Modulation of GABAA receptor channel gating by pentobarbital

We have studied the kinetic properties of channel gating of recombinant α1β2γ2L GABAA receptors transiently expressed in human embryonic kidney 293 cells, using the cell-attached, single-channel patch-clamp technique. The receptors were activated by GABA, β-alanine or piperidine-4-sulfonic acid (P4S), and the effects of pentobarbital (PB) on single-channel activity were examined. At relatively high concentrations of agonist, single-channel activity occurred in well-defined clusters. In global terms, PB increased the mean open time for events in clusters, without changing the mean closed time. The addition of PB shifted the curve relating the probability of being open in a cluster (Po) to lower agonist concentrations, and that shift could be accounted for by the changes in mean open time. The intracluster closed-time histograms contained four components. The durations and relative frequencies of these closed-dwell components were not affected by the presence of 40 μm PB, at any agonist concentration. The duration of one component was dependent upon the concentration of agonist used to activate the receptor. Accordingly, the inverse of the mean duration of this component will be called the effective opening rate. The channel-opening rate constant (β) was determined from the value of the effective opening rate at a saturating agonist concentration. β was about 1900 s−1 when the receptors were activated by GABA, 1500 s−1 when activated by β-alanine, and too low to be determined when P4S was administered. In the presence of 40 μm PB, β was about 1500 s−1 when the receptors were activated by GABA, 1400 s−1 when activated by β-alanine, and 50 s−1 when activated by P4S. Hence, the potentiating effect of PB is not mediated by a change in β. The concentration of agonist producing a half-maximal effective opening rate also remained unaffected in the presence of PB, indicating that receptor affinity for agonists is not influenced by PB. The distributions of the intracluster open durations elicited by GABA could be described by the sum of three exponentials, with mean durations of about 0.4, 2.4 and 6.3 ms. The duration and relative frequency of the components did not change with GABA concentration (20 μm to 1 mm). In the presence of 40 μm PB, however, the mean duration of the longest of the open times increased (mean durations of about 0.4, 2.0 and 13 ms). The intracluster open durations elicited by β-alanine could be described by the sum of two exponential components (1.1 and 3.5 ms). However, in the presence of 40 μm PB the open-time distribution contained three exponential components (0.2, 2 and 10 ms). Finally, openings elicited by P4S exhibited two components (0.3 and 0.9 ms). In the presence of 40 μm PB, three components could be distinguished (0.5, 2.5 and 13 ms). These observations indicate that the potentiating effect of PB on GABA type A (GABAA) receptors reflects effects on the open state(s) of the receptors. In the case of receptors activated by GABA, the observations are consistent with the idea that the action is the result of PB stabilizing one of the open states. The actions on receptors activated by P4S or β-alanine are also broadly consistent with this idea. However, the changes in open-time distributions caused by PB appear to be more complex. Possible explanations of the effects of PB on gating by different agonists are considered. The GABA type A (GABAA) receptor is a ligand-gated ion channel whose activation leads to cellular inhibition in the central nervous system. This receptor is normally activated by GABA and can be modulated by a number of compounds including benzodiazepines, barbiturates and steroidal anaesthetics (reviewed in, for example, Olsen & Tobin, 1990). The sedative and hypnotic effects of barbiturates are thought to result, at least in part, from actions on GABAA receptors. However, barbiturates have several distinct effects that have complicated efforts to reach a mechanistic understanding of their actions. Depending upon the dose, pentobarbital (PB) can have three effects on GABAA receptor function. At low micromolar concentrations, PB potentiates the activation of the receptor by GABA (Akaike et al. 1990). At high micromolar concentrations, PB directly gates the GABAA receptor (Akaike et al. 1985, 1990; Rho et al. 1996; Akk & Steinbach, 2000; Serafini et al. 2000), and at millimolar concentrations PB blocks the receptors (Akaike et al. 1985, 1990; Rho et al. 1996; Akk & Steinbach, 2000; Serafini et al. 2000). The effects can be separated functionally, and appear to be mediated via different interaction sites (for example, Dalziel et al. 1999; Serafini et al. 2000). In spite of the plethora of information available on the potentiating effect of PB from whole-cell experiments, relatively few studies have examined the actions of PB on single-channel currents from GABAA receptors. Macdonald et al. (1989) showed that in mouse spinal cord neurones, co-application of 50 μm PB with 2 μm GABA leads to an increase in the mean open duration of the channel. In both the presence and absence of PB, the open-time distribution was described by three exponential components; the increase in mean open time resulted from an increase in the relative contribution of the long-lived openings, while the time constants were unaffected by PB. In contrast, none of the five closed-time components were altered in the presence of PB. Porter et al. (1992) found that in Chinese hamster ovary cells stably expressing bovine α1 and β1 subunits, 50 μm PB mainly increased the proportion of long-duration openings elicited by 5 μm GABA. However, a small increase in the duration of openings was also observed. Of the five closed-time components, PB prolonged the durations of the four longer-duration components. In both of these studies, it was concluded that the major action of PB was to increase the relative opening rate for the long-duration open component, with minimal effects on channel closing rates. In the present work, we have used the single-channel patch-clamp technique to investigate the gating mechanisms of recombinant GABAA receptors (α1β2γ2L) transiently expressed in human embryonic kidney (HEK) 293 cells. We used three agonists, GABA, β-alanine and piperidine-4-sulfonic acid (P4S) to activate the GABAA receptor, to allow us to study more accurately the kinetic steps in activation that are affected by pentobarbital. P4S is a high-affinity, low-efficacy agonist for the GABAA receptor (Ebert et al. 1994). The maximal response to P4S is significantly lower than that obtained in the presence of GABA, suggesting that channel gating is impaired in the presence of P4S. A reduction in the maximal response can be achieved by a reduction in the channel-opening rate constant and/or an increase in the channel-closing rate constant. β-Alanine, on the other hand, is a low-affinity, high-efficacy agonist for the GABAA receptor (Jones et al. 1998). The maximal response to β-alanine is comparable to the maximal response to GABA, but the concentration dependence is shifted to higher concentrations of agonist. In order to overcome some of the obstacles to interpreting single-channel data, we examined receptor function under conditions where the activity takes place in clusters. Clusters can be elicited by high, desensitizing concentrations of the agonist, and each cluster originates from the activity of a single receptor (Sakmann et al. 1980). Thus, the uncertainty in the number of active channels is eliminated. Single channel clusters are groups of channel openings and closings reflecting rapid kinetic events such as agonist association and dissociation, channel opening and closing, and dwells in various short-lived desensitized and blocked states. The cluster is terminated by entry of the receptor into a long-lived desensitized state.