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Br J Pharmacol
2024 Nov 08;18121:4229-4244. doi: 10.1111/bph.16490.
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Direct measurements of neurosteroid binding to specific sites on GABAA receptors.
Chintala SM
,
Tateiwa H
,
Qian M
,
Xu Y
,
Amtashar F
,
Chen ZW
,
Kirkpatrick CC
,
Bracamontes J
,
Germann AL
,
Akk G
,
Covey DF
,
Evers AS
.
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BACKGROUND AND PURPOSE: Neurosteroids are allosteric modulators of GABAA currents, acting through several functional binding sites although their affinity and specificity for each site are unknown. The goal of this study was to measure steady-state binding affinities of various neurosteroids for specific sites on the GABAA receptor.
EXPERIMENTAL APPROACH: Two methods were developed to measure neurosteroid binding affinity: (1) quenching of specific tryptophan residues in neurosteroid binding sites by the neurosteroid 17-methylketone group, and (2) FRET between MQ290 (an intrinsically fluorescent neurosteroid) and tryptophan residues in the binding sites. The assays were developed using ELIC-α1GABAAR, a chimeric receptor containing transmembrane domains of the α1-GABAA receptor. Tryptophan mutagenesis was used to identify specific interactions.
KEY RESULTS: Allopregnanolone (3α-OH neurosteroid) was shown to bind at intersubunit and intrasubunit sites with equal affinity, whereas epi-allopregnanolone (3β-OH neurosteroid) binds at the intrasubunit site. MQ290 formed a strong FRET pair with W246, acting as a site-specific probe for the intersubunit site. The affinity and site-specificity of several neurosteroid agonists and inverse agonists was measured using the MQ290 binding assay. The FRET assay distinguishes between competitive and allosteric inhibition of MQ290 binding and demonstrated an allosteric interaction between the two neurosteroid binding sites.
CONCLUSIONS AND IMPLICATIONS: The affinity and specificity of neurosteroid binding to two sites in the ELIC-α1GABAAR were directly measured and an allosteric interaction between the sites was revealed. Adaptation of the MQ290 FRET assay to a plate-reader format will enable screening for high affinity agonists and antagonists for neurosteroid binding sites.
FIGURE 2. Spectral and functional properties of MQ290. (a) Structure of MQ290. (b) Fluorescence excitation and emission spectra for ELIC-α1GABAAR 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 α1β3 GABAA receptors expressed in Xenopus oocytes. (c) Representative traces of currents elicited by low concentrations of GABA (0.02–0.03 μM; PA = 0.03–0.10 for WT and 0.3–1.0 μM; PA = 0.08 for Q241L) in α1β3 (top) and α1Q242Lβ3 (bottom) GABAA receptors. (d) Enhancement of currents elicited with low GABA (n = 3–8) by MQ290 (0.1–30 μM) in α1β3 (○: open circles) and α1Q241Lβ3 (□: 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 α1β3, but not α1Q242Lβ3 receptors, 10- and 30-μM MQ290 enhance (P < 0.05) the currents elicited by GABA.
FIGURE 3. Tryptophan–MQ290 FRET signal reflects a specific interaction with W246 in the intersubunit binding pocket in ELIC-α1GABAAR. (a) Fluorescence emission spectra (Ex = 280 nm) of ELICα1-GABAAR (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 Figure S6). (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 Figure 4a). Subtraction of the non-specific signal from total signal yields the specific FRET binding signal (▲) with a Kd=2.60±0.35μM. 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-α1GABAAR and ELIC-α1W246LGABAAR (n = 5 ± SD for each data point). * indicates that the maximal FRET signal is different (P < 0.05) between WT and W246L receptors.
FIGURE 4. AlloP and Epi-AlloP inhibit FRET between ELIC-α1GABAAR 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-α1Q246LGABAAR in the absence or presence of 30-μM concentrations of AlloP and Epi-AlloP. (d) Concentration-dependence of extracted MQ290-ELIC-α1GABAAR FRET in the presence of 0.3-μM 3×Kd and 30-μM AlloP (n = 5 ± SD at each point). (e) Concentration-dependence of extracted MQ290-ELIC-α1GABAAR FRET in the presence of 1.5-μM 3×Kd and 30-μM Epi-AlloP (n = 5 ± SD at each point).
FIGURE 5. Inhibition of ELIC-α1GABAAR–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). IC50±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.
FIGURE 6. Inhibition of ELIC-α1GABAAR–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). IC50±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-α1GABAAR. The figure shows the extracted FRET spectra of MQ290 in ELIC-α1GABAAR 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 α1β3 GABAA 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; PA = 0.03–0.10). Epi-PREG potentiation is absent in α1Q242Lβ3 GABAA receptors ([GABA] = 0.3– 1.0 μM; PA = 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 α1β3 and α1Q242Lβ3 GABAA 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.
FIGURE 7. The effect of α1Q242L mutation on ELIC-α1GABAAR-MQ290 FRET and AlloP binding to the intersubunit site on ELIC-α1GABAAR. (a) Concentration–dependent MQ290 FRET signal in ELIC-α1GABAAR (WT) and ELIC-α1Q242LGABAAR (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-α1Q242LGABAAR; 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 IC50 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.
