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. 2002 Dec 6;546(Pt 3):641–646. doi: 10.1113/jphysiol.2002.032300

Low doses of ethanol and a neuroactive steroid positively interact to modulate rat GABAA receptor function

Gustav Akk 1, Joe Henry Steinbach 1
PMCID: PMC2342575  PMID: 12562992

Abstract

Fast inhibitory responses in the central nervous system are mediated by the GABAA receptor. The activation and function of the GABAA receptor can be modulated by a variety of compounds including benzodiazepines, barbiturates and neuroactive steroids. Modulation of the GABAA receptor function by ethanol has been observed in some but not all studies. We have studied the effect of ethanol at concentrations corresponding to light intoxication on the function of the recombinant GABAA receptor containing α1β2γ2 subunits. The experiments were performed both in the absence and presence of low, subthreshold concentrations of a neuroactive steroid. The results demonstrate that, in the presence of the steroid, 0.05 % (9 mm) ethanol potentiates the GABAA receptor function by increasing the channel mean open duration. No effect was observed on the channel closed time durations. The data suggest that ethanol influences channel closing with no effect on the affinity of the receptor for GABA or the channel opening rate constant.

The GABAA receptor constitutes a major inhibitory force in the central nervous system. Normally activated following the binding of two molecules of GABA to its extracellular domain, the GABAA receptor also contains binding sites for and is modulated by a number of compounds including benzodiazepines, barbiturates, neuroactive steroids and, possibly, ethanol.

Ethanol is one of the most widely used psychoactive substances, exposure to which affects a variety of cellular processes including the function of ligand-gated ion channels (Harris, 1999). The link between changes in the GABA-activated currents and exposure to ethanol may involve a number of mechanistically distinct pathways. First, systemic administration of ethanol can lead to changes in the expression of GABAA receptor subunits. The nature of this effect depends on the type of exposure (acute vs. chronic) and is brain region-specific (Wu et al. 1995; Grobin et al. 2000).

A second possibility is that ethanol could affect the levels of endogenous modulators. The GABAA receptor can be modulated by a number of steroid compounds including ones produced in the brain (Baulieu, 1997). It is known that ethanol administration can lead to an increase in the levels of neurosteroids that may be sufficient to potentiate the GABAA receptor function and could underlie some of the behavioural effects following exposure to ethanol (Morrow et al. 2001).

The simplest link between ethanol and the GABAA receptor, direct interaction between the two, has been the most difficult to prove. While high concentrations (>100 mm, 0.6 %) of ethanol are known to potentiate α2β1 and α2β1γ2 subunit-containing receptors expressed in Xenopus oocytes (Mihic et al. 1997; Ueno et al. 1999), studies utilizing cell cultures have produced mixed results.

In terms of effects on electrophysiological responses, a potentiating action of ethanol on GABAA receptors has been observed in some studies (Celentano et al. 1988; Aguayo, 1990; Harris et al. 1995) but not in others (White et al. 1990; Sapp & Yeh, 1998; Zhai et al. 1998).

The discrepancy between results obtained from different expression systems even within the same laboratory has led to the hypothesis that the effect of ethanol on GABAA receptors requires the presence of additional factors or alterations. Potential candidates include receptor phosphorylation, interaction with another GABAA receptor modulator or even a mechanism involving microtubule formation, which may modify the receptor and turn it into an alcohol-sensitive isoform (Wafford & Whiting 1992; Whatley et al. 1996; Criswell et al. 1999).

Analysis of single-channel currents is a powerful tool that can be used to distinguish between seemingly similar effects on whole-cell currents and to establish the mechanism of action. However, to date, there has been only one report on the effect of ethanol on GABAA receptor single-channel currents. Tatebayashi et al. (1998) found that, in rat dorsal root ganglion neurons, addition of 100 mm (0.6 %) ethanol resulted in an increase in the frequency of openings and a slight (≈20 %) prolongation of the channel openings elicited by 1 µm GABA. Such changes suggest that the action of ethanol bears certain similarities with the action of barbiturates and steroid anaesthetics (increase in open time durations) as well as benzodiazepines (increase in frequency of openings). Studying the effect of a modulator in the presence of low concentrations of GABA (e.g. 1 µm) has its advantages and disadvantages. The advantage is an increased dynamic range. It is easier to see potentiation of submaximal currents. The disadvantage is that the number of active channels at any given time in the patch is unknown. So, an increase in the frequency of openings may result from an increased affinity of the receptor for GABA, an increased channel opening rate constant, an increase in the number of active receptors in the patch or a combination of these. Such uncertainty can be avoided by examining the effect of a modulator on single-channel clusters which represent activity from a single ion channel.

In the present manuscript, we have applied single-channel kinetic analysis in studies on ethanol-mediated potentiation of the recombinant GABAA receptor function. We conclude that ethanol does not affect the affinity of the receptor for GABA, or how fast the receptor channel opens. The potentiating effect of ethanol is mediated by a stabilization of one of the open states of the receptor.

Methods

Rat GABAA receptor α1, β2 and γ2L subunit cDNAs subcloned into pcDNAIII (Invitrogen Corp., San Diego, CA, USA) were transiently expressed in human embryonic kidney (HEK) 293 cells using a calcium phosphate precipitation-based transfection technique (Ausubel et al. 1992). The single-channel currents were recorded using a patch clamp technique in the cell-attached configuration (Hamill et al. 1981). The bath solution contained (mm): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose and 10 Hepes; pH 7.4. The pipette solution contained (mm): 120 NaCl, 5 KCl, 10 MgCl2, 0.1 CaCl2, 20 tetraethylammonium, 5 4-aminopyridine, 10 glucose, 10 Hepes; pH 7.4. GABA, ethanol and ACN (when needed) were added to the pipette solution and no correction for osmolarity was made. The concentration of ethanol is given as a percentage (v/v). For reference, 0.1 % ethanol equals 17 mm. The pipette potential was held at +60 to +80 mV. In most patches, the cell membrane potential was ≈-40 mV (data not shown), thus the total potential across the patch membrane was between −100 and −120 mV. The channel activity was recorded with an Axopatch 200B amplifier, low-pass filtered at 10 kHz, acquired with a Digidata 1200 series interface at 50 kHz using pCLAMP 7 software (Axon Instruments, Foster City, CA, USA) and stored on a PC hard drive.

The kinetic analysis was performed on single-channel clusters. A cluster is defined as a series of openings and closings of a single ion channel which starts as the channel returns from the long-lived desensitized state(s) and ends when the channel enters the long-lived desensitized state(s). The procedure for cluster isolation has been described elsewhere (Steinbach & Akk, 2001). Due to a low number of receptors in the patch and the relatively short lifetime of clusters compared to the dwells in the long-lived desensitized state(s), the activity in most patches consisted of episodes of intense activity of a single channel (a cluster) separated from other clusters by intervals lasting up to tens of seconds.

The isolated clusters were low-pass filtered at 3 kHz and idealized using the segmented-k-means algorithm (program SKM, Qin et al. 1996). The intracluster open and closed times were estimated using maximum likelihood methods which incorporate a correction for missed events (program MIL, Qin et al. 1997). The error limits in Tables 1 and 2 were estimated from the curvature of the likelihood surface at its maximum using the approximation of parabolic shape (Qin et al. 1996). The optimal fit for intracluster open times was obtained with a sum of three exponentials, and for intracluster closed times with a sum of three or four exponentials. For simpler presentation, the intracluster open and closed times are sometimes presented as weighted mean durations calculated from the durations and amplitudes of the fitted components. Open probability was defined as the fraction of time the receptor spends in the open states within a cluster.

Table 1.

Single-Channel properties in the presence of 50 μm GABA and various concentrations of ethaol

Ethanol OT1 OT2 OT3 CT1 CT2 CTβ
(%) (ms) (%) (ms) (%) (ms) (%) (ms) (%) (ms) (%) (ms) (%)
0 0.3 ± 0.1 26 ± 1 3.0 ± 0.3 58 ± 6 7.1 ± 0.9 16 ± 6 0.2 ± 0.1 64 ± 1 1.6 ± 0.1 16 ± 1 21.2 ± 0.5 21 ± 1
0.1 0.4 ± 0.1 31 ± 3 3.8 ± 0.6 57 ± 12 8.2 ± 2.8 12 ± 13 0.2 ± 0.1 54 ± 3 1.5 ± 0.4 15 ± 2 16.9 ± 2.6 30 ± 2
0.5 0.3 ± 0.1 18 ± 2 1.7 ± 0.1 57 ± 3 5.7 ± 0.3 24 ± 3 0.2 ± 0.1 59 ± 1 1.4 ± 0.1 27 ± 1 13.2 ± 0.5 14 ± 1
2 0.2 ± 0.1 21 ± 3 2.4 ± 0.3 59 ± 13 4.6 ± 1.4 20 ± 24 0.1 ± 0.1 54 ± 3 1.4 ± 0.3 24 ± 2 11.0 ± 1 23 ± 3
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The intracluster open and closed time histograms were fitted using program MIL. Error limits were calculated as described in Methods. In the absence of ethanol, the dataset contained 3 patches (total of 10 949 events). In the presence of ethanol, the datasets (one patch at each ethanol concentration) contained 2352 events (0.1%), 14 541 events (0.5%) or 2268 events (2% ethanol).

Table 2.

Single-Channel properties in the presence of 50 μm GABA and 10nm ACN various concentrations of ethanol

Ethanol OT1 OT2 OT3 CT1 CT2 CTβ
(%) (ms) (%) (ms) (%) (ms) (%) (ms) (%) (ms) (%) (ms) (%)
0 0.3 ± 0.1 35 ± 1 2.7 ± 0.1 55 ± 2 8.8 ± 0.8 10 ± 2 0.2 ± 0.1 68 ± 1 1.5 ± 0.1 14 ± 1 18.5 ± 0.7 18 ± 1
0.01 0.4 ± 0.1 40 ± 2 3.2 ± 0.5 33 ± 4 9.1 ± 0.7 26 ± 5 0.2 ± 0.1 55 ± 2 1.7 ± 0.2 30 ± 2 14.7 ± 0.9 15 ± 1
0.05 0.4 ± 0.1 35 ± 2 3.0 ± 0.3 37 ± 3 10.8 ± 2.0 28 ± 3 0.2 ± 0.1 62 ± 1 1.9 ± 0.1 29 ± 1 14.1 ± 1.0 9 ± 1
0.1 0.5 ± 0.1 29 ± 1 8.1 ± 0.3 63 ± 2 35.7 ± 3.8 8 ± 2 0.2 ± 0.1 65 ± 2 1.6 ± 0.2 19 ± 1 18.5 ± 0.7 16 ± 1
0.5 0.4 ± 0.1 31 ± 1 4.8 ± 0.2 52 ± 2 21.7 ± 0.9 17 ± 2 0.2 ± 0.1 59 ± 1 1.5 ± 0.1 24 ± 1 21.7 ± 0.5 17 ± 1
1 0.4 ± 0.1 20 ± 1 3.9 ± 0.2 61 ± 3 11.8 ± 0.7 20 ± 3 0.2 ± 0.1 66 ± 1 1.5 ± 0.1 17 ± 1 11.0 ± 0.5 17 ± 1
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The intracluster open and closed time histograms were fitted using program MIL. Error limits were calculated as described in Methods. The datasets contained 16 515 events (0%, 4 Patches), 5949 events (0.01 %, 3 patches), 9915 events (0.05 %, 3 patches), 7956 events (0.1 %, 3 patches), 16 103 events (0.5 %, 4 patches) or 21 140 events (1% ethanol, 3 patches)

Data in Fig. 2 and throughout the text are represented as means ± s.e.m. Analysis by Student's t test was used, and P values of < 0.05 were considered statistically significant.

Figure 2. The increase in cluster open probability in the presence of ethanol and ACN is caused by an increase in the open time durations.

Figure 2

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The intracluster mean open duration (A) and open probability (B) of clusters elicited by 50 µm GABA and various concentrations of ethanol (EtOH) in the absence (○) or presence (•) of 10 nm ACN. For comparison, data representing mean open times and open probability at 50 µm GABA (continuous line) are presented in both graphs. The dotted lines show error limits for the 50 µm GABA data. The mean open duration was calculated as a weighted mean from the open time values presented in Table 2. The cluster open probability (Po) was calculated from the open and closed time durations presented in Table 2 according to: Po = mean open duration/(mean open duration + mean closed duration). Each point represents the mean ±s.e.m. of data from 3 to 4 patches (*P < 0.05 GABA vs. GABA + ACN + ethanol).

Dr D. Covey (Washington University in St Louis) kindly provided (+)3α-hydroxy-5α-androstan-17β-carbonitrile (ACN). Ethanol was purchased from Equistar Chemicals (Tuscola, IL, USA). All other drugs and chemicals were purchased from Sigma Chemical Company (St Louis, MO, USA).

Results

Activation of the receptor by GABA

The characterization of single-channel activity of the α1β2γ2 subunit-containing receptor has been performed previously (Steinbach & Akk, 2001). In brief, at [GABA] above 20 µm, the activity takes place in single-channel clusters. In the present work, receptor function and its modulation were examined under two different conditions. While the majority of the experiments were performed in the presence of 50 µm GABA (≈EC40), specific aspects of the mechanism of ethanol action were investigated using data obtained in the presence of 1 mm (saturating concentration) GABA.

At 50 µm GABA, the best fit for the intracluster closed time histograms was obtained with the sum of three exponentials. The shortest component, CT1 (closed time 1), has a mean duration of 0.17 ms and constitutes 64 % of all intracluster closed events. CT2 has a mean duration of 1.6 ms and a relative frequency of 16 %. Finally, CTβ has a mean duration of 21.2 ms and makes up 21 % of the intracluster closed. The durations of CT1 and CT2 are not affected by agonist concentration. The duration of CTβ decreases as the agonist concentration is increased suggesting that CTβ represents dwells in mono- and unliganded states. The relative frequencies of the closed time components are not affected by the concentration of GABA. The intracluster open time histograms were fitted to a sum of three exponentials with mean durations of 0.28 ms (26 %, OT1 (open time 1)), 3.0 ms (58 %, OT2) and 7.1 ms (16 %, OT3). The durations and the relative fractions of the intracluster open time components are relatively unaffected by the concentration of GABA (Steinbach & Akk, 2001).

Activation of the receptor in the presence of GABA and ethanol

We first examined the ethanol concentration-response relationship in the presence of 50 µm GABA (in the absence of ACN). The addition of ethanol does not lead to changes in the durations of intracluster open or closed intervals. The weighted mean open duration in the absence of modulators is 3.2 ms. In the presence of ethanol, the mean open durations were 3.3, 2.4 and 2.4 ms for 0.1, 0.5 and 2 % ethanol, respectively. The mean intracluster closed times were 5.7, 5.4, 2.3 and 2.9 ms in the absence and presence of 0.1, 0.5 and 2 % ethanol, respectively. The breakdown into components is given in Table 1. Hence, the data demonstrate that the application of ethanol alone is not sufficient to affect the kinetic properties of the α1β2γ2 subunit-containing GABAA receptor expressed in HEK cells.

Activation of the receptor in the presence of GABA and ACN

ACN is a synthetic neuroactive steroid which potentiates responses of the GABAA receptor to GABA (Wittmer et al. 1996) and has a complex effect on the GABAA receptor kinetic behaviour. First, the open channel properties are affected. The presence of ACN leads to an increase in both the prevalence and duration of OT3, the longest-lived component in the open time histograms. Second, the presence of ACN influences the intracluster closed time distributions. ACN leads to a reduction in the relative weight of one of the components in the closed time histograms (CTβ). A sample cluster elicited by 50 µm GABA, in the presence of 1 µm ACN is shown in Fig. 1. The effect of the steroid is concentration-dependent with half-maximal effect on the open durations at approximately 100 nm ACN (G. Akk & J. H. Steinbach, unpublished observations).

Figure 1. Coapplication of ethanol and ACN increases cluster open probability.

Figure 1

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Single-channel clusters obtained in the presence of 50 µm GABA and in the absence or presence of 0.1 % ethanol, 10 nm ACN, 0.1 % ethanol + 10 nm ACN or 1 µm ACN. Channel openings are shown downward. The intracluster closed and open time histograms for each set of conditions are given next to the representative cluster. In the presence of GABA and 1 µm ACN, the mean closed durations were 0.12 (67 %), 1.1 (17 %), 21.2 (11 %) and 91 ms (5 %). The mean open durations were 0.42 (26 %), 3.4 (33 %) and 27.8 (42 %). For other sets of data, the parameters of the fit are given in Tables 1 and 2.

In the presence of 10 nm ACN no appreciable effect can be observed on single-channel currents elicited by 50 µm GABA. A sample single-channel cluster obtained in the presence of 50 µm GABA and 10 nm ACN is shown in Fig. 1. The results from fitting the intracluster open and closed time histograms are given in Table 2.

Activation of the receptor in the presence of GABA and ethanol + ACN

We then studied the modulation of the GABAA receptor function by ethanol in the presence of 10 nm ACN. The effect was examined over the range of 0.01–1 % ethanol. A sample cluster recorded in the presence of 0.1 % ethanol is shown in Fig. 1. The addition of ethanol in the presence but not in the absence of 10 nm ACN resulted in an increase in the cluster open probability (Fig. 2).

The increase in the open probability was mediated by an increase in the channel mean open duration. In the absence of ethanol, the mean open duration of the GABAA receptor activated by 50 µm GABA (in the presence of 10 nm ACN) is 3.2 ms. Upon the addition of ethanol, the mean open duration increases to 3.8, 4.6, 6.4, 6.8 or 5.2 ms in the presence of 0.01, 0.05, 0.1, 0.5 or 1 % ethanol in the pipette medium.

The underlying mechanism for this effect is an increase in the channel mean open duration caused by increases in the durations of OT3, and possibly OT2 (see Table 2). The effect on channel open times resembles what we observed previously in pentobarbital-mediated potentiation of the GABAA receptor function (Steinbach & Akk, 2001) and is similar to the effect seen in the presence of higher concentrations of ACN (Fig. 2). However, it is unlikely that ethanol acts solely to shift the ACN dose-response curve towards lower steroid concentrations. Not all effects that ACN at higher doses exerts on the GABAA receptor functional properties were replicated in the presence of ethanol and low concentrations of ACN. For example, the coapplication of ethanol and 10 nm ACN did not affect the frequency of CTβ as ACN did at higher doses (see above and Fig. 2). Table 2 gives the durations and the relative weights of the three closed time components in the absence and presence of 0.01–1 % ethanol.

We have previously associated the closed time component CTβ with dwells in the vacant and monoliganded states (Steinbach & Akk, 2001). The mean duration of this component under non-saturating conditions is sensitive to changes in receptor affinity and the channel opening rate constant. Thus, the absence of changes in the duration of CTβ suggests that the addition of ethanol does not lead to changes in the affinity of the receptor for GABA or the opening rate constant of the channel. The durations and relative weights of the remaining two closed time components were similarly unaffected by ethanol.

The lack of effect of ethanol on the channel opening rate constant was confirmed by experiments carried out in the presence of 1 mm GABA. Here, the high concentration of GABA in the medium fully saturates the agonist binding sites, and the inverse of the mean duration of CTβ is essentially equal to the channel opening rate constant. The mean value for CTβ in the presence of 0.1 % ethanol (and 10 nm ACN) was 0.67 ± 0.10 ms (total of 11278 events, two patches), while under control conditions the duration of this component was 0.71 ms (Steinbach & Akk, 2001). Hence, the application of ethanol does not affect the channel opening rate constant.

Discussion

The aim of these experiments was to establish conditions under which ethanol at concentrations corresponding to light intoxication affects the functional properties of the recombinant GABAA receptor, and to examine the mechanism of action responsible for such effect. Our data show that ethanol, in the presence of a neuroactive steroid, ACN, potentiates the GABAA receptor function. The effect is mediated by changes in the gating kinetics leading to an increase in the mean open duration and, hence, the open probability of the channel. No changes in the receptor affinity or channel opening were observed.

The physiological effect of the GABAA receptor activation in the central nervous system is determined mainly by the magnitude (peak response) and the shape (decay profile) of the synaptic currents in response to the release of GABA from the presynaptic nerve terminal. The peak of the response is regulated by a number of factors, the most important of which in the context of the present study is the open probability of the receptor channel. In the presence of saturating concentrations of GABA, the open probability of the α1β2γ2 subunit-containing GABAA receptor is ≈0.8 (Steinbach & Akk, 2001). It is apparent that not even a significant increase in the mean open duration can considerably affect the maximal open probability and, hence, the amplitude of peak current.

On the other hand, the amplitude and time constant of the fast decay components of the IPSC are strongly influenced by the durations of bursts of openings. An increase in the channel open duration would lead to an increase in the duration of a burst and, hence, affect the shape of the synaptic current. In addition, during the synaptic response a portion of receptors may be exposed to subsaturating concentrations of GABA contributing to the IPSC decay profile (Hill et al. 1998). An increase in the open probability of such receptors is likely to be induced by the application of ethanol. This could lead to an additional prolongation in the decay time course and increase the total charge transferred during the synaptic event.

The potentiating effect of ethanol was noted only in the presence of ACN. As far as we can tell, there is a requirement for a steroidal compound because coapplication of ethanol with other GABAA receptor potentiators, diazepam (1–100 nm) or pentobarbital (0.5–40 µm), did not lead to an additional potentiating effect in receptor function (data not shown). Benzodiazepines act on the receptor function by increasing the affinity of the receptor for GABA leading to an enhancement in the frequency of bursts of openings (Rogers et al. 1994; Lavoie & Twyman, 1996). In contrast, the mechanism of action of pentobarbital involves an increase in the durations of openings (Steinbach & Akk, 2001), more closely resembling the action of neuroactive steroids. In our study, neither diazepam nor pentobarbital could replace ACN as a cofactor in the ethanol-mediated potentiation of the GABAA receptor function. We believe that these findings support the notion of separate binding sites for potentiating steroids and pentobarbital or benzodiazepines. The data also suggest that there exists a potential interaction between the ethanol binding site and a site for potentiating steroids, but not for pentobarbital or diazepam. Whether the occupation of the ethanol site leads to a stronger binding of steroid in its site (a binding mechanism) or to an enhanced coupling between the steroid site and its effector machinery (a gating mechanism) is not clear. Our present knowledge about the mechanisms of steroid interactions with the GABAA receptor and the data presented in this study would not allow a distinction between these two mechanisms.

The mechanism by which ethanol acts on the GABAA receptor is not a simple leftward shift of the ACN potentiation curve. The application of steroids leads to a number of changes in the kinetic properties of the GABAA receptor. In addition to the increase in the mean open duration, the presence of potentiating steroids can also affect the channel closed times (Twyman & Macdonald, 1992; also see Fig. 2). We did not observe changes in the intracluster closed times in the presence of ethanol and 10 nm ACN suggesting that ethanol affects only one of the actions that ACN has on the receptor function. This may suggest the presence of two (or more) interaction sites for neurosteroids on the receptor. Ethanol, directly, or via occupation of a separate ethanol binding site, would then interact only with the steroid binding site for which the occupation is responsible for changes in the channel open times, but it would not interact with the steroid binding site which is coupled to changes in channel closed times.

Acknowledgments

We thank Jessie Zhang for tissue culture work and John Bracamontes for preparing the subunit constructs. J.H.S. is the Russell and Mary Shelden Professor of Anesthesiology. This work was supported by the Alcoholic Beverage Medical Research Foundation (G.A.) and NIH PO1 GM47969 (J.H.S.).

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