Rapid Throughput Analysis of GABAA Receptor Subtype Modulators and Blockers Using DiSBAC1(3) Membrane Potential Red Dye

Abstract

Fluorometric imaging plate reader membrane potential dye (FMP-Red-Dye) is a proprietary tool for basic discovery and high-throughput drug screening for G-protein-coupled receptors and ion channels. We optimized and validated this potentiometric probe to assay functional modulators of heterologous expressed GABA_A receptor (GABA_AR) isoforms (synaptic α1β3γ2, extrasynaptic α4β3δ, and β3 homopentomers). High-resolution mass spectrometry identified FMP-Red-Dye as 5,5'-(1-propen-1-yl-3-ylidene)bis[1,3-dimethyl-2-thio-barbituric acid]. GABA_AR-expressing cells equilibrated with FMP-Red-Dye exhibited depolarized equilibrium membrane potentials compared with GABA_AR-null cells. The channel blockers picrotoxin, fipronil, and tetramethylenedisulfotetramine, and the competitive antagonist bicuculline reduced fluorescence near the levels in GABA_AR-null cells indicating that FMR-Red-Dye, a barbiturate derivative, activates GABA_AR-mediated outward Cl^- current in the absence of GABA. GABA caused concentration-dependent increases in fluorescence with rank order of potencies among GABA_AR isoforms consistent with results from voltage-clamp experiments (EC₅₀ values for α4β3δ, α1β3γ2, and β₃ homopentamers were 6 ± 1, 40 ± 11, and >18 mM, respectively), whereas GABA_AR-null cells were unresponsive. Neuroactive steroids (NAS) increased fluorescence of GABA_AR expressing cells in the absence of GABA and demonstrated positive allosteric modulation in the presence of GABA, whereas benzodiazepines only exhibited positive allosteric modulator (PAM) activity. Of 20 NAS tested, allopregnanolone, (3α,5α,20E)-3-hydroxy-13,24-cyclo-18-norcholan-20-ene-21-carbonitrile, eltanolone, 5β-pregnan-3α,21-diol-20-one, and ganaxolone showed the highest potency. The FMP-Red-Dye-based assay described here provides a sensitive and quantitative method of assessing the activity of GABA_AR agonists, antagonists, and PAMs on diverse GABA_AR isoforms. The assay has a wide range of applications, including screening for antiseizure agents and identifying channel blockers of interest to insecticide discovery or biosecurity.

Fig. 1.

FMP-Red-Dye modulates α1β3γ2 and α4β3δ GABA_ARs expressed in HEK 293 and L-tk cells, respectively. (A and B) FMP-Red-Dye causes a slow increase in fluorescence [arbitrary florescence units (AFUs)] in cells expressing both receptor types over a 30-minute period that is blocked by 1 µM PTX. The AFU value in GABA_AR-null cells minimally changes during the 30 minutes after onset of FMP-Red-Dye exposure. (C and D) GABA (1 µM) causes an instantaneous increase in AFUs in cells expressing both receptors types compared with GABA_AR-null cells; the AFU continues to slowly rise over 30 minute. GABA has no effect on AFUs in GABA_AR-null cells.

Fig. 2.

Current clamp experiments demonstrating that incubation with FMP-Red-Dye leads to a depolarization of GABA_AR-expressing cells. Membrane potential (V_m) values of α1β3γ2 expressing HEK 293 cells and GABA_AR-null HEK 293 cells measured by current clamp under three conditions: 1) no treatment; 2) incubation with FMP-Red-Dye for 30 minutes prior to recording membrane potential in the absence or presence of 50 µM fipronil (FIP); and 3) incubation with FMP-Red-Dye for 30 minutes followed by 10 minutes of UV irradiation prior to recording membrane potential; n = 8–10 cells per condition. Unpaired t test was used to compare the treatments with control (white bar). *P < 0.05, **P < 0.01. Each bar represents mean ± S.D.

Fig. 3.

Comparison of GABA responses in cells expressing GABA_AR as assessed with the FMP-Red-Dye technique and by voltage-clamp recording. (A) Both α1β3γ2 and α4β3δ GABA_AR-expressing cells exhibit fluorescence responses of increasing amplitude following exposure to increasing concentrations of GABA in the range of 0.1 nM to 30 μM. In these experiments, cells were equilibrated with FMP-Red-Dye for 30 minutes. Then, baseline fluorescence was recorded for 2 minutes followed by exposure to vehicle [(VEH); 0.01% dimethylsulfoxide] or GABA. The ΔF/F₀ values were determined at the peak of the fluorescence response. The black arrow indicates the time of GABA addition; GABA remained for the duration of the recording. GABA_AR-null cells do not respond to GABA. (B, left) Concentration-response curves for GABA based on fluorescence responses reveals that α4β3δ GABA_ARs expressed in L-tk cells are significantly more sensitive to GABA than α1β3γ2 GABA_ARs expressed in HEK 293 cells for EC₅₀ values of 6 nM [95% confidence interval (CI): 4–8 nM (n_H = 0.7; n = 10)] and 40 nM [95% CI: 33–54 nM (n_H = 1.1; n = 10)]. The β3 homopentamers transiently expressed in HEK 293 cell are largely insensitive to GABA (EC₅₀ > 1 mM). Dose-response curves were plotted using nonlinear regression with a four-parameter logistic equation and independent F test was applied to determine the statistical differences for the EC₅₀ values and slopes between α1β3γ2 and α4β3δ GABA_AR-expressing cell lines. Each data point represents mean ± S.D. of data from 10 wells. (B, right) Concentration-response curves for GABA activation of α1β3γ2 receptors in HEK 293 cells and α4β3δ receptors in L-tk cells from whole-cell voltage-clamp recordings for EC₅₀ values of 6.67 μM [95% CI: 5.30–8.04 μM (n_H = 1.8; n = 13)] and 549 μM [95% CI: 435–663 nM (n_H = 1.8; n = 10] Each data point represents mean ± S.D. *P < 0.0001 for α1β3γ2 versus α4β3δ.

Fig. 4.

PTX and TETS block α₁β₃γ₂ GABA_AR-dependent FMP-Red-Dye fluorescence. HEK 293 cells stably transfected with α1β3γ2 GABA_AR were exposed to FMP-Red-Dye for 30 minutes to activate the receptors. GABA_AR-null cells were used as the control. PTX (A) or TETS (C) caused slow, concentration-dependent inhibition of the fluorescence in cells expressing α1β3γ2 GABA_AR; minimal effects were obtain in GABA_AR-null cells. Arrows indicate time of addition of PTX and TETS to the wells; the blockers were not removed. (B and D) Plots of ΔF/F₀ from experiments similar to those illustrated in (A) and (C). Each plot represents eight experiments. The IC₅₀ values are 6.5 µM [95% confidence interval (CI): 3.2–12 μM] and 3.8 µM (95% CI: 2.8–5.2 μM) for PTX and TETS, respectively. TETS is significantly more potent than PTX (P < 0.0001). Dose-response curves were plotted using nonlinear regression with a four-parameter logistic equation and independent F test was applied to determine the statistical differences for the EC₅₀ values between TETS and PTX. Each data point represents mean ± S.D. of data from eight wells. Red traces represent responses to vehicle (0.01% dimethylsulfoxide). *P < 0.0001.

Fig. 5.

GABA potentiation and PTX inhibition of β₃ homopentameric GABA_AR-dependent FMP-Red-Dye fluorescence. HEK 293 cells transiently transfected with β3 homopentameric GABA_AR were exposed to FMP-Red-Dye for 30 minutes to activate the receptors. GABA_AR-null cells were used as the control. (A) GABA caused slow, concentration-dependent potentiation of the fluorescence in cells expressing β3 homopentameric GABA_AR; minimal effects were obtained in GABA_AR-null cells. (B) PTX caused slow, concentration-dependent inhibition of the fluorescence in cells expressing β3 homopentameric GABA_AR; minimal effects were obtained in GABA_AR-null cells. Arrows indicate time of addition of GABA and PTX, which was not removed. Red traces represent the responses to vehicle (0.01% dimethylsulfoxide). (B and D) Plots of ΔF/F₀ from experiments similar to those illustrated in (A) and (C). Each data point represents mean ± S.D. of data from 10 wells. The EC₅₀ value for GABA could not be determined since the response did not plateau. The IC₅₀ value for PTX is 1.8 µM (95% confidence interval: 1.4–2.2 μM). Dose-response curves were plotted for PTX using nonlinear regression with a four-parameter logistic equation.

Fig. 6.

Fipronil blocks β3-homomeric GABA_ARs, while allpregnanolone fails to affect the fluorescence signal even at concentrations ≥ 10 µM. (A) Fipronil blocks β3 receptors in a concentration-dependent manner but does not have any significant effects on GABA_AR-null cells. Dose-response curves were plotted using nonlinear regression with a four-parameter logistic equation. Each data point represents mean ± S.D. of data from eight wells. (B) Allopregnanolone does not have any effect even at 10 µM.

Fig. 7.

NAS-induced FMP-Red-Dye fluorescence responses of α1β3γ2 GABA_AR in HEK 293 cells. (A–D) Representative traces of responses to 0.01 nM to 10 μM of eltanolone, allopregnanolone, XJ-42, and ganaxolone, respectively. The arrow indicates time of addition of the test compound, which was not removed. Red traces represent the responses to vehicle (0.01% dimethylsulfoxide). (E) Concentration-response curves for each NAS and cortisol. Dose-response curves were plotted using nonlinear regression with a four-parameter logistic equation. Separate one-way analysis of variance with additional correction (Tukey) for post hoc multiple comparisons was applied for EC₅₀ and slope to determine the statistical differences within a subunit composition—not across subtypes (Table 2). Each data point represents mean ± S.D. of data from 10 wells.*P < 0.0001.

Fig. 8.

NAS-induced FMP-Red-Dye fluorescence responses of α4β3δ GABA_ARs in L-tk cells. (A–D) Representative traces of responses to 0.01 nM to 10 μM of eltanolone, allopregnanolone, XJ-42, and ganaxolone, respectively. The arrow indicates time of addition of the test compound, which was not removed. Red traces represent the responses to vehicle (0.01% dimethylsulfoxide). (E) Concentration-response curves for each NAS. Dose-response curves were plotted using nonlinear regression with a four-parameter logistic equation. Separate one way analysis of variance with additional correction (Tukey) for post hoc multiple comparisons was applied for EC₅₀ and slope to determine the statistical differences within a subunit composition—not across subtypes (Table 2). Each data point represents mean ± S.D. of data from 10 wells.*P < 0.0001.

Fig. 9.

Allopregnanolone and ganaxolone potentiation of GABA responses of α1β3γ2 GABA_AR in HEK 293 cells. (A) Concentration-response curves for allopregnanolone and ganaxolone potentiation of FMP-Red Dye responses to 10 nM GABA. The graph plots mean ± S.D. fold increase in peak response in the presence the NAS compared with the response to 10 nM GABA alone. Each data point is the mean ± S.D. of measurements of 10 wells. The EC₅₀ values for allopregnanolone and ganaxolone are 1.7 nM [95% confidence interval (CI): 1–3.1 nM (n_H = 1.5)] and 20 nM [95% CI: 14–55 nM (n_H = 1.4)], respectively (P < 0.0001). Dose-response curves were plotted using nonlinear regression with a four-parameter logistic equation and independent F test was applied to determine the statistical differences for EC₅₀ values and slopes between allopregnanolone and ganaxolone in combination with 10 nM GABA in α1β3γ2 GABA_AR in HEK 293 cells. Inset shows representative traces with addition of GABA alone and GABA plus allopregnanolone at concentrations of 1 and 10 nM. (B) Concentration-response curves for allopregnanolone and ganaxolone potentiation of peak inward Cl⁻ current responses to 1 μM GABA (EC₁₀ value) in patch-clamp recordings. Each data point is the mean ± S.D. of measurements of 3–6 cells. The EC₅₀ values for allopregnanolone and ganaxolone are 71.3 nΜ [95% CI: 57.1–85.5 nM (n_H = 1.8)] and 114.8 nΜ [95% CI: 99.2–130.4 nM (n_H = 2.2)], respectively. Inset shows representative traces with application of GABA alone and GABA plus allopregnanolone at 100 and 175 nM.

Fig. 10.

Effects of midazolam on FMP-Red-Dye fluorescence in cells expressing α1β3γ2 GABA_AR. In the absence of GABA, midazolam fails to generate substantial fluorescence signals compared with vehicle (Veh) except at high concentrations (>1 μM). GABA (10 nM) induces a small fluorescence signal. Combination of midazolam and GABA results in potentiation of the signal [EC₅₀, 51 nM (95% confidence interval: 30–83 nM)]. Dose-response curves were plotted using nonlinear regression with a four-parameter logistic equation. Each data point represents the mean ± S.D. of measurements in eight cells.