Analysis of the Ligand Binding Site of the 5-HT 3 Receptor Using Site Directed Mutagenesis: Importance of Glutamate 106

Boess, Frank; Steward, Lucinda J.; Steele, Joy A.; Liu, D.; Reid, John L.; Glencorse, Thora A.; Martin, Ian L.

doi:10.1016/s0028-3908(97)00044-0

Cited by 76 publications

(60 citation statements)

References 36 publications

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“…Given that radioligand binding was completely ablated when Glu-129 was replaced with the chemically similar aspartate, yet the receptors reached the membrane, we propose that this residue is critical for the local structure of the binding pocket. Our data are somewhat surprising given previous experiments that reported both radioligand binding (of a different antagonist GR65630) and function of an E129D mutant 5-HT 3 receptor (Glu-106 by their numbering) (35). These authors reported poor expression of Glu-129 mutants (E129A and E129Q receptors expressed too poorly to characterize them) and used a different splice variant of the receptor, which might explain these differences.…”

Section: Fig 2 Representative Examples Of the Three Model Typescontrasting

confidence: 82%

Locating an Antagonist in the 5-HT3 Receptor Binding Site Using Modeling and Radioligand Binding

Thompson

Price

Reeves

et al. 2005

Journal of Biological Chemistry

119

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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...

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Section: Fig 2 Representative Examples Of the Three Model Typescontrasting

confidence: 82%

Locating an Antagonist in the 5-HT3 Receptor Binding Site Using Modeling and Radioligand Binding

Thompson

Price

Reeves

et al. 2005

Journal of Biological Chemistry

119

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“…In the AChBP and, by inference, the other members of the cys-loop ligand-gated ion channel family, the ligand-binding domain is at the interface between subunits and is made up of a series of six domains (A-F), three from one subunit (A-C), and three from the adjacent subunit (D-F) (Brejc et al, 2001;Celie et al, 2004). Consistent with this notion, studies from several laboratories have identified a number of residues at or near the 5-HT 3 R ligand-binding site (Boess et al, 1997;Hope et al, 1999;Yan et al, 1999;Spier and Lummis, 2000;Venkataraman et al, 2002;Yan and White, 2002;Schreiter et al, 2003;Price and Lummis, 2004) that are located in the various binding-site domains in the model.…”

Section: Resultsmentioning

confidence: 82%

Spatial Orientation of the Antagonist Granisetron in the Ligand-Binding Site of the 5-HT₃ Receptor

Dong¹,

White²

2005

Mol Pharmacol

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The serotonin type 3 receptor (5-HT 3 R) is a member of the cys-loop ligand-gated ion channel (LGIC) superfamily. Like almost all membrane proteins, high-resolution structural data are unavailable for this class of receptors. We have taken advantage of the high degree of homology between LGICs and the acetylcholine binding protein (AChBP) from the freshwater snail Lymnea stagnalis, for which high-resolution structural data are available, to create a structural model for the extracellular (i.e., ligand-binding) domain of the 5-HT 3 R and to perform a series of ligand docking experiments to delineate the architecture of the ligand-binding site. Structural models were created using homology modeling with the AChBP as a template. Docking of the antagonist granisetron was carried out using a Lamarckian genetic algorithm to produce models of ligand-receptor complexes. Two energetically similar conformations of granisetron in the binding site were obtained from the docking simulations. In one model, the indazole ring of granisetron is near Trp90 and the tropane ring is near Arg92; in the other, the orientation is reversed. We used double-mutant cycle analysis to determine which of the two orientations is consistent with experimental data and found that the data are consistent with the model in which the indazole ring of granisetron interacts with Arg92 and the tropane ring interacts with Trp90. The combination of molecular modeling with double-mutant cycle analysis offers a powerful approach for the delineation of the architecture of the ligand-binding site.The serotonin type 3 receptor (5-HT 3 R) is a member of the cys-loop ligand-gated ion channel gene family, which includes the muscle and neuronal nicotinic acetylcholine receptors, the glycine receptor, and the GABA A receptor (Connolly and Wafford, 2004;Lester et al., 2004). Two different subunits, termed 5-HT 3A and 5-HT 3B , have been described previously (Reeves and Lummis, 2002). The 5-HT 3A subunit forms functional receptors with the appropriate pharmacological properties when expressed in Xenopus laevis oocytes or mammalian cells. However, there are some differences between the properties of the expressed homomeric receptors and 5-HT 3 Rs in some, but not all, neurons. Perhaps the most significant difference is that the single-channel conductance of the expressed receptors is in the subpicosiemen range, whereas that of the receptors in many (but not all) neurons is in the range of 10 to 20 pS (Yang et al., 1992;Hussy et al., 1994). This difference, along with the fact that other members of the cys-loop LGIC family are composed of several different subunits, led to the search and subsequent discovery of an additional 5-HT 3 R subunit, now termed the 5-HT 3B subunit (Davies et al., 1999;Dubin et al., 1999).When expressed by itself, the 5-HT 3B subunit does not form functional receptors. When the 5-HT 3B subunit is coexpressed with the 5-HT 3A subunit, the ligand-binding properties of the expressed receptors are identical to those resulting from expressi...

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“…Initial radioligand binding studies were performed by MDS Pharma Services (Bothell, WA) as previously described for serotonin receptor subtypes 1A, 1B, 2B, 2C, 3, 5A, 6, 7, serotonin transporter [5,6,8,23,31,32,[37][38][39][40][41][43][44][45]47]. Significant binding affinity was only observed at the 5-HT 5a receptor (>50%) for the PE and DCM extracts at a concentration of 50 μg/ml [12].…”

Section: Serotonin Receptor Binding Assaysmentioning

confidence: 99%

Valerian extract and valerenic acid are partial agonists of the 5-HT5a receptor in vitro

Dietz

Mahady

Pauli

et al. 2005

Molecular Brain Research

126

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Insomnia is the most frequently encountered sleep complaint worldwide. While many prescription drugs are used to treat insomnia, extracts of valerian (Valeriana officinalis L., Valerianaceae) are also used for the treatment of insomnia and restlessness. To determine novel mechanisms of action, radioligand binding studies were performed with valerian extracts (100% methanol, 50% methanol, dichloromethane [DCM], and petroleum ether [PE]) at the melatonin, glutamate, and GABA A receptors, and 8 serotonin receptor subtypes. Both DCM and PE extracts had strong binding affinity to the 5-HT 5a receptor, but only weak binding affinity to the 5-HT 2b and the serotonin transporter. Subsequent binding studies focused on the 5-HT 5a receptor due to the distribution of this receptor in the suprachiasmatic nucleus of the brain, which is implicated in the sleep-wake cycle. The PE extract inhibited [ 3 H]lysergic acid diethylamide (LSD) binding to the human 5-HT 5a receptor (86% at 50 μg/ml) and the DCM extract inhibited LSD binding by 51%. Generation of an IC 50 curve for the PE extract produced a biphasic curve, thus GTP shift experiments were also performed. In the absence of GTP, the competition curve was biphasic (two affinity sites) with an IC 50 of 15.7 ng/ml for the high-affinity state and 27.7 μg/ml for the lowaffinity state. The addition of GTP (100 AM) resulted in a right-hand shift of the binding curve with an IC 50 of 11.4 μg/ml. Valerenic acid, the active constituent of both extracts, had an IC 50 of 17.2 AM. These results indicate that valerian and valerenic acid are new partial agonists of the 5-HT 5a receptor.

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Analysis of the Ligand Binding Site of the 5-HT 3 Receptor Using Site Directed Mutagenesis: Importance of Glutamate 106

Cited by 76 publications

References 36 publications

Locating an Antagonist in the 5-HT3 Receptor Binding Site Using Modeling and Radioligand Binding

Locating an Antagonist in the 5-HT3 Receptor Binding Site Using Modeling and Radioligand Binding

Spatial Orientation of the Antagonist Granisetron in the Ligand-Binding Site of the 5-HT₃ Receptor

Valerian extract and valerenic acid are partial agonists of the 5-HT5a receptor in vitro

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Analysis of the Ligand Binding Site of the 5-HT 3 Receptor Using Site Directed Mutagenesis: Importance of Glutamate 106

Cited by 76 publications

References 36 publications

Locating an Antagonist in the 5-HT3 Receptor Binding Site Using Modeling and Radioligand Binding

Locating an Antagonist in the 5-HT3 Receptor Binding Site Using Modeling and Radioligand Binding

Spatial Orientation of the Antagonist Granisetron in the Ligand-Binding Site of the 5-HT3 Receptor

Valerian extract and valerenic acid are partial agonists of the 5-HT5a receptor in vitro

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Spatial Orientation of the Antagonist Granisetron in the Ligand-Binding Site of the 5-HT₃ Receptor