Article Figures & Data
Figures
- Fig. 1.
Depolarization of the membrane potentiates TP receptor activation. (A) The scheme illustrates the experimental setup used for FRET measurements under voltage clamp conditions in HEK293 cells expressing the TP receptor-sensor. Cells were continuously superfused with buffer or agonist-containing buffer. (B) The emission of CFP and YFP in a single cell stably expressing TP receptor-sensor superfused with 3 µM U46619 was recorded over time, corrected for photobleaching, and plotted as indicated by the colors as shown in the representative trace (out of n = 6). The corresponding emission ratio YFP:CFP is shown in red (normalized to initial values). (C) In experiments similarly described in (B), cells were exposed to 100 nM U46619 at −90 mV. Subsequently, cells were depolarized to +60 mV for 20 seconds as indicated; after wash-out, a high concentration of 3 µM U46619 was applied (obtained emission ratio was normalized to the negative amplitude evoked by 3 µM U46619) n = 5; means ± S.D.). (D) Representative recording and (E) show the voltage-dependence of the TP receptor-sensor for indicated voltages. Zero and −90 mV were always included as reference potentials. (E) Summarized data (n = 5–15 cells per data point; means ± S.D.) of the voltage-induced alterations in the emission ratio after normalization to values obtained at 0 mV. These data were fitted to a Boltzmann function giving rise to value for V0.5 = −46 mV.
- Fig. 2.
Propagation of voltage effect on TP receptor downstream signals. Cells transfected with indicated FRET-based biosensor for TP receptor-Gα13 (representative recording n = 3) (A), TP receptor induced Gα13-p115 RhoGEF (representative recording n = 7) interactions (B), and TP receptor induced Gαq activation (representative recording n = 5) (C) were subjected to single-cell FRET recording under voltage clamp conditions using the indicated voltage and superfusion protocol. (D) Values in the presence of agonist of ΔF535/F480 at +60 mV were divided by values of ΔF535/F480 at −90 mV; the values for the effect voltage had on TP receptor-sensor activity, TP receptor-Gα13 interaction, TP receptor induced Gα13-p115 RhoGEF interaction, and TP receptor induced Gαq activation were compared: no significant difference could be detected (analyzed by Kruskal-Wallis-Test, P value = 0.10; means ± S.D., TP receptor-sensor = 2.21 ± 0.85, n = 29; TP receptor-Gα13 = 2.23 ± 0.24, n = 3; TP receptor induced Gα13-p115 RhoGEF interaction = 2.53 ± 0.69, n = 15, and TP receptor induced Gαq activation = 1.72 ± 0.42, n = 5).
- Fig. 3.
Voltage effect on U46619 mediated TP receptor-sensor activation. (A) Means ± S.D. concentration-response curves of TP receptor-sensor at −90 (EC50 93 nM) and at +60 mV (EC50 21 nM,) respectively (n = 7–15 per data point), were recorded. All amplitudes of agonist-evoked declines in FRET were normalized to the amplitude of a reference concentration (3 µM U46619 at −90 mV). (B) Voltage effect on TP receptor-sensor activity in presence of a saturating agonist concentration (n = 6). (C) Means ± S.D. of the time course of TP receptor-sensor activation induced upon depolarization from −90 to +60 mV in the presence of U46619 (50 nM [blue]: n = 7, 200 nM [red] n = 15). Left: Averaged data were fitted to a monoexponential function. Right: Half times determined by fitting of individual experiments are illustrated as means ± S.D. (t1/2 (50 nM) = 2.2 ± 0.33 seconds, t1/2 (200 nM) = 1.2 ±0.4 seconds; ***P = 0.0001, Mann Whitney test).
- Fig. 4.
Voltage effect on TP receptor activated with U46619 analogs. (A) Structures of tested U46619 analogs (https://www.caymanchem.com/; From Cayman Chemicals). Left, from top to bottom: U46619, I-BOP, 8-iso PGE2, 8-iso PGE1. Right, from top to bottom: PGE2 ethanolamide, PGE2 methyl ester, 15-keto PGE2, 15(S)-15-methyl PGE2. Ligands were tested in similar experiments as shown in (B). (B) PGE2 Ethanolamide tested in TP receptor induced Gα13-p115 RhoGEF interaction assay under voltage clamp conditions (representative recording n = 3). (C) Summary of ligands tested in terms of voltage dependence. “+” indicates an increase of at least 50% in ΔF535/ΔF480 between −90 and +60 mV in presence of a nonsaturating agonist concentration.
- Fig. 5.
TP receptor R295 is involved in the voltage sensing process. (A) Left: Snakeplot created with GPCRdb.org of TP receptor indicating positions of mutated amino acids either in blue or in green. Right: Crystal structure of TP receptor cocrystallized with ramatroban (Protein Data Bank: 6IIU). The black numbers indicate minimum heavy atom distances (in angstrom) between the carboxyl group of ramatroban and R7.40. (B) Summary of mutants tested in terms of voltage dependence. “+” indicates an increase of at least 50% in ΔF535/ΔF480 between −90 and +60 mV in presence of a nonsaturating agonist concentration. (C–E) HEK293T cells transiently transfected with Gα13-p115 RhoGEF interaction assay and as indicated with either TP receptor wt or TP receptor R295K or R295A. (C) Left: Means ± S.D. cells were depolarized from −90 to +60 mV in the presence of U46619 (TP receptor wt [blue]: n = 6, TP receptor R295K [green] n = 5). Right: Cells were selected for a similar response to U46619 at −90 mV (included: ΔF535/F480 in presence of U46619 at −90 mV: 0.02 ≤ ΔF535/ΔF480 ≤ 0.08). The responses of the selected groups to +60 mV were then compared showing a significantly stronger activation upon depolarization for TP receptor R295K compared with wt (TP receptor wt = 3.13 ± 0.30, n = 7; TP receptor R295K = 5.31 ± 1.85, n = 6; Mann Whitney test **P = 0.0012). (D) Means ± S.D. TP receptor R295A induced Gα13-p115-interaction (n = 6). (E) Summarized data (n = 3–14 cells per data point) of the voltage-induced alterations in the emission ratio after subtraction of the −90 mV response for TP receptor wt, TP receptor R295K, and R295A recorded in the TP receptor induced Gα13-p115-interaction in presence of U46619. For −45 mV values TP receptor R295K and TP receptor R295A showed significant difference against wt (means ± S.D., wt n = 7, ΔF535/F480: 0.07 ± 0.01; R295K n = 5, ΔF535/F480: 0.02 ± 0.01; R295A n = 5, ΔF535/F480: 0.04 ± 0.03; Mann Whitney test R295K vs. wt: *P = 0.03, R295A vs. wt **P = 0.003); 60 mV (means ± S.D., wt n = 6, ΔF535/F480: 0.09 ± 0.03; R295K n = 7, ΔF535/F480: 0.17 ± 0.04; R295A n = 7, ΔF535/F480: 0.16 ± 0.06; Mann Whitney test R295K vs. wt: *P = 0.04, R295A vs. wt **P = 0.001); 100 mV (means ± S.D., wt n = 5, ΔF535/F480: 0.11 ± 0.04; R295K n = 6, ΔF535/F480: 0.21 ± 0.05; Mann Whitney test R295K vs. wt: **P = 0.009).
- Fig. 6.
Voltage dependence of FP receptor means ± S.D. of HEK293T cells transfected with FP receptor and FRET Gαq–activation assay, depicted in the scheme on top. The single traces were normalized to the negative response on stimulation with 10 µM U46619 and averaged (n = 6). Cells were activated with 333 nM U46619. Agonist-activated cells were clamped from −90 to +60 mV and vice versa.
- Fig. 7.
Voltage dependence of IP receptor. (A) Left: Means ± S.D. of HEK293T cells transfected with mcherry-IP receptor and FRET based cAMP-Sensor Epac1-camps. The single traces were normalized to the response on stimulation with 0.1 µM iloprost and averaged (0 mV: n = 5; −90 mV n = 4). A nonsaturating agonist concentration of iloprost was applied to cells that were held at −90 mV, and after 2 minutes the mean cAMP production was measured (T1). Subsequently, cells were either clamped to 0 mV (blue) or kept at −90 mV (black traces) for 2 minutes. The mean cAMP production was measured for both groups (T2). Right: T1 blue: 30.5% ± 14.1%; black: 26.2% ± 5.8%; T2 blue 49.1% ± 12.1%; black 48.6% ± 8.6%. No significant difference between the two conditions in the production of cAMP was observed (n.s., not significant). (B) Measurements of HEK293T cells transfected with TP receptor wt and Epac1-camps. Cells were stimulated with U46619 and clamped to the indicated potentials; traces were normalized on response to 3 µM U46619. To the nonsaturating U46619 concentration the Gαq inhibitor FR900359 with a final concentration of 1 µM has been added (means ± S.D.; n = 5). (C) Measurement of β2-AR voltage dependence in cAMP assay. Cells were stimulated with isoproterenol (Iso) and clamped to the indicated potentials; traces were normalized on response to 0.1 µM isoproterenol (means ± S.D.; n = 5).
- Fig. 8.
Voltage dependence of EP3 receptor. Left: Representative inward K+ current of HEK293T cells expressing wild-type EP3 receptor and GIRK channels evoked by 10 nM or 1 µM iloprost, measured at −90 or −30 mV (n = 6). Right: The GIRK current ratio (10 nM:1 µM) indicates that the response to 10 nM iloprost is potentiated at −30 mV (current ratio [10 nM/1 µM] at −90 mV = 0.13 ± 0.10; at −30 mV = 0.44 ± 0.20; *P = 0.016. Wilcoxon matched-pairs signed rank test).
Additional Files
Data Supplement
- Supplemental Figures -
Supplementary Figure 1 - Correction for photobleaching, localization and depolarization in absence of agonist of TP receptor-sensor.
Supplementary Figure 2 - TP receptor- Gα13 and Gα13-p115 RhoGEF interaction depolarization in absence of agonist and localization of TP receptor-eYFP.
Supplementary Figure 3 - Voltage effect on TP receptor`s affinity.
Supplementary Figure 4 - Voltage effect on different agonists activating TP receptor.
Supplementary Figure 5 - Voltage effect on different mutants activating TP receptor.
Supplementary Figure 6 - Signal specificity for FP receptor.
Supplementary Figure 7 - Signal specificity for IP receptor.
Supplementary Figure 8 - Signal specificity for EP3 receptor and EP3 receptor D124R voltage dependence.
- Supplemental Figures -
