1-Oxo-5-hydroxytryptamine: A Surprisingly Potent Agonist of the 5-HT_3(Serotonin) Receptor

A novel synthetic route to 1-oxo-5-hydroxytryptamine, the benzofuran analogue of serotonin, has been developed. The new synthesis proceeds via the [3+2] cycloaddition of p-benzoquinone and 2,3-dihydrofuran, followed by a Lewis acid-catalyzed isomerization. This molecule proves to be a competent agonist (equipotent to serotonin) of the 5-HT_3 receptor, demonstrating that the indolic proton of serotonin is not essential to its activation of the receptor

A Hydrogen Bond in Loop A Is Critical for the Binding and Function of the 5-HT3 Receptor†

The binding sites of Cys-loop receptors are formed from at least six loops (A−F). Here we have used mutagenesis, radioligand binding, voltage clamp electrophysiology, and homology modeling to probe the role of two residues in loop A of the 5-HT_3 receptor: Asn128 and Glu129. The data show that substitution of Asn128, with a range of alternative natural and unnatural amino acids, changed the EC_(50) (from ∼10-fold more potent to ∼10-fold less potent than that of the wild type), increased the maximal peak current for mCPBG compared to 5-HT (R_(max)) 2−19-fold, and decreased n_H, indicating this residue is involved in receptor gating; we propose Asn128 faces away from the binding pocket and plays a role in facilitating transitions between conformational states. Substitutions of Glu129 resulted in functional receptors only when the residue could accept a hydrogen bond, but with both these and other substitutions, no [^3H]granisetron binding could be detected, indicating a role in ligand binding. We propose that Glu129 faces into the binding pocket, where, through its ability to hydrogen bond, it plays a critical role in ligand binding. Thus, the data support a modified model of the 5-HT_3 receptor binding site and show that loop A plays a critical role in both the ligand binding and function of this receptor

A Hydrogen Bond in Loop A Is Critical for the Binding and Function of the 5-HT_3 Receptor

The binding sites of Cys-loop receptors are formed from at least six loops (A−F). Here we have used mutagenesis, radioligand binding, voltage clamp electrophysiology, and homology modeling to probe the role of two residues in loop A of the 5-HT_3 receptor: Asn128 and Glu129. The data show that substitution of Asn128, with a range of alternative natural and unnatural amino acids, changed the EC_(50) (from ∼10-fold more potent to ∼10-fold less potent than that of the wild type), increased the maximal peak current for mCPBG compared to 5-HT (R_(max)) 2−19-fold, and decreased n_H, indicating this residue is involved in receptor gating; we propose Asn128 faces away from the binding pocket and plays a role in facilitating transitions between conformational states. Substitutions of Glu129 resulted in functional receptors only when the residue could accept a hydrogen bond, but with both these and other substitutions, no [^3H]granisetron binding could be detected, indicating a role in ligand binding. We propose that Glu129 faces into the binding pocket, where, through its ability to hydrogen bond, it plays a critical role in ligand binding. Thus, the data support a modified model of the 5-HT_3 receptor binding site and show that loop A plays a critical role in both the ligand binding and function of this receptor

Construction, Verification and Experimental Use of Two Epitope-Tagged Collections of Budding Yeast Strains

A major challenge in the post-genomic era is the development of experimental approaches to monitor the properties of proteins on a proteome-wide level. It would be particularly useful to systematically assay protein subcellular localization, post-translational modifications and protein–protein interactions, both at steady state and in response to environmental stimuli. Development of new reagents and methods will enhance our ability to do so efficiently and systematically. Here we describe the construction of two collections of budding yeast strains that facilitate proteome-wide measurements of protein properties. These collections consist of strains with an epitope tag integrated at the C-terminus of essentially every open reading frame (ORF), one with the tandem affinity purification (TAP) tag, and one with the green fluorescent protein (GFP) tag. We show that in both of these collections we have accurately tagged a high proportion of all ORFs (approximately 75% of the proteome) by confirming expression of the fusion proteins. Furthermore, we demonstrate the use of the TAP collection in performing high-throughput immunoprecipitation experiments. Building on these collections and the methods described in this paper, we hope that the yeast community will expand both the quantity and type of proteome level data available

A Coupled Array of Noncovalent Interactions Impacts the Function of the 5‑HT_3A Serotonin Receptor in an Agonist-Specific Way

The serotonin type 3A (5-HT_3A) receptor is a Cys-loop (pentameric) neurotransmitter-gated ion channel found in the central and peripheral nervous systems and implicated in numerous diseases. In previous studies with the endogenous agonist serotonin, we identified two interactions critical for receptor function: a cation−π interaction with W183 in loop B (TrpB) and a hydrogen bond to E129 in loop A. Here we employ mutant cycle analyses utilizing conventional and unnatural amino acid mutagenesis to demonstrate that a third residue, D124 of loop A, forms two functionally important hydrogen bonds to the backbone of loop B. We also show that these three interactions, the cation−π interaction, the backbone hydrogen bonds, and the E129 hydrogen bond, are tightly coupled to each other, suggesting they function as a single unit. We also identify key functional differences between serotonin and the competitive partial agonist m-chlorophenyl biguanide (mCPBG) at these residues. mCPBG displays no cation−π at TrpB and extreme sensitivity to the positioning of E129, on which it is reliant for initiation of channel gating

5-Fluorotryptamine is a partial agonist at 5-HT3 receptors, and reveals that size and electronegativity at the 5 position of tryptamine are critical for efficient receptor function

Antagonists, but not agonists, of the 5-HT3 receptor are useful therapeutic agents, and it is possible that partial agonists may also be potentially useful in the clinic. Here we show that 5-fluorotryptamine (5-FT) is a partial agonist at both 5-HT3A and 5-HT3AB receptors with an Rmax (Imax / Imax5-HT) of 0.64 and 0.45 respectively. It is about 10 fold less potent than 5-HT: EC50 = 16 and 27 μM, and Ki for displacement of [3H]granisetron binding = 0.8 and 1.8 μM for 5-HT3A and 5-HT3AB receptors respectively. We have also explored the potencies and efficacies of tryptamine and a range of 5-substituted tryptamine derivatives. At 5-HT3A receptors tryptamine is a weak (Rmax = 0.15), low affinity (EC50 = 113 μM; Ki = 4.8 μM) partial agonist, while 5-chlorotryptamine has a similar affinity to 5-FT (EC50 = 8.1 μM; Ki = 2.7 μM) but is a very weak partial agonist (Rmax = 0. 0037). These, and data from 5-methyltryptamine and 5-methoxytryptamine, reveal the importance of size and electronegativity at this location for efficient channel opening