Distinct activities of GABA agonists at synaptic- and extrasynaptic-type GABAA receptors

Abstract

The activation characteristics of synaptic and extrasynaptic GABA(A) receptors are important for shaping the profile of phasic and tonic inhibition in the central nervous system, which will critically impact on the activity of neuronal networks. Here, we study in isolation the activity of three agonists, GABA, muscimol and 4,5,6,7-tetrahydoisoxazolo[5,4-c]pyridin-3(2H)-one (THIP), to further understand the activation profiles of alpha 1 beta 3 gamma 2, alpha 4 beta 3 gamma 2 and alpha 4 beta 3 delta receptors that typify synaptic- and extrasynaptic-type receptors expressed in the hippocampus and thalamus. The agonists display an order of potency that is invariant between the three receptors, which is reliant mostly on the agonist dissociation constant. At delta subunit-containing extrasynaptic-type GABA(A) receptors, both THIP and muscimol additionally exhibited, to different degrees, superagonist behaviour. By comparing whole-cell and single channel currents induced by the agonists, we provide a molecular explanation for their different activation profiles. For THIP at high concentrations, the unusual superagonist behaviour on alpha 4 beta 3 delta receptors is a consequence of its ability to increase the duration of longer channel openings and their frequency, resulting in longer burst durations. By contrast, for muscimol, moderate superagonist behaviour was caused by reduced desensitisation of the extrasynaptic-type receptors. The ability to specifically increase the efficacy of receptor activation, by selected exogenous agonists over that obtained with the natural transmitter, may prove to be of therapeutic benefit under circumstances when synaptic inhibition is compromised or dysfunctional.

Figure 1. Activation of recombinant synaptic and extrasynaptic GABA_A receptors by three agonists

A, structures of the selective GABA_A receptor agonists: GABA (square), muscimol (triangle) and THIP (circle). The agonists are labelled G (GABA), M (muscimol) and T (THIP) in this and subsequent figures. B, agonist concentration–response relationships for GABA, muscimol and THIP on recombinant α1β3γ2 (a), α4β3γ2 (b) and α4β3δ (c) receptors expressed in L-tk cells (n= 5–7; mean ±s.e.m.). C, whole cell currents induced by saturating concentrations of GABA, muscimol and THIP on recombinant α1β3γ2 (a), α4β3γ2 (b) and α4β3δ (c).

Figure 2. Single channels have three identical conductance levels for synaptic- and extrasynaptic-type GABA_A receptors A–C, single channel currents induced by GABA

(A), muscimol (B) and THIP (C) on recombinant α1β3γ2 (a), α4β3γ2 (b) and α4β3δ (c) receptors. D, typical current amplitude histogram (α1β3γ2, 3 μm GABA) showing curve fits with three Gaussians each (dotted lines) and their sum (continuous line). E, three conductance levels (high, 26–31 pS (squares); intermediate, 17–22 pS (circles); low, 11–14 pS (triangles)) were resolved for all three agonists at three concentrations (low EC_5-20 (white); intermediate EC_40-60 (grey); and high EC_80-100 (black)) (n= 5–7; mean ±s.e.m.). See Table 1 for agonist concentrations. F, relative areas of the single channel conductances for all three agonists at each receptor isoform, at three concentrations.

Figure 3. Low external pH affects the single channel conductance

A and B, single channel currents induced by high concentrations of THIP at recombinant synaptic-type α1β3γ2 receptors exposed to pH 4 or 7.4 external solution. C, displacement of the single channel current amplitude histogram by pH 4 (left) from pH 7.4 (right). D and E, single channel main state conductances for synaptic-type α1β3γ2 and extrasynaptic-type α4β3δ GABA_A receptors for both GABA (10 mm) and THIP (10 mm) at pH 4.0 and 7.4. Sample currents are shown in the insets.

Figure 4. Channel open probability is affected by the receptor isoform and agonist concentration

Open probability was measured by analysing clusters of channel activity induced by the three concentrations (see Table 1) of GABA (G), muscimol (M) and THIP (T). Data are means ±s.e.m. n= 5–7.

Figure 5. Open times vary with the receptor isoform and agonist concentration

A, mean open times for GABA (G), muscimol (M) and THIP (T) at three concentrations for α1β3γ2, α4β3γ2 or α4β3δ receptors. B, open time distributions for GABA (3 μm; a), muscimol (3 μm; b) and THIP (300 μm; c) on α4β3δ receptors, each showing two exponentials (dotted lines) and their sum (continuous line). C, open time constants (a; short τ_O1, squares; and long τ_O2, circles) and their relative areas (b) determined from exponential fits to the open time constant distributions. D, bar graph of the long open times (τ_O2) for THIP compared with GABA and muscimol on α4β3δ receptors (*P= 0.0136; ANOVA, n= 6–7).

Figure 6. Intraburst closed time relationships for synaptic- and extrasynaptic-type GABA_A receptors

A, closed time constants determined from distributions of short closed times (τ_C1 (squares) and τ_C2 (circles)) representing closed times within bursts of channel activity induced by GABA (G), muscimol (M) and THIP (T) at low, mid and high concentrations. B, relative areas for the intraburst closed times, τ_C1 and τ_C2. n= 5–7 experiments.

Figure 7. Channel burst analysis for synaptic- and extrasynaptic-type GABA_A receptors

A, sample single channel recordings of bursts and single open events induced by GABA (30 μm) on α1β3γ2 (a), GABA (3 μm) on α4β3δ (b), and THIP (300 μm) on α4β3δ (c). B, burst duration distributions corresponding to the receptor isoforms shown in (A). Individual (dotted lines) and summed exponential fits (continuous line) are shown. C, mean burst durations for GABA (G), muscimol (M) and THIP (T) for three concentrations at each receptor isoform. D, bar graph of the mean burst durations for α4β3δ GABA_A receptors with the highest concentrations of GABA, muscimol and THIP (*P= 0.0005, ANOVA, n= 6–7). E, mean number of openings per burst for three concentrations of GABA, muscimol and THIP on each receptor isoform. n= 5–7.

Figure 8. Binding and conformation constants for GABA, muscimol and THIP for synaptic- and extrasynaptic-type receptors

These constants were determined from the single channel rate constants determined from fitting the linear/branched model to the single channel data for all three receptors: α1β3γ2, α4β3γ2 and α4β3δ receptors. A, GABA_A receptor kinetic model. B, dissociation constants for monoliganded (K₁; a) and bilganded (K₂; b) receptor states. C, efficacies for the monoliganded (E₁; a) and bilganded (E₂;b) gating transitions. D, desensitisation equilibrium constants (D) calculated for GABA, muscimol and THIP.

Figure 9. Theoretical whole-cell agonist concentration–response curves

The curves were generated from the state function for the linear/branched receptor model. The transition constants in the model were determined from the single channel rate constants following the predictive fitting of the single channel data with the linear/branched receptor model using QuB. The data points represent the peaks of simulated currents that were subsequently generated in ChanneLab using the linear/branched model and the best fit single channel rate constants for each agonist. Simulated concentration–response curves for GABA (squares), muscimol (triangles) and THIP (circles) are shown for α1β3γ2 (A), α4β3γ2 (B) and α4β3δ (C) GABA_A receptors.

Figure 10. Extrasynaptic-type α4β3δ receptors are desensitised by low GABA concentrations

Simulated agonist-activated currents are presented for the linear/branched receptor model. The transition constants used in the model to generate the currents were determined after predictive fitting of the single channel data for GABA and muscimol. Two simulations were used; one in which access to the desensitised state (D) was allowed (+D state) and one in which desensitisation was absent (–D state). Notably, the receptor is substantially desensitised by low concentrations of GABA (A) or muscimol (B) that do not cause overt fading of the current response.