A NanoBRET-Based H3R Conformational Biosensor to Study Real-Time H3 Receptor Pharmacology in Cell Membranes and Living Cells

Abstract

Conformational biosensors to monitor the activation state of G protein-coupled receptors are a useful addition to the molecular pharmacology assay toolbox to characterize ligand efficacy at the level of receptor proteins instead of downstream signaling. We recently reported the initial characterization of a NanoBRET-based conformational histamine H₃ receptor (H₃R) biosensor that allowed the detection of both (partial) agonism and inverse agonism on living cells in a microplate reader assay format upon stimulation with H₃R ligands. In the current study, we have further characterized this H₃R biosensor on intact cells by monitoring the effect of consecutive ligand injections in time and evaluating its compatibility with photopharmacological ligands that contain a light-sensitive azobenzene moiety for photo-switching. In addition, we have validated the H₃R biosensor in membrane preparations and found that observed potency values better correlated with binding affinity values that were measured in radioligand competition binding assays on membranes. Hence, the H₃R conformational biosensor in membranes might be a ready-to-use, high-throughput alternative for radioligand binding assays that in addition can also detect ligand efficacies with comparable values as the intact cell assay.

Keywords: BRET; GPCR; H3R; conformational biosensor; histamine.

Figure 1

Ligand-induced changes in Δicl3-H₃R_{Nluc/Halo(618)} biosensor conformation detected by BRET in intact HEK293A cells. (A) Scheme of H₃R biosensor configuration with the self-labeling HaloTag protein inserted in the truncated IL3 between Thr²²⁹ and Phe³⁴⁸ and Nluc fused to the C-terminal tail as the BRET acceptor and donor, respectively. (B) Conformational changes in Δicl3-H₃R_{Nluc/Halo(618)} upon stimulation with 10 μM H₃R ligands measured as ΔBRET ratio in time. (C) Concentration-response curves measured after 30 min stimulation of the H₃R biosensor with H₃R ligands. Data are displayed as mean ± SD from 4 independent experiments performed in duplicate. (D–E) the photo-switchable agonist VUF15000 (D) and inverse agonist VUF14738 (E) switch from trans (cyan) to cis (magenta) upon illumination with 360 nm and from cis to trans by illumination with 430 nm. (F) Concentration-response curves measured after 20 min stimulation of the H₃R biosensor with dark (trans) or pre-illuminated (cis) photo-switchable VUF15000 and VUF14738. Data are displayed as the mean ± SD from 3 independent experiments performed in duplicate.

Figure 2

Dynamic changes in Δicl3-H₃R_{Nluc/Halo(618)} biosensor conformation detected by BRET in intact HEK293A cells. (A) Injection of different concentrations pitolisant attenuates the histamine-induced (10 μM) conformational change in the H₃R biosensor. (B) Consecutive injection of increasing (log) concentrations of histamine in the same three wells. Data are displayed as mean ± SD from one representative experiment performed in triplicate. (C) Concentration-response curve of histamine generated from Figure 2B, 15 min after each consecutive injection of increasing concentrations histamine in triplicate (3 wells/exp) or 15 min after stimulation of individual wells with increasing concentrations histamine in triplicate (21 wells/exp). Data are displayed as mean ± SD from 3 independent experiments performed in triplicate.

Figure 3

BRET responses of H₃R ligands determined on Δicl3-H₃R_{Nluc/Halo(618)} cell membrane prepared in 50 mM Tris-HCl (pH 7.4). (A) ΔBRET time course of eight H₃R ligands at 10 μM concentration. (B) ΔBRET ratio measurements in 24 wells of a 96-well plate containing H₃R biosensor-expressing cell membranes treated with either vehicle (10 and 60 min), 10 μM histamine (10 min) or 10 μM pitolisant (60 min) to calculate the Z-factor. One representative graph from three independent experiments is shown. (C) Z-factors over time of cell membrane treated with 10 μM histamine or pitolisant in 96-well plate. (D) Concentration-response curves measured after 30 min stimulation of H₃R biosensor-expressing membranes with H₃R ligands. Data are displayed as mean ± SD from at least 3 independent experiments performed in duplicate.

Figure 4

Comparison of H₃R biosensor pharmacology on intact cells versus membrane preparations in response to H₃R ligands. (A,B) Comparison of intrinsic activity (IA) values (A) and pEC₅₀ (B) obtained from H₃R biosensor in intact cells versus membrane preparations upon stimulation with increasing ligand concentrations for 30 min (see Figure 1C and Figure 3D; Table 1). (C) Comparison of pK_i values obtained from radioligand competition binding experiments on H₃R biosensor-expressing membranes in 50 mM Tris-HCl (pH 7.4) with pEC₅₀ values obtained from H₃R biosensors in intact cells (in HBSS) and membrane preparations (in 50 mM Tris-HCl (pH 7.4)) upon stimulation with increasing ligand concentrations for 30 min (see Figure 1C and Figure 3D; Table 1). Differences between pEC₅₀ values obtained from H₃R biosensor in intact cells versus membrane preparations are indicated with grey arrows. Data are displayed as mean ± SD from at least 3 independent experiments performed in duplicate. Deming linear regression was used to compare the fitted affinity and/or potency values between the different assay formats, the dotted line represents line of unity (B,C). HA = histamine; ime = imetit; pit = pitolisant; clob = clobenpropit; thio = thioperamide; bav = bavisant; ABT = ABT-239; PF = PF-3654746.