Ondansetron and granisetron binding orientation in the 5-HT(3) receptor determined by unnatural amino acid mutagenesis

Abstract

The serotonin type 3 receptor (5-HT(3)R) is a ligand-gated ion channel found in the central and peripheral nervous systems. The 5-HT(3)R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT(3)A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket.

Figure 1

Basic layout of a Cys-loop (pentameric) receptor. The structure is that of the GluCl α subunit (pdb 3RIF). Left: The full receptor, with two subunits are highlighted; one (blue) contributes principal agonist binding site residues, found on loops A (red), B (green), and C (blue). The complementary subunit (black) contributes loops D (purple), E (orange), and F (yellow). The α-helical region corresponds to the transmembrane domain; the region above it is extracellular. Right: Detail of agonist binding site, noting approximate locations of key residues considered here.

Figure 2

Chemical structures of drugs and amino acids used in this study. (a) 5-HT₃A receptor antagonists. (b) 5-HT₃A receptor agonists. (c) Tryptophan analogs.

Figure 3

Dose-response curves of wild-type 5-HT₃A receptor. Responses to 5-HT with increasing concentrations of: (a) ondansetron, and (b) granisetron. Data fit to the Hill equation. Fit parameters: (a) Mean maximal current: 8 ± 4 μA. [ondansetron] = 0 nM, EC₅₀: 1.3 ± 0.2 μM, n_H: 2.4 ± 0.6; [ondansetron] = 0.64 nM, Imax: 63%, EC₅₀: 1.1 ± 0.4 μM, n_H: 2 ± 1; [ondansetron] = 4.5 nM, Imax = 61%, EC₅₀: 9 ± 29 μM, n_H: 2 ± 8. (b) Mean maximal current: 9 ± 4 μA. [granisetron] = 0 nM, EC₅₀: 1.5 ± 0.1 μM, n_H: 2.0 ± 0.1; [granisetron] = 0.6 nM, Imax: 25%, EC₅₀: 0.7 ± 0.7 μM, n_H: 2 ± 7; [granisetron] = 1.2 nM, Imax: 4%, EC₅₀: 1 ± 1 μM, n_H: 1 ± 2.

Figure 4

Representative voltage-clamp traces and dose-inhibition curves for antagonists for wild type and Trp183 mutant 5-HT₃A receptors. Representative traces for inhibition of (a) wild-type and (b) W183F₃W receptors by increasing doses of granisetron. Time of application and concentration noted by black bars. Channels opened by addition of mCPBG (red bars) at (a) 1 μM and (b) 13 μM. Hashes indicate wash times. Ondansetron inhibition curves shown for receptors activated by (c) mCPBG, and (d) 5-HT. Granisetron inhibition curves shown for receptors activated by (e) mCPBG, and (f) 5-HT. Data fit to the Hill equation; fit parameters (IC₅₀ and n_H) are in Table 2.

Figure 5

Fluorination plots. Calculated cation-π binding ability versus log[IC₅₀/IC₅₀(wt)] for a series of fluorinated tryptophan derivatives at Trp183. Ondansetron fluorination plots for receptors activated by (a) mCPBG, and (b) 5-HT. Granisetron fluorination plots for receptors activated by (a) mCPBG, and (b) 5-HT. Red lines are linear fits (y = mx + b) inclusive of all points. Fit parameters (a) m: −0.15 ± 0.03, b: 5.1 ± 0.6, R: 0.94; (b) m: −0.18 ± 0.03, b: 6.0 ± 0.8, R: 0.96; (c) m: −0.11 ± 0.04, b: 3 ± 1, R: 0.77; (d) m: −0.14 ± 0.06, b: 5 ± 2, R: 0.82.

Scheme 1