Molecular mechanism of setron-mediated inhibition of full-length 5-HT3A receptor

Abstract

Serotonin receptor (5-HT_3AR) is the most common therapeutic target to manage the nausea and vomiting during cancer therapies and in the treatment of irritable bowel syndrome. Setrons, a class of competitive antagonists, cause functional inhibition of 5-HT_3AR in the gastrointestinal tract and brainstem, acting as effective anti-emetic agents. Despite their prevalent use, the molecular mechanisms underlying setron binding and inhibition of 5-HT_3AR are not fully understood. Here, we present the structure of granisetron-bound full-length 5-HT_3AR solved by single-particle cryo-electron microscopy to 2.92 Å resolution. The reconstruction reveals the orientation of granisetron in the orthosteric site with unambiguous density for interacting sidechains. Molecular dynamics simulations and electrophysiology confirm the granisetron binding orientation and the residues central for ligand recognition. Comparison of granisetron-bound 5-HT_3AR with the apo and serotonin-bound structures, reveals key insights into the mechanism underlying 5-HT_3AR inhibition.

Fig. 1

Cryogenic electron microscopy (cryo-EM) structure of granisetron-bound full-length serotonin 3A receptor (5-HT_3AR). a A schematic showing three fundamental conformations that constitute the gating cycle in pentameric ligand-gated ion channel (pLGIC) function: a resting state, a transient open state, and a desensitized state. Agonist-binding shifts the equilibrium towards the open state and then to the high-agonist affinity, desensitized state. Orthosteric (competitive) antagonists exert their effect by shifting the equilibrium towards the resting (or inhibited) state. b Trace showing a continuous recording of 5-HT_3AR currents (−60 mV) in oocytes measured by two-electrode voltage clamp (TEVC) in the presence of serotonin (marked by red line) and pre-applied granisetron (marked by orange line). The effect of granisetron inhibition was fully reversible as seen in the third pulse. c Map of full-length 5-HT_3AR-granisetron reconstructed from 46,757 particles at 2.92 Å resolution. Side-view parallel to the membrane and extracellular view are shown in left and right panels, respectively. Each monomer is shown in a different color for clarity. Density corresponding to granisetron (left panel, circle) and glycans (right panel, arrow) are indicated. d Three-dimensional cartoon model of 5-HT_3AR-granisetron structure generated from EM reconstruction (side view). For each subunit, three sets of glycans are shown as stick representation. e Top-view of 5-HT_3AR-granisetron map sliced at the binding site to show all five granisetron molecules, each bound at the interface of two subunits (indicated by arrows)

Fig. 2

The granisetron binding site. a The density map of granisetron contoured at 9σ (left) and map around the residues at the binding site located at the intersubunit interface (right). The residue labels on the principal subunit are marked in black and those on the complementary subunit are marked in brown. b A comparison of the serotonin 3A receptor-apo (5-HT_3AR-apo), 5-HT_3AR-granisetron, and 5-HT_3AR-serotonin structures shows that residues involved in ligand-binding undergo rotameric reorientation. c Alignment of the three structures reveals an inward motion of loop C in 5-HT_3AR-granisetron relative to 5-HT_3AR-apo, which is in the direction toward activation as seen in the 5-HT_3AR-serotonin structure

Fig. 3

Conformational differences between the apo and ligand-bound states. a An extracellular view of the extracellular domain (ECD) upon global alignment of serotonin 3A receptor-apo (5-HT_3AR-apo) structure with 5-HT_3AR-granisetron (left) and 5-HT_3AR-serotonin (right). Only ECDs from two non-adjacent subunits is shown for clarity. A counter-clockwise motion of the ECD is observed as indicated by the arrows. The serotonin-induced motion is of larger magnitude compared to that of granisetron, highlighted by the solid and dotted arrows, respectively. b A comparison of the transmembrane domains (TMDs) (viewed from the extracellular side) in 5-HT_3AR-granisetron structure (left) and 5-HT_3AR-serotonin (right) when aligned with respect to 5-HT_3AR-apo. Only two non-adjacent TMD subunits are shown for clarity. In both panels, a clockwise rotation of the TMD is observed with 5-HT_3AR-serotonin revealing a larger change. c Pathway of ion permeation of 5-HT_3AR-apo and 5-HT_3AR-granisetron generated with HOLE. The cartoon representation of two subunits are shown for clarity. The locations of pore constrictions are shown as sticks. The pore radius is plotted as a function of distance along the pore axis. The dotted line indicates the approximate radius of a hydrated Na⁺ ion, which is estimated at 2.76 Å (right)

Fig. 4

Assessment of the overall stability of the granisetron-serotonin 3A receptor (5-HT_3AR) structure. a Time evolution of the root mean squared deviations (RMSD) of Cα atoms of secondary structure elements of the extracellular domain (ECD) and all Cα atoms of the 5-HT_3AR pentamer (left panel). The RMSD of each of the granisetron molecules (labeled CWB) in each subunit A to E (right panel) calculated with respect to the cryo-EM-derived structure during 100 ns production simulations. b Two possible granisetron poses with the bicyclic ring in boat/chair or chair/chair conformation and the N-methyl group in axial or equatorial positions in the piperidine chair conformation. These two poses were used as input for metadynamics-based ranking. c Ranking of the two granisetron poses shown in b using metadynamics. Error bars represent the standard error of the mean of RMSD estimates from 10 metadynamics simulations. Source data are provided as a Source Data file

Fig. 5

Effects of mutations at the ligand-binding pocket on granisetron inhibition. a Granisetron interactions with Trp156 and Tyr207 from the principal subunit and Trp63, Arg65, Tyr126 from the complementary subunit are depicted as stick representation. b Serotonin dose response measured by two-electrode voltage clamp (TEVC) recordings (at −60 mV) for wild-type (WT) 5-HT_3AR, W63Y, R65A, Y126F, W156Y, and Y207F mutants, expressed in oocytes. The half-maximal effective concentration (EC₅₀), the Hill coefficient (nH), and the number of independent oocyte experiments for WT and mutants are: WT (EC₅₀: 2.70 + 0.09 μM; nH: 2.3 + 0.17; n: 3), W63Y (EC₅₀: 9.93 + 0.77 μM; nH: 3.1 + 0.72; n: 4), R65A (EC₅₀: 13.79 + 0.50 μM; nH: 4.4 + 0.59; n: 4), Y126F (EC₅₀: 42.8 + 4.4 μM; nH: 2.6 + 0.71; n: 4), W156Y (EC₅₀: 306 + 44 μM; nH: 1.58 + 0.24; n: 4), and Y207F (EC₅₀: 20.35 + 1.7 μM; nH: 1.9 + 0.27; n: 5). c Currents were elicited in response to serotonin (concentrations used near EC₅₀ values of WT and mutants). The following concentrations of serotonin were used: WT-1 μM, W63Y-10 μM, R65A-10 μM; Y126F-40 μM, W156Y-200 μM, and Y207F-20 μM. Currents were measured in response to serotonin (marked by red line) and pre-application of granisetron (marked by orange line). Dotted arrows show the extent of granisetron inhibition. d A plot of the ratio of peak current in the presence of granisetron to peak current in the absence of granisetron is shown for WT and mutants. Data are shown as mean ± s.d. (n is indicated within parentheses). Significance at p = 0.001 (***) and p = 0.05 (**) calculated by two-sample t test for WT and mutants. Source data are provided as a Source Data file