role. The glycine receptor binding site, however, contains predominantly Phe residues. Homology models suggest that two of these Phe side chains, Phe159 and Phe207, and possibly a third, Phe63, are positioned such that they could contribute to a cation-{pi} interaction with the primary amine of glycine. Here, we test this hypothesis by incorporation of a series of fluorinated Phe derivatives using unnatural amino acid mutagenesis. The data reveal a clear correlation between the glycine EC50 value and the cation-{pi} binding ability of the fluorinated Phe derivatives at position 159, but not at positions 207 or 63, indicating a single cation-{pi} interaction between glycine and Phe159. The data thus provide an anchor point for locating glycine in its binding site, and demonstrate for the first time a cation-{pi} interaction between Phe and a neurotransmitter

Proline Residues in the Transmembrane/Extracellular Domain Interface Loops Have Different Behaviors in 5-HT_3 and nACh Receptors

Cys-loop receptors are important drug targets that are involved in signaling in the nervous system. The binding of neurotransmitters in the extracellular region of these receptors triggers an allosteric activation mechanism, the full details of which remain elusive, although structurally flexible loops in the interface between the extracellular region of Cys-loop receptors and the pore-forming transmembrane domain are known to play an important role. Here we explore the roles of three largely conserved Pro residues in two of these loops, the Cys-loop and M2-M3 loop, in 5-HT_3A and α7 nACh receptors. The data from natural and noncanonical amino acid mutagenesis suggest that in both proteins a Pro is essential in the Cys-loop, probably because of its enhanced ability to form a cis peptide bond, although other factors are also involved. The important characteristics of Pros in the M2-M3 loop, however, differ in these two receptors: in the 5-HT_3 receptor, the Pros can be replaced by some charged amino acids resulting in EC_(50)s similar to those of wild-type receptors, while such substitutions in the nACh receptor ablate function. Ala substitution at one of these Pros also has different effects in the two receptors. Thus, our data show that even highly conserved residues can have distinct behaviors in related Cys-loop receptors

5-HT3 receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The 5-HT3 receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on 5-Hydroxytryptamine (serotonin) receptors [66]) is a ligand-gated ion channel of the Cys-loop family that includes the zinc-activated channels, nicotinic acetylcholine, GABAA and strychnine-sensitive glycine receptors. The receptor exists as a pentamer of 4TM subunits that form an intrinsic cation selective channel [5]. Five human 5-HT3 receptor subunits have been cloned and homo-oligomeric assemblies of 5-HT3A and hetero-oligomeric assemblies of 5-HT3A and 5-HT3B subunits have been characterised in detail. The 5-HT3C (HTR3C, Q8WXA8), 5-HT3D (HTR3D, Q70Z44) and 5-HT3E (HTR3E, A5X5Y0) subunits [83, 122], like the 5-HT3B subunit, do not form functional homomers, but are reported to assemble with the 5-HT3A subunit to influence its functional expression rather than pharmacological profile [124, 63, 157]. 5-HT3A, -C, -D, and -E subunits also interact with the chaperone RIC-3 which predominantly enhances the surface expression of homomeric 5-HT3A receptor [157]. The co-expression of 5-HT3A and 5-HT3C-E subunits has been demonstrated in human colon [82]. A recombinant hetero-oligomeric 5-HT3AB receptor has been reported to contain two copies of the 5-HT3A subunit and three copies of the 5-HT3B subunit in the order B-B-A-B-A [7], but this is inconsistent with recent reports which show at least one A-A interface [96, 150]. The 5-HT3B subunit imparts distinctive biophysical properties upon hetero-oligomeric 5-HT3AB versus homo-oligomeric 5-HT3A recombinant receptors [32, 41, 56, 85, 139, 129, 79], influences the potency of channel blockers, but generally has only a modest effect upon the apparent affinity of agonists, or the affinity of antagonists ([17], but see [41, 30, 35]) which may be explained by the orthosteric binding site residing at an interface formed between 5-HT3A subunits [96, 150]. However, 5-HT3A and 5-HT3AB receptors differ in their allosteric regulation by some general anaesthetic agents, small alcohols and indoles [138, 135, 71]. The potential diversity of 5-HT3 receptors is increased by alternative splicing of the genes HTR3A and E [64, 19, 124, 123, 120]. In addition, the use of tissue-specific promoters driving expression from different transcriptional start sites has been reported for the HTR3A, HTR3B, HTR3D and HTR3E genes, which could result in 5-HT3 subunits harbouring different N-termini [152, 79, 120]. To date, inclusion of the 5-HT3A subunit appears imperative for 5-HT3 receptor function

5-HT3 receptors in GtoPdb v.2021.3

The 5-HT3 receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on 5-Hydroxytryptamine (serotonin) receptors [69]) is a ligand-gated ion channel of the Cys-loop family that includes the zinc-activated channels, nicotinic acetylcholine, GABAA and strychnine-sensitive glycine receptors. The receptor exists as a pentamer of 4 transmembrane (TM) subunits that form an intrinsic cation selective channel [7]. Five human 5-HT3 receptor subunits have been cloned and homo-oligomeric assemblies of 5-HT3A and hetero-oligomeric assemblies of 5-HT3A and 5-HT3B subunits have been characterised in detail. The 5-HT3C (HTR3C, Q8WXA8), 5-HT3D (HTR3D, Q70Z44) and 5-HT3E (HTR3E, A5X5Y0) subunits [86, 125], like the 5-HT3B subunit, do not form functional homomers, but are reported to assemble with the 5-HT3A subunit to influence its functional expression rather than pharmacological profile [127, 66, 161]. 5-HT3A, -C, -D, and -E subunits also interact with the chaperone RIC-3 which predominantly enhances the surface expression of homomeric 5-HT3A receptor [161]. The co-expression of 5-HT3A and 5-HT3C-E subunits has been demonstrated in human colon [85]. A recombinant hetero-oligomeric 5-HT3AB receptor has been reported to contain two copies of the 5-HT3A subunit and three copies of the 5-HT3B subunit in the order B-B-A-B-A [9], but this is inconsistent with recent reports which show at least one A-A interface [99, 154]. The 5-HT3B subunit imparts distinctive biophysical properties upon hetero-oligomeric 5-HT3AB versus homo-oligomeric 5-HT3A recombinant receptors [35, 44, 59, 88, 143, 132, 82], influences the potency of channel blockers, but generally has only a modest effect upon the apparent affinity of agonists, or the affinity of antagonists ([19], but see [44, 33, 38]) which may be explained by the orthosteric binding site residing at an interface formed between 5-HT3A subunits [99, 154]. However, 5-HT3A and 5-HT3AB receptors differ in their allosteric regulation by some general anaesthetic agents, small alcohols and indoles [142, 139, 73]. The potential diversity of 5-HT3 receptors is increased by alternative splicing of the genes HTR3A and HTR3E [67, 21, 127, 126, 123]. In addition, the use of tissue-specific promoters driving expression from different transcriptional start sites has been reported for the HTR3A, HTR3B, HTR3D and HTR3E genes, which could result in 5-HT3 subunits harbouring different N-termini [156, 82, 123]. To date, inclusion of the 5-HT3A subunit appears imperative for 5-HT3 receptor function

The structural basis of function in Cys-loop receptors

Cys-loop receptors are membrane-spanning neurotransmitter-gated ion channels that are responsible for fast excitatory and inhibitory transmission in the peripheral and central nervous systems. The best studied members of the Cys-loop family are nACh, 5-HT_3, GABA_A and glycine receptors. All these receptors share a common structure of five subunits, pseudo-symmetrically arranged to form a rosette with a central ion-conducting pore. Some are cation selective (e.g. nACh and 5-HT_3) and some are anion selective (e.g. GABA_A and glycine). Each receptor has an extracellular domain (ECD) that contains the ligand-binding sites, a transmembrane domain (TMD) that allows ions to pass across the membrane, and an intracellular domain (ICD) that plays a role in channel conductance and receptor modulation. Cys-loop receptors are the targets for many currently used clinically relevant drugs (e.g. benzodiazepines and anaesthetics). Understanding the molecular mechanisms of these receptors could therefore provide the catalyst for further development in this field, as well as promoting the development of experimental techniques for other areas of neuroscience. In this review, we present our current understanding of Cys-loop receptor structure and function. The ECD has been extensively studied. Research in this area has been stimulated in recent years by the publication of high-resolution structures of nACh receptors and related proteins, which have permitted the creation of many Cys loop receptor homology models of this region. Here, using the 5-HT_3 receptor as a typical member of the family, we describe how homology modelling and ligand docking can provide useful but not definitive information about ligand interactions. We briefly consider some of the many Cys-loop receptors modulators. We discuss the current understanding of the structure of the TMD, and how this links to the ECD to allow channel gating, and consider the roles of the ICD, whose structure is poorly understood. We also describe some of the current methods that are beginning to reveal the differences between different receptor states, and may ultimately show structural details of transitions between them

Tyrosine Residues That Control Binding and Gating in the 5-Hydroxytryptamine₃ Receptor Revealed by Unnatural Amino Acid Mutagenesis

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-HT₃ 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-HT₃ 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