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. 2010 May;160(1):130-41.
doi: 10.1111/j.1476-5381.2010.00679.x.

Structurally Diverse Amphiphiles Exhibit Biphasic Modulation of GABAA Receptors: Similarities and Differences With Neurosteroid Actions

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Free PMC article

Structurally Diverse Amphiphiles Exhibit Biphasic Modulation of GABAA Receptors: Similarities and Differences With Neurosteroid Actions

M Chisari et al. Br J Pharmacol. .
Free PMC article

Abstract

Background and purpose: Some neurosteroids, notably 3alpha-hydroxysteroids, positively modulate GABA(A) receptors, but sulphated steroids negatively modulate these receptors. Recently, other lipophilic amphiphiles have been suggested to positively modulate GABA receptors. We examined whether there was similarity among the actions of these agents and the mechanisms of neurosteroids. Significant similarity would affect theories about the specificity of steroid actions.

Experimental approach: Xenopus laevis oocytes were challenged with Triton X-100, octyl-beta-glucoside, capsaicin, docosahexaenoic acid and sodium dodecyl sulphate (SDS), along with different GABA concentrations.

Key results: These compounds have both positive and negative effects on GABA currents, which can be accentuated according to the degree of receptor activation. A low GABA concentration (1 microM) promoted potentiation and a high concentration (20 microM) promoted inhibition of current, except for SDS that inhibited function even at low GABA concentrations. Amphiphile inhibition was characterized by enhanced apparent desensitization and by weak voltage dependence, similar to pregnenolone sulphate antagonism. We then tested amphiphile effects on mutated receptor subunits that are insensitive to negative (alpha1V256S) and positive (alpha1Q241L or alpha1N407A/Y410F) steroid modulation. Negative regulation by amphiphiles was nearly abolished in alpha1V256S-mutated receptors, but potentiation was unaffected. In alpha1Q241L- or alpha1N407A/Y410F-mutated receptors, potentiation by amphiphiles remained intact.

Conclusions and implications: Structurally diverse amphiphiles have antagonist actions at GABA(A) receptors very similar to those of sulphated neurosteroids, while the potentiating mechanisms of these amphiphiles are distinct from those of neurosteroid-positive modulators. Thus, such antagonism at GABA(A) receptors does not have a clear pharmacophore requirement.

Figures

Figure 1
Figure 1
GABA current is typically potentiated by amphiphiles at low GABA concentration. (A) Representative traces of oocytes stimulated by 1 µM GABA (black trace, left) and by co-application of 50 µM Triton X-100 (red trace, middle). After Triton washout, GABA was reapplied to measure current recovery (grey trace, right). (B) Representative traces of other tested amphiphiles (as indicated). (C) Summary of peak current in oocytes treated as in (A and B). GABA responses were averaged (dotted line). Values are means ± SE [Triton, n= 8; octyl-β-glucoside (OG), n= 9, capsaicin, n= 10; docosahexaenoic acid (DHA), n= 10; sodium dodecyl sulphate (SDS), n= 7], *P < 0.05, **P < 0.01 ***P < 0.0001 (paired t-test), significantly different from responses to GABA alone (dotted line). (D) Increasing concentrations of Triton (10–300 µM, n= 7), OG (0.1–10 mM, n= 3), capsaicin (10–300 µM, n= 4) and DHA (3–300 µM, n= 6) were applied to Xenopus laevis oocytes expressing rat α1β2γ2L GABAA receptor subunits. Concentration–response curves were obtained in the presence of 1 µM GABA, and potentiation was expressed relative to the GABA response (expressed as 1) in the absence of amphiphiles. For display purposes, the lines represent fits of the Hill equation (adjusted to account for the lower asymptote of 1.0) to the averaged data points shown in each graph. Parameters from the summary fits for Triton, OG, capsaicin and DHA, respectively, were: EC50 32 µM, 3160 µM, 77 µM and 28 µM; Hill coefficient: 7.7, 2.4, 2.1 and 1.6. (E) Increasing concentrations of SDS (1–100 µM, n= 5) on α1β2γ2L GABAA receptors expressed in X. laevis oocytes. This concentration–response curve was obtained in the presence of 20 µM GABA, and inhibition was expressed relative to the GABA response (treated as 1) in the absence of SDS. Limiting factors that prevented the use higher concentrations of amphiphiles were solution saturation (for OG and capsaicin) and plasma membrane damage (for Triton X-100 and SDS).
Figure 3
Figure 3
The effects of Triton X-100 at multiple GABA concentrations. (A1–A4) Representative responses from one oocyte to increasing GABA concentrations (0.3–100 µM) in the presence and absence (as indicated) of 50 µM Triton X-100. For these experiments GABA and Triton X-100 were simultaneously co-applied; there was no pre-application of modulator. (B and C) Concentration–response curves of current at the peak and at the end of drug application (as indicated) resulting from increasing GABA concentrations alone (0.3–300 µM) and in the presence of 50 µM Triton X-100 (n= 10–14). Responses were expressed relative to that of the highest GABA concentration applied (300 µM). For display purposes, the lines represent fits of the Hill equation to the averaged data points shown in the figure. Note that because of the mixture of effects, a single Hill equation is likely not an appropriate model, so the parameters from these fits are of empirical value only. Parameters from the summary fits for GABA alone and in presence of Triton X-100, respectively, were: for the peak, EC50 52.2 µM and 2.7 µM, Hill coefficient 0.9 and 1.7; for the end, EC50 12.4 µM and 0.7 µM, Hill coefficient 1.7 and 1.8.
Figure 2
Figure 2
GABA current is inhibited by amphiphiles at high GABA concentration. (A) Representative traces of oocytes stimulated by 20 µM GABA (black trace) and by co-application of amphiphiles (red traces, as indicated). GABA was reapplied after each modulator to measure current recovery (grey traces). (B) Summary of normalized current (peak and end of drug application) in oocytes treated as in (A). Dotted horizontal line represents averaged GABA responses before and after drug application. Values are means ± SE [Triton, n= 14; sodium dodecyl sulphate (SDS), n= 6; octyl-β-glucoside (OG), n= 10; capsaicin, n= 16; docosahexaenoic acid (DHA), n= 16], ***P < 0.0001 (paired t-test), significantly different from responses to GABA alone (dotted line).
Figure 4
Figure 4
Inhibition is modestly voltage-dependent. (A and C) Representative oocyte currents to 100 µM GABA (black trace) and to co-application of Triton or sodium dodecyl sulphate (SDS) (red traces, as indicated). (B and D) Summary of normalized current (peak and end of drug application) in oocytes treated as in (A and C). Dotted horizontal line represents averaged GABA responses before and after drug application. Values are means ± SE (Triton, n= 5; SDS, n= 6), *P < 0.05, **P < 0.01, ***P < 0.0001 (paired t-test), significantly different from responses to GABA alone (dotted line).
Figure 5
Figure 5
α1Q241L-mutated receptors show similar potentiation (and inhibition) as wild-type (WT) receptors. (A) Representative traces of GABA potentiation by amphiphiles (as indicated). (B) Representative traces of inhibited GABA current by sodium dodecyl sulphate (SDS) 25 µM. (C) Summary of peak current in oocytes treated as in (A and B). Dotted horizontal line represents averaged GABA responses before and after drug application. Values are means ± SE [Triton, n= 6; octyl-β-glucoside (OG), n= 7, capsaicin, n= 6; docosahexaenoic acid (DHA), n= 7; SDS, n= 7], **P < 0.01, ***P < 0.0001 (paired t-test), significantly different from responses to GABA alone (dotted line).
Figure 6
Figure 6
α1V256S mutation reduces amphiphile inhibition but not potentiation compared with wild-type (WT). (A) Representative responses of oocytes to 5 µM GABA (black traces) and to co-application of amphiphiles (red traces, as indicated). (B) Summary of normalized current (peak and steady state) in oocytes treated as in panel (A). Dotted horizontal line represents averaged GABA responses before and after drug application. Values are means ± SE [Triton, n= 5; sodium dodecyl sulphate (SDS), n= 6; octyl-β-glucoside (OG), n= 5, capsaicin, n= 5; docosahexaenoic acid (DHA), n= 8], *P < 0.05, **P < 0.01, ***P < 0.0001 (paired t-test), significantly different from responses to GABA alone (dotted line). (C) Representative traces of GABA potentiation by amphiphiles (as indicated) in presence of 0.05 µM GABA. (D) Summary of peak current in oocytes treated as in (A). Dotted horizontal line represents averaged GABA responses before and after drug application. Values are means ± SE (Triton, n= 6; OG, n= 6, capsaicin, n= 7; DHA, n= 7; SDS n= 7), *P < 0.05, **P < 0.01, ***P < 0.0001 (paired t-test), significantly different from responses to GABA alone (dotted line).

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