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

Abstract

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...

Binding Sites for Bilobalide, Diltiazem, Ginkgolide, and Picrotoxinin at the 5-HT3 Receptor

“…Residues were mostly mutated to amino acids with similar chemical properties to prevent complications arising from changes in channel gating. Conserva- tive changes minimize the impact on receptor structure and function, but still reveal specific interactions between the receptor and antagonist (e.g., Thompson et al, 2005). Wildtype and mutant receptors (except for S2ЈT and S12ЈT, which were nonfunctional) responded to 5-HT in a concentrationdependent manner, with EC 50 and n H values of mutants differing Ͻ6-fold from wild type (Table 1; Fig.…”

A Cation-π Interaction at a Phenylalanine Residue in the Glycine Receptor Binding Site Is Conserved for Different Agonists

“…This was performed as described previously (Reeves et al, 2003;Thompson et al, 2005). Three-dimensional models of the extracellular region of the glycine receptor were built using MODELLER 6, version 2 (http://salilab.org/modeller/) based on the crystal structure of acetylcholine-binding protein in the agonistbound state (carbamylcholine; Protein Data Bank ID 1UV6).…”

Locating the Carboxylate Group of GABA in the Homomeric rho GABAA Receptor Ligand-binding Pocket

“…This approach has been made possible by the availability of the high resolution structure of the acetylcholine binding protein (AChBP), which is homologous to the extracellular domain of the nicotinic ACh receptor (6). Using this as a template, computer-generated models of ligandbinding pockets of Cys loop receptors, combined with previous data from structure-activity studies, have identified important features of these pockets and on the orientation of agonists and antagonists when located in their binding sites (7)(8)(9)(10)(11)(12)(13). Generating an accurate representation of the orientation of GABA in the GABA C receptor-binding site could identify the processes involved in ligand recognition at this receptor and would also assist in drug design; current data indicate that drugs acting on GABA C receptors could possibly be used to treat visual, sleep, and cognitive disorders (4,14).…”