Loop B is a major structural component of the 5-HT3 receptor

The 5-HT3 receptor belongs to a family of therapeutically important neurotransmitter-gated receptors whose ligand binding sites are formed by the convergence of six peptide loops (A-F). Here we have mutated 15 amino acid residues in and around loop B of the 5-HT3 receptor (Ser-177 to Asn-191) to Ala or a residue with similar chemical properties. Changes in [3H]granisetron binding affinity (Kd) and 5-HT EC50 were determined using receptors expressed in human embryonic kidney 293 cells. Substitutions at all but one residue (Thr-181) altered or eliminated binding for one or both mutants. Receptors were nonfunctional or EC50 values were altered for all but two mutants (S182T, I190L). Homology modeling indicates that loop B contributes two residues to a hydrophobic core that faces into the β-sandwich of the subunit, and the experimental data indicate that they are important for both the structure and the function of the receptor. The models also show that close to the apex of the loop (Ser-182 to Ile-190), loop B residues form an extensive network of hydrogen bonds, both with other loop B residues and with adjacent regions of the protein. Overall, the data suggest that loop B has a major role in maintaining the structure of the region by a series of noncovalent interactions that are easily disrupted by amino acid substitutions

An Efficient and Information Rich Biochemical Method Design for Fragment Library Screening on Ion Channels

Drug discovery requires a simple, rapid, and cost-effective method for the early identification of novel leads and elimination of poor candidates. Here we present an experimental design that fulfils these criteria, using a ligand-gated ion channel expressed in a mammalian cell line, whose function can be probed using a voltage-sensitive dye. The experimental design is novel, as it uses the same screen to identify hit fragments and to characterize them as agonists or antagonists. The results were independently validated using radioligand binding, although the new technique has several advantages over radioligand methods. A number of novel high-affinity ligands were found. The method is broadly applicable to a wide range of receptor types including ligand-gated ion channels (LGICs), voltage-gated ion channels (VGICs), and G protein-coupled receptors (GPCRs), all of which are important drug targets

VUF10166, a novel compound with differing activities at 5-HT₃A and 5-HT₃AB receptors

The actions of a novel, potent 5-HT₃ receptor ligand, [2-chloro-(4-methylpiperazine-1-yl)quinoxaline (VUF10166)], were examined at heterologously expressed human 5-HT₃A and 5-HT₃AB receptors. VUF10166 displaced [³H]granisetron binding to 5-HT₃A receptors expressed in human embryonic kidney cells with high affinity (K(i) = 0.04 nM) but was less potent at 5-HT₃AB receptors (K(i) = 22 nM). Dissociation of [³H]granisetron in the presence of VUF10166 was best fit with a single time constant (t(1/2) = 53 min) at 5-HT₃A receptors, but with two time constants (t(1/2) = 55 and 2.4 min) at 5-HT₃AB receptors. Electrophysiological studies in oocytes revealed that VUF10166 inhibited 5-HT-induced responses at 5-HT₃A receptors at nanomolar concentrations, but inhibition and recovery were too slow to determine an IC₅₀. At 5-HT₃AB receptors, inhibition and recovery were faster, yielding an IC₅₀ of 40 nM. Cysteine substitutions in the complementary (-), but not the principal (+), face of the 5-HT₃B subunit produced heteromeric receptors in which the actions of VUF10166 resembled those at homomeric receptors. At 5-HT₃A receptors, VUF10166 at higher concentrations also behaved as a partial agonist (EC₅₀ = 5.2 μM; R(max) = 0.24) but did not elicit significant responses at 5-HT₃AB receptors at ≤100 μM. Thus, we propose that VUF10166 binds to the common A+A- site of both receptor types and to a second A+B- modulatory site in the heteromeric receptor. The ability of VUF10166 to distinguish between 5-HT₃A and 5-HT₃AB receptors could help evaluate differences between these receptor types and has potential therapeutic value

Modulation of the erwinia ligand-gated ion channel (ELIC) and the 5-HT3 receptor via a common vestibule site

Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to channel opening. In this study, we developed nanobodies (single domain antibodies), which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote (Erwinia) channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functional importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulation in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Agonists and antagonists bind to an A-A interface in the heteromeric 5-HT(3)AB receptor

The 5-HT3 receptor is a member of the Cys-loop family of transmitter receptors. It can function as a homopentamer (5-HT3A-only subunits) or as a heteropentamer. The 5-HT(3)AB receptor is the best characterized heteropentamer. This receptor differs from a homopentamer in its kinetics, voltage dependence, and single-channel conductance, but its pharmacology is similar. To understand the contribution of the 5-HT3B subunit to the binding site, we created homology models of 5-HT(3)AB receptors and docked 5-HT and granisetron into AB, BA, and BB interfaces. To test whether ligands bind in any or all of these interfaces, we mutated amino acids that are important for agonist and antagonist binding in the 5-HT3A subunit to their corresponding residues in the 5-HT3B subunit and vice versa. Changes in [H-3]granisetron binding affinity (K-d) and 5-HT EC50 were determined using receptors expressed in HEK-293 cells and Xenopus oocytes, respectively. For all A-to-B mutant receptors, except T181N, antagonist binding was altered or eliminated. Functional studies revealed that either the receptors were nonfunctional or the EC50 values were increased. In B-to-A mutant receptors there were no changes in K-d, although EC50 values and Hill slopes, except for N170T mutant receptors, were similar to those for 5-HT(3)A receptors. Thus, the experimental data do not support a contribution of the 5-HT3B subunit to the binding pocket, and we conclude that both 5-HT and granisetron bind to an AA binding site in the heteromeric 5-HT(3)AB receptor