Activation of GABA(A) receptors containing the alpha4 subunit by GABA and pentobarbital

Abstract

The activation properties of GABA(A) receptors containing alpha4beta2gamma2 and alpha4beta2delta subunits were examined in the presence of GABA or pentobarbital. The receptors were expressed transiently in HEK 293 cells, and the electrophysiological experiments were carried out using cell-attached single-channel patch clamp or whole-cell macroscopic recordings. The data show that GABA is a stronger activator of alpha4beta2gamma2 receptors than alpha4beta2delta receptors. Single-channel clusters were recorded from alpha4beta2gamma2 receptors in the presence of 10-5000 microm GABA. The maximal intracluster open probability was 0.35, with a half-maximal response elicited by 32 microm GABA. Simultaneous kinetic analysis of single-channel currents obtained at various GABA concentrations yields a channel opening rate constant of 250 s(-1), and a K(D) of 20 microm. In contrast, only isolated openings were observed in the presence of GABA for the alpha4beta2delta receptor. Pentobarbital was a strong activator of both alpha4beta2gamma2 and alpha4beta2delta receptors. The maximal cluster open probability, recorded in the presence of 100 microm pentobarbital, was 0.7. At higher pentobarbital concentrations, the cluster open probability was reduced, probably due to channel block. The results from single-channel experiments were confirmed by macroscopic recordings from HEK cells in the presence of GABA or pentobarbital.

Figure 1. Single-channel clusters from the α4β2γ2 GABA_A receptor

The clusters were elicited by 20 μm (upper traces) or 1000 μm (lower traces) GABA. High resolution fragments are shown below the clusters. Channel openings are shown downward. GABA at 1000 μm elicits a maximal response from this receptor. The intracluster open and closed time distributions are given in Fig. 2 and Table 1, respectively.

Figure 2. The properties of currents from the α4β2γ2 GABA_A receptor elicited by GABA

A, mean open durations of the three open time components at 10–5000 μm GABA. The durations of open times are not affected by GABA concentration. The averaged open times are: 0.35 ms for OT₁, 1.3 ms for OT₂ and 6.3 ms for OT₃. B, relative weights of the three open time components. The relative weights were not affected by the concentration of GABA. The averaged weights are: 34% for OT₁, 63% for OT₂ and 3% for OT₃. The inset key applies to A and B. C, the effective opening rate at various GABA concentrations. The effective opening rate is calculated as an inverse of CT_β (see Table 1). The curve was fitted using a Hill equation. The fitting parameters are : β′_max = 399 s⁻¹, EC₅₀ = 204 μm, n_H = 1.3. D, the intracluster open probability at various GABA concentrations. The curve was fitted using a Hill equation. The fitting parameters are : P_o,max = 0.35, EC₅₀ = 32 μm, n_H = 0.9. In all panels, each data point corresponds to data from one patch. The numbers of events and clusters used in the analysis are given in Table 1.

Figure 3. The intracluster closed time histograms from α4β2γ2 subunit receptors activated by 20, 100 and 5000 μm GABA

The continuous lines were calculated according to Model 1 and the rate constants given in the text. The numbers of events and clusters used in the analysis are given in the text.

Figure 4. Single-channel clusters from the α4β2γ2 GABA_A receptor

The clusters were elicited by 200 μm (A) or 1000 μm (B) pentobarbital. Higher resolution fragments are shown below the clusters. Channel openings are shown downward. PB at 200 μm elicits a maximal response from this receptor; at higher concentrations PB inhibits the receptor function. C, the intracluster open probability at various PB concentrations. D, the calculated mean open interval duration at various PB concentrations. The curve was fitted using an equation: open time = 1/(closing rate +k_+B[PB]). The fitting parameters are: closing rate = 134 ± 24 s⁻¹, k_+B = 0.59 ± 0.27 μm⁻¹ s⁻¹. Only data obtained at 30–2000 μm PB were used in the analysis. E, the calculated mean closed interval durations at various PB concentrations.

Figure 5. Macroscopic currents from HEK cells expressing α4β2γ2 subunit receptors

A, the currents were elicited by 100 μm PB, 1 mm PB or 1 mm GABA. Rebound current in the end of the 1 mm PB application is caused by removal of PB-mediated block. B, normalized to peak current levels in the presence of 100 μm PB, the relative peak current for 1 mm PB was 0.54 ± 0.23 (n = 5), for 1 mm PB the rebound current was 0.83 ± 0.19 and the peak current for 1 mm GABA was 0.85 ± 0.21 (n = 6). C, residual current measured 5 s after the peak response is plotted. The residual current level for 100 μm PB was 0.61 ± 0.11, for 1 mm PB it was 0.71 ± 0.14 and for 1 mm GABA it was 0.73 ± 0.05. D, comparison of single-channel cluster open probability and peak macroscopic responses in the presence of pentobarbital. The currents were normalized to the levels at 100 μm PB.

Figure 6. Single-channel currents from the α4β2δ GABA_A receptor

The currents were elicited by 1000 μm GABA (A), 40 μm pentobarbital (B) or 1000 μm GABA + 40 μm PB (C). Channel openings are shown downward. The patch closed and open interval duration histograms are given next to the traces. The results of multi exponential fits are [duration (relative frequency)]: A: closed, 0.38 ms (26%), 11.1 ms (32%) and 37 ms (42%); open, 0.12 ms (22%) and 3.2 ms (78%); B: closed, 0.15 ms (24%), 2.5 ms (58%), 27 ms (12%) and 167 ms (7%); open, 0.1 ms (9%), 1.4 ms (13%) and 11 ms (77%); C: closed, 0.22 ms (39%), 2.3 ms (37%), 23 ms (14%) and 111 ms (10%); open, 0.15 ms (23%), 2 ms (30%) and 8.2 ms (48%). Due to rounding, the sum of weights may not equal 100%.

Figure 7. Macroscopic currents from HEK cells expressing α4β2δ subunit receptors

A, the currents were elicited by 100 μm PB, 1 mm PB or 1 mm GABA. Rebound current in the end of the 1 mm PB application is caused by removal of PB-mediated block. B, normalized to peak current levels in the presence of 100 μm PB, the peak current for 1 mm PB was 0.5 ± 0.19 (n = 4), for 1 mm PB the rebound current was 0.79 ± 0.13 and the peak current for 1 mm GABA was 0.35 ± 0.03 (n = 4). C, residual current measured 5 s after the peak response is plotted, normalized to the peak response in that trace. The residual current level for 100 μm PB was 0.12 ± 0.06, for 1 mm PB it was 0.43 ± 0.11 and for 1 mm GABA it was 0.54 ± 0.06.