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Novel Molecule Exhibiting Selective Affinity for GABAA Receptor Subtypes.
Borghese CM, Herman M, Snell LD, Lawrence KJ, Lee HY, Backos DS, Vanderlinden LA, Harris RA, Roberto M, Hoffman PL, Tabakoff B.
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Aminoquinoline derivatives were evaluated against a panel of receptors/channels/transporters in radioligand binding experiments. One of these derivatives (DCUK-OEt) displayed micromolar affinity for brain γ-aminobutyric acid type A (GABAA) receptors. DCUK-OEt was shown to be a positive allosteric modulator (PAM) of GABA currents with α1β2γ2, α1β3γ2, α5β3γ2 and α1β3δ GABAA receptors, while having no significant PAM effect on αβ receptors or α1β1γ2, α1β2γ1, α4β3γ2 or α4β3δ receptors. DCUK-OEt modulation of α1β2γ2 GABAA receptors was not blocked by flumazenil. The subunit requirements for DCUK-OEt actions distinguished DCUK-OEt from other currently known modulators of GABA function (e.g., anesthetics, neurosteroids or ethanol). Simulated docking of DCUK-OEt at the GABAA receptor suggested that its binding site may be at the α + β- subunit interface. In slices of the central amygdala, DCUK-OEt acted primarily on extrasynaptic GABAA receptors containing the α1 subunit and generated increases in extrasynaptic "tonic" current with no significant effect on phasic responses to GABA. DCUK-OEt is a novel chemical structure acting as a PAM at particular GABAA receptors. Given that neurons in the central amygdala responding to DCUK-OEt were recently identified as relevant for alcohol dependence, DCUK-OEt should be further evaluated for the treatment of alcoholism.
Figure 2. DCUK effect on GABA responses. (a) Effect of DCUK compounds on submaximal (EC10) GABA responses of α1β2γ2 GABAA receptors (n = 4–5 at each concentration of DCUK compound). (b) Effect of DCUK-OEt on submaximal (EC10) GABA responses of α1β3δ GABAA receptors (n = 5–6 at each concentration of DCUK-OEt). (c) Effect of DCUK-OEt (0.3 µM) and escalating GABA concentrations applied to α1β2γ2 and α1β3δ GABAA receptors (n = 9 each). GABA concentrations used: 3 and 1 µM for α1β2γ2 and α1β3δ, respectively (~EC10); 30 µM (~EC60); 3 mM (~EC100). (d) DCUK-OEt (0.3 μM) effect in the absence and presence of 20 μM flumazenil (Flu) (n = 5 for each condition). Data represent mean ± SEM. **p < 0.01 compared to α1β2γ2 (one-way ANOVA and post-hoc contrasts).
Figure 3. Predicted docking of DCUK-OEt and DCUKA within extracellular domain interfaces of GABAA receptor subunits. The α subunit is shaded in green, β in cyan and γ in yellow. (a) Extracellular (top down) view of the pentameric GABAA receptor. The interfaces illustrated are α + β- (alternative site), and α + γ- (benzodiazepine site). (b) DCUK-OEt and (c) DCUKA within the alternative site (α + β-). DCUK-OEt is represented by orange sticks and DCUKA is represented by pink sticks. (d) DCUKA and (e) DCUK-OEt within the benzodiazepine site (α + γ-). Dashed lines indicate predicted non-bond interactions (green = H-bonds, orange = electrostatic or π-cation/anion, magenta = π-π, purple = π-σ, pink = hydrophobic).
Figure 4. DCUK-OEt potentiates tonic currents in medialCeA neurons. (a and b) Focal application of DCUK-OEt (0.5 µM) significantly increased the holding current in medialCeA neurons (*p < 0.05, paired t-test). (c) No change was evident in frequency, amplitude, rise and decay of mIPSPs with focal application of DCUK-OEt. (d) Correlation of magnitude of increase in tonic current produced by 0.5 μM DCUK-OEt with reduction of current by subsequent application of 100 μM gabazine. To demonstrate that changes in holding current were due to increases in tonic signaling, the GABAA receptor antagonist gabazine (GBZ) (100 μM) was focally applied following DCUK-OEt application. For all graphs, n = 11 cells.
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