Activation and block of recombinant GABA(A) receptors by pentobarbitone: a single-channel study

Abstract

Recombinant GABA(A) receptors (alpha1beta2gamma2L) were transiently expressed in HEK 293 cells. We have investigated activation and block of these receptors by pentobarbitone (PB) using cell-attached single-channel patch clamp. Clusters of single-channel activity elicited by 500 microM PB were analysed to estimate rate constants for agonist binding and channel gating. The minimal model able to describe the kinetic data involved two sequential binding steps, followed by channel opening. The estimated channel opening rate constant is approximately 1500 s(-1), and the estimated equilibrium dissociation constants for the binding steps involved in activation are approximately 2 mM. Our results show a dose-dependent block of receptors at millimolar concentrations of PB that results in reduced open interval durations. The reduction in mean open time is linearly proportional to PB concentration, indicating that block can be produced by binding of a single PB molecule. Addition of millimolar concentrations of PB in the presence of GABA also produces a reduction of open channel lifetime in addition to a progressive increase in the closed interval durations within a cluster. The data suggest that the receptor contains two or more blocking sites while occupancy of only one of the sites is sufficient for channel block. Neither the blocking rate constant nor return rate from the blocked state(s) is affected by pH (ionization status of the PB molecule) demonstrating that both neutral and anionic forms of PB cause channel block.

Figure 1

Sample currents recorded from cell-attached patches on HEK cells transiently transfected with α1β2γ2 subunits, and open interval duration histograms. The average open interval duration in the presence of 200 μM PB is 1.9 ms (A). When PB concentration is increased to 10 mM, the open interval duration is reduced to 0.16 ms (B). Outward chloride flux is shown downward. Each histogram is from one patch, 735 events in (A), 407 events in (B). For determination of open time, currents were filtered at 2 kHz (A) or 4 kHz (B).

Figure 2

The mean open interval duration of α1β2γ2 receptors is reduced at high PB concentrations (A). Each data point represents data from one patch. The channel open durations were obtained after filtering the data at 2–4 kHz. The data recorded in the presence of PB concentrations over 1 mM were filtered at 4 kHz. The curve was fitted using Equation 1. The results of the analysis are shown in Table 1. The mean open interval duration is not affected by voltage (B). Data for each PB concentration come from a separate patch. Data for different V_pip at each concentration come from the same patch. The regression coefficient demonstrated voltage-sensitivity of >1000 mV per e-fold change.

Figure 3

Sample clusters of α1β2γ2 receptors elicited by 500 μM PB. The clusters were defined as series of openings separated by closed intervals shorter than 500 ms (see Methods). Outward chloride flux is shown downward.

Figure 4

Kinetic analysis of single-channel data obtained at 500 μM PB. The analysis was performed on data from three patches containing in total 3373 openings. The currents were analysed using program MIL and different kinetic schemes. In each model: C, closed receptor, O, open receptor, B, blocked receptor; rate constants for left to right transitions are shown above the transition symbol. Rate constants that were constrained are shown in parentheses (C–E). All rates are given in units of s⁻¹. The goodness of the fit is shown as a log-likelihood next to each model (LL). The closed and open time histograms also contain a solid line that shows the interval distribution predicted by the respective model and rate constants. Data was filtered at 2.5 kHz, dead time was 0.07 ms.

Figure 5

The closed interval duration distribution in the presence of 100 μM PB. The mean duration of the briefer component is 1/(β+k₋₂) and the relative area is β/(β+k₋₂). For the slower component, the relative area is 1-(β/(β-k₋₂)), and the mean duration is the best fit. The solid line is calculated using the gating rate constants shown in Figure 4D. Histogram corresponds to one patch with 424 events.

Figure 6

Sample clusters and open and closed interval duration histograms of α1β2γ2 receptors activated by GABA and varying concentrations of PB. The currents were recorded at pH 7.4 (A, B) or 9.1 (C), and activated by 50 μM GABA and 200 μM PB (A) or 50 μM GABA and 2 mM PB (B, C). The time constants for closed and open dwell intervals are shown in histograms. The results show that increasing concentrations of PB result in channel block and cause shorter openings and longer closed interval durations (compare A to B). Elevated pH at the same concentrations of GABA and PB results in shorter openings but has little effect on the slowest component of the closed interval duration histogram (B and C). The histograms correspond to the patch from which a representative cluster is shown.

Figure 7

The slowest components of the open and closed interval duration histograms of clusters recorded in the presence of 50 μM GABA and varying PB concentrations at pH 7.4. (A) The mean open duration is reduced at increasing PB concentrations. The curve was fitted using Equation 1. The results are shown in Table 1. (B) The mean duration of the longest component of closed intervals within a cluster is increased at high PB concentrations. We assume that this component is associated with the blocked state (see Results). Each point corresponds to data from one patch.