CGP7930 - An allosteric modulator of GABABRs, GABAARs and inwardly-rectifying potassium channels

Abstract

Type-A and -B GABA receptors (GABA_ARs/GABA_BRs) control brain function and behaviour by fine tuning neurotransmission. Over-time these receptors have become important therapeutic targets for treating neurodevelopmental and neuropsychiatric disorders. Several positive allosteric modulators (PAMs) of GABARs have reached the clinic and selective targeting of receptor subtypes is crucial. For GABA_BRs, CGP7930 is a widely used PAM for in vivo studies, but its full pharmacological profile has not yet been established. Here, we reveal that CGP7930 has multiple effects not only on GABA_BRs but also GABA_ARs, which for the latter involves potentiation of GABA currents, direct receptor activation, and also inhibition. Furthermore, at higher concentrations, CGP7930 also blocks G protein-coupled inwardly-rectifying K⁺ (GIRK) channels diminishing GABA_BR signalling in HEK 293 cells. In male and female rat hippocampal neuron cultures, CGP7930 allosteric effects on GABA_ARs caused prolonged rise and decay times and reduced the frequency of inhibitory postsynaptic currents and potentiated GABA_AR-mediated tonic inhibition. Additional comparison between predominant synaptic- and extrasynaptic-isoforms of GABA_AR indicated no evident subtype selectivity for CGP7930. In conclusion, our study of CGP7930 modulation of GABA_ARs, GABA_BRs and GIRK channels, indicates this compound is unsuitable for use as a specific GABA_BR PAM.

Keywords: CGP7930; GABA; GABA(A) receptors; GABA(B) receptors; Neuronal inhibition; Positive allosteric modulator; Potassium channels.

Fig. 1

Modulatory effects of CGP7930 on GABA_A and GABA_B receptors A) Comparative overlay for CGP7930 (blue) and propofol (red) structures reveals a high degree of conformational similarity. B) Whole cell GABA current profiles recorded from GIRK cells expressing GABA_B receptors activated by 0.01–1000 μM GABA. C) Normalised GABA concentration-response relationship for GABA_B receptor activated currents in GIRK cells. (EC₅₀ = 2.0 ± 0.7 μM; n = 6). D) Representative whole-cell currents activated by ∼ EC₂₀ GABA (black bar) in the absence and presence (following pre-incubation) of CGP7930 (blue bar). E) Concentration-response relationship for CGP7930 modulating EC₂₀ GABA_B receptor activated currents normalised (%) to the control maximal GABA response in the absence of CGP7930 (EC₅₀ = 11.1 ± 7.0 μM; IC₅₀ = 10.5 ± 2.0 μM; n = 4). Note the bell-shaped nature of the curve. In this and succeeding figures, concentration response data points represent the mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

CGP7930 modulation of neuronal GABA_A and GABA_B receptors A) Whole-cell voltage-clamp baclofen- and muscimol-activated currents recorded from hippocampal neurons in culture. B) Normalised concentration-response relationships for baclofen and muscimol in hippocampal neurons. Baclofen EC₅₀ = 5.6 ± 0.4 μM (n = 12) and muscimol EC₅₀ = 2.7 ± 0.1 μM (n = 9). C) Whole-cell currents in hippocampal cultured neurons evoked by maximum and ∼EC_10-15 baclofen (1 μM, top panel, blue bar) and muscimol (0.5 μM, middle, black bar) showing potentiation by CGP7930 (pre-incubation, grey bar), and direct activation by CGP7930 when applied alone (bottom). Note that cells were bathed in high external K⁺ for recording baclofen-activated currents and for the direct activation by CGP7930. D) Baclofen and muscimol agonist potentiation-response curves in the presence of CGP7930, normalised (%) to the respective control maximum agonist response (I_max) (Muscimol: EC₅₀ = 2.0 ± 1.1 μM; IC₅₀ = 7.9 ± 1.8 μM; n = 10; Baclofen: EC₅₀ = 0.9 ± 0.6 μM; n = 7). E) Whole-cell voltage-clamp currents directly activated by 10 μM CGP7930 in high K⁺ external solution. The inward current is blocked by either 100 μM picrotoxin (PTX) or bicuculline (Bic). Note that the downward deflection after picrotoxin application is a rebound current arising post wash-off. F) Bar chart showing inhibition of CGP7930 direct activation current by PTX and Bic (both 100 μM). Bar graph represents the mean ± S.E.M. n = 4–12 cells; ***P < 0.001; one-way ANOVA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Inhibition of K⁺ currents by CGP7930 in GIRK cells A) Representative, basally-active, whole-cell currents for GIRK channels bathed in high K⁺ solution, showing inhibition by CGP7930 (0.1–100 μM). Note, the inward basal K⁺ current is revealed following CGP7930 block. B) Concentration response curve for CGP7930 blocking basally-active GIRK channels shown as increasing outward K⁺ currents that are normalised to the maximal (%) CGP7930 block (= maximum outward current response) from the same cell. EC₅₀ = 9.7 ± 0.6 μM; n = 4. C) Current-voltage relationships (I–V) for basally active whole-cell K⁺ currents in GIRK cells under the following conditions: control/basal I–V (black); +3 mM Ba²⁺ (blue); +10 μM CGP7930 (red open symbols); and +100 μM CGP7930 (red filled symbols); n = 5–6. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Modulatory effects of CGP7930 at recombinant GABA_A receptor currents in HEK-293 cells A) Whole-cell GABA-activated current profiles recorded from HEK-293 cells expressing α1β2γ2L (top) or α4β3δ (bottom) receptors. B) GABA concentration-response relationships for α1β2γ2L and α4β3δ receptors expressed in HEK-293 cells. EC₅₀ α1β2γ2L = 5.1 ± 1.7 μM; n = 9; α4β3δ = 1.2 ± 0.1 μM; n = 7. C) Directly-activated CGP7930 currents (1–100 μM, red bars) recorded from HEK-293 cells expressing either α1β2γ2L or α4β3δ receptors. D) Concentration-response curves for CGP7930 direct activation of α1β2γ2L or α4β3δ receptors (normalised to the respective maximum GABA currents). E) Control maximum and ∼EC₂₀ GABA (black) currents recorded from HEK-293 cells expressing α1β2γ2L and α4β3δ receptors, followed by GABA EC₂₀ currents in the pre-applied presence (red bar) of 0.01–30 μM CGP7930. Note direct activation currents for 3, 10 and 30 μM CGP7930 are also shown, prior to co-application with GABA EC₂₀. F) GABA EC₂₀ modulation curves for α1β2γ2L and α4β3δ in the presence of CGP7930 normalised to the respective maximum GABA currents; (α1β2γ2L: EC₅₀ = 1.7 ± 1.3 μM; IC₅₀ = 6.6 ± 2.3 μM; n = 5; α4β3δ: EC₅₀ = 1.0 ± 0.3 μM; IC₅₀ = 19.6 ± 13.1 μM n = 7). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Modulatory effects of CGP7930 on phasic and tonic inhibition in cultured hippocampal neurons A) Representative sIPSCs recorded from a hippocampal cultured neuron in control conditions and in the presence (bars) of CGP7930 (0.1–1 μM). Note sIPSC amplitudes are ‘clipped’ in some panels to show details of CGP7930 direct effects. Bicuculline (Bic, 50 μM) was applied at the end of each experiment to block all IPSCs. B, C) Frequency (B) and amplitude (C) bar graphs of sIPSCs in hippocampal neurons in the absence (control) or presence (0.1–1 μM) of CGP7930. Individual data points are shown including the mean ± SEM. D) Individual and mean (bold) peak-scaled sIPSC waveforms recorded from hippocampal neurons are shown for control and in the presence of CGP7930 (0.1–1 μM). E, F) Bar graphs for IPSC exponential decay times (E) and 10–90% rise times (F) for hippocampal neurons. G, H) CGP7930-induced changes to the baseline holding currents (G) and mean RMS current noise (H) for hippocampal neurons. Relative changes to the baseline holding current and RMS noise are shown on moving sequentially from one condition, ie, control, to another condition, ie, 0.1 μM CGP7930. Individual data points and mean ± SEM are shown. n = 7–9 cells, ns – not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; One-way ANOVA with Dunnett's multiple comparison test.