Journal: British journal of pharmacology

Article Title: Direct measurements of neurosteroid binding to specific sites on GABA A receptors

doi: 10.1111/bph.16490

Figure Lengend Snippet: NS quenching of tryptophan residues defines two distinct binding sites in ELIC-α1GABA A R. (a) Docking poses of AlloP in the intersubunit and α1-intrasubunit binding sites in the X-ray crystallographic structure of a homo-pentameric ELIC-α1GABA A R (PDB: 6CDU). Adjacent subunits are coloured in blue and lavender to emphasize the interface between two identical subunits. The side chains of tryptophan residues (W246, W412, W288 and W161) and glutamine 242 (Q242) are shown as ball and stick drawings. AlloP is shown as a stick drawing in olive with the lowest energy poses in the intersubunit site adjacent to W246 and the α1-intrasubunit site adjacent to W412. (b) Structures of AlloP and Epi-AlloP. The 17-methylketone on the D-ring is responsible for quenching of tryptophan by photo-induced electron transfer. (c, d) Fluorescence emission spectra of ELIC-α1GABA A R (0.3 μM) following excitation at 280 nm in the presence of indicated concentrations of AlloP and Epi-AlloP. (e, f) Concentration–dependent quenching of tryptophan emission by AlloP and Epi-AlloP in wild-type receptors and receptors with a W246L mutation. Data are normalized such that peak emission in the absence of NS is set to a value of one. The extent of quenching by AlloP (panel e) is significantly different between WT and W246L; * P < 0.05.

Article Snippet: One hundred microlitres of purified ELIC-α1GABA A R (0.3 μM in Buffer A + 0.015% DDM) was transferred into a 50-μl window quartz cuvette (Starna Cells, Atascadero, CA, USA), which was placed in a cuvette holder maintained at 4°C.

Techniques: Binding Assay, Fluorescence, Concentration Assay, Mutagenesis

Journal: British journal of pharmacology

Article Title: Direct measurements of neurosteroid binding to specific sites on GABA A receptors

doi: 10.1111/bph.16490

Figure Lengend Snippet: Spectral and functional properties of MQ290. (a) Structure of MQ290. (b) Fluorescence excitation and emission spectra for ELIC-α1GABA A R tryptophan and MQ290. The overlap of tryptophan emission (violet) and MQ290 excitation (cyan) is shown in blue. (c and d) Effects of MQ290 on GABA-elicited currents in α 1 β 3 GABA A receptors expressed in Xenopus oocytes. (c) Representative traces of currents elicited by low concentrations of GABA (0.02–0.03 μM; P A = 0.03–0.10 for WT and 0.3–1.0 μM; P A = 0.08 for Q241L) in α 1 β 3 (top) and α 1 Q242L β 3 (bottom) GABA A receptors. (d) Enhancement of currents elicited with low GABA (n = 3–8) by MQ290 (0.1–30 μM) in α 1 β 3 (○: open circles) and α 1 Q241L β 3 (□: open squares). The y-axis shows the ratio of the response of GABA + MQ290 to GABA alone, with a value of 1 indicating no MQ290 enhancement. * indicates that in α 1 β 3 , but not α 1 Q242L β 3 receptors, 10- and 30-μM MQ290 enhance ( P < 0.05) the currents elicited by GABA.

Article Snippet: One hundred microlitres of purified ELIC-α1GABA A R (0.3 μM in Buffer A + 0.015% DDM) was transferred into a 50-μl window quartz cuvette (Starna Cells, Atascadero, CA, USA), which was placed in a cuvette holder maintained at 4°C.

Techniques: Functional Assay, Fluorescence

Journal: British journal of pharmacology

Article Title: Direct measurements of neurosteroid binding to specific sites on GABA A receptors

doi: 10.1111/bph.16490

Figure Lengend Snippet: Tryptophan–MQ290 FRET signal reflects a specific interaction with W246 in the intersubunit binding pocket in ELIC-α 1 GABA A R. (a) Fluorescence emission spectra (Ex = 280 nm) of ELICα 1 -GABA A R (0.3 μM) in the presence of varying concentrations of MQ290 (0–30 μM). (b) Extracted tryptophan-MQ290 FRET signal from spectra in panel a; the FRET signal is isolated by subtraction of the emission spectra of MQ290 without protein and of the contribution of tryptophan emission to the spectra (see ). (c) FRET intensity as a function of MQ290 concentration. The figure plots peak intensity of the FRET spectra (370 nm) in the absence (●: total) or presence (■: non-specific) of 30-μM AlloP, where AlloP is a competitive inhibitor of MQ290 binding (see ). Subtraction of the non-specific signal from total signal yields the specific FRET binding signal (▲) with a K d = 2.60 ± 0.35 μM . All values are normalized to the total FRET signal at 30 μM with n = 5 for each data point. (d) Specific FRET signal plotted as a function of MQ290 concentration in wild-type (WT) ELIC-α1GABA A R and ELIC-α 1 W246L GABA A R (n = 5 ± SD for each data point). * indicates that the maximal FRET signal is different ( P < 0.05) between WT and W246L receptors.

Article Snippet: One hundred microlitres of purified ELIC-α1GABA A R (0.3 μM in Buffer A + 0.015% DDM) was transferred into a 50-μl window quartz cuvette (Starna Cells, Atascadero, CA, USA), which was placed in a cuvette holder maintained at 4°C.

Techniques: Binding Assay, Fluorescence, Isolation, Concentration Assay

Journal: British journal of pharmacology

Article Title: Direct measurements of neurosteroid binding to specific sites on GABA A receptors

doi: 10.1111/bph.16490

Figure Lengend Snippet: AlloP and Epi-AlloP inhibit FRET between ELIC-α1GABA A R and MQ290. (a) Extracted FRET spectra of MQ290 (3 μM) in the absence or presence of AlloP (0.01–30 μM). Inhibition of the FRET signal saturates at AlloP concentrations ≥ 1 μM. (b) Extracted FRET spectra of MQ290 (1 μM) in the absence or presence of Epi-AlloP (0.01–30 μM). Inhibition of the FRET signal saturates at Epi-AlloP concentrations ≥ 10 μM. (c) Extracted FRET spectra of MQ290 (1 μM) with ELIC-α 1 Q246L GABA A R in the absence or presence of 30-μM concentrations of AlloP and Epi-AlloP. (d) Concentration-dependence of extracted MQ290-ELIC-α1GABA A R FRET in the presence of 0.3-μM 3 × K d and 30-μM AlloP (n = 5 ± SD at each point). (e) Concentration-dependence of extracted MQ290-ELIC-α1GABA A R FRET in the presence of 1.5-μM 3 × K d and 30-μM Epi-AlloP (n = 5 ± SD at each point).

Article Snippet: One hundred microlitres of purified ELIC-α1GABA A R (0.3 μM in Buffer A + 0.015% DDM) was transferred into a 50-μl window quartz cuvette (Starna Cells, Atascadero, CA, USA), which was placed in a cuvette holder maintained at 4°C.

Techniques: Inhibition, Concentration Assay

Journal: British journal of pharmacology

Article Title: Direct measurements of neurosteroid binding to specific sites on GABA A receptors

doi: 10.1111/bph.16490

Figure Lengend Snippet: Inhibition of ELIC-α1GABA A R–MQ290 FRET by 3α-OH NS. (a) Structures of 3α-OH NS. (b) Inhibition of MQ290 (3 μM) FRET signal. FRET intensity for each sample was normalized to the maximum signal (370 nm) in the absence of competitor (n = 5 ± SE for each data point). IC 50 ± SE values for the NS are AlloP = 0.23 ± 0.02 μM; GX = 0.43 ± 0.05; PREG = 1.92 ± 0.64; and YX03 = 0.91 ± 0.35.

Article Snippet: One hundred microlitres of purified ELIC-α1GABA A R (0.3 μM in Buffer A + 0.015% DDM) was transferred into a 50-μl window quartz cuvette (Starna Cells, Atascadero, CA, USA), which was placed in a cuvette holder maintained at 4°C.

Techniques: Inhibition

Journal: British journal of pharmacology

Article Title: Direct measurements of neurosteroid binding to specific sites on GABA A receptors

doi: 10.1111/bph.16490

Figure Lengend Snippet: Inhibition of ELIC-α1GABA A R–MQ290 FRET by 3β-OH NS. (a) Structures of 3β-OH NS. (b) Inhibition of MQ290 (1 μM) FRET signal. FRET intensity for each sample was normalized to the maximum signal (370 nm) in the absence of competitor (n = 5 ± SD for each data point). IC 50 ± SE values for the NS are Epi-AlloP = 0.36 ± 0.30 μM; Epi-GX = 1.92 ± 0.40 μM; Epi-PREG = 1.31 ± 0.42 μM. (c) Inhibition of MQ290 (0.5 μM) FRET signal in ELIC-α1GABA A R. The figure shows the extracted FRET spectra of MQ290 in ELIC-α1GABA A R in the presence of various combinations of Epi-PREG, Epi-AlloP and AlloP. Epi-PREG adds to the inhibition of the FRET signal produced by a saturating concentration of Epi-AlloP (30 μM) but does not add to the inhibition produced by saturating concentrations of either AlloP (30 μM) or AlloP + Epi-AlloP. (d) Epi-PREG potentiates GABA-elicited currents in α 1 β 3 GABA A receptors expressed in Xenopus oocytes. (Left) Representative traces showing that Epi-PREG, but not Epi-AlloP or Epi-GX (all 10 μM) potentiates the currents elicited by GABA (0.02–0.03 μM; P A = 0.03–0.10). Epi-PREG potentiation is absent in α 1 Q242L β 3 GABA A receptors ([GABA] = 0.3– 1.0 μM; P A = 0.08), indicating that its action is mediated by the intersubunit binding site. (Right) Enhancement of currents elicited with low GABA (n = 3–8) by 3β-OH NS (10 μM) in wild-type α 1 β 3 and α 1 Q242L β 3 GABA A receptors. The y-axis shows the ratio of the response to GABA + NS to GABA alone, with a value of 1 indicating no enhancement. * indicates a significant difference ( P < 0.05) between (NS + GABA) and GABA alone.

Article Snippet: One hundred microlitres of purified ELIC-α1GABA A R (0.3 μM in Buffer A + 0.015% DDM) was transferred into a 50-μl window quartz cuvette (Starna Cells, Atascadero, CA, USA), which was placed in a cuvette holder maintained at 4°C.

Techniques: Inhibition, Produced, Concentration Assay, Binding Assay

Journal: British journal of pharmacology

Article Title: Direct measurements of neurosteroid binding to specific sites on GABA A receptors

doi: 10.1111/bph.16490

Figure Lengend Snippet: The effect of α 1 Q242L mutation on ELIC-α1GABA A R-MQ290 FRET and AlloP binding to the intersubunit site on ELIC-α1GABA A R. (a) Concentration–dependent MQ290 FRET signal in ELIC-α1GABA A R (WT) and ELIC-α1 Q242L GABA A R (n = 3). The significantly reduced FRET indicates that MQ290 binds to the intersubunit site with a different pose than in WT. (b) AlloP (0.01–30 μM) quenching of tryptophan emission (Ex280) in WT and ELIC-α1 Q242L GABA A R; experiments were conducted in the presence of 30 μM Epi-AlloP to occlude quenching of intrasubunit site tryptophan residues. The data for each sample are normalized to the maximal tryptophan emission (330 nm) in the absence of AlloP (n = 5 ± SE for each data point). The extent of quench is reduced by the Q242L mutation (−17% in Q242L vs. −28% in WT), and there is no change in IC 50 values (0.73 ± 0.12 for WT; 0.70 ± 0.18 for Q242L), indicating that Q242L changes the binding pose of AlloP in the intersubunit site with only a modest change in binding affinity. * indicates a difference ( P < 0.05) in the maximal FRET intensity between WT and Q242L receptors.

Article Snippet: One hundred microlitres of purified ELIC-α1GABA A R (0.3 μM in Buffer A + 0.015% DDM) was transferred into a 50-μl window quartz cuvette (Starna Cells, Atascadero, CA, USA), which was placed in a cuvette holder maintained at 4°C.

Techniques: Mutagenesis, Binding Assay, Concentration Assay