Introduction

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The -aminobutyric acid type A (GABA_A) receptor mediates the majority of inhibitory synaptic responses in the mammalian brain (Sivilotti and Nistri, 1991) and is the site of action for many drugs (Burt and Kamatchi, 1991; Macdonald and Olsen, 1994). Ethanol has been reported to potentiate GABA-induced responses in several preparations (Aguayo 1990; Nishio and Narahashi, 1990; Nakahiro et al., 1991; Reynolds et al., 1992; Sigel et al., 1993; Marszalec et al., 1994; Weiner et al., 1994; Harris et al., 1995). In most of these studies, the focus was on the alcohol-induced potentiation of GABA-induced peak current. However, alcohol-mediated changes in other properties of GABA-induced current have not received much attention.

In rat dorsal root ganglion (DRG) neurons, alcohols have been shown to modify GABA currents in several ways (Nakahiro et al., 1991; Arakawa et al., 1992). The effects include: 1) potentiation of GABA-induced peak currents with a brief alcohol coapplication; 2) inhibition of peak currents with extended alcohol preperfusions; 3) apparent acceleration of desensitization of GABA-induced currents; 4) inhibition of the steady-state currents that follow desensitization; and 5) direct generation of current by alcohols. The interpretation of alcohol-GABA channel interactions is difficult because of a variety of GABA_A receptor subtypes that exist in the native neurons, each composed of different combinations of receptor subtypes (Burt and Kamatchi, 1991; Macdonald and Olsen, 1994). Thus, it remains to be seen whether alcohols exert these multiple actions on a single type of GABA_A receptor.

To explore whether alcohols exert multiple effects on a single type of GABA_A receptor, we have initiated our study by using human embryonic kidney (HEK) cells transfected with cDNAs encoding for the 1, 2, and 2S rat GABA_A receptor subunits. The 1, 2, and 2S subunit combination has been reported as the most prevalent form of the GABA_A receptor in the mammalian brain (Burt and Kamatchi, 1991). In our previous studies with this combination of GABA_A receptor subunits, we found that alcohols potentiate peak currents without changing the maximal GABA response. In other words, alcohols shift the GABA dose-response curve toward lower concentrations (Kurata et al., 1993; Marszalec et al., 1994).

All experiments reported here made use of the eight-carbon alcohol n-octanol, because ethanol has a weak effect on GABA-induced currents in 122S-transfected HEK cells (Marszalec et al., 1994). However, the previous study with DRG neurons indicates that n-alcohols having less than 10 carbons (including ethanol) produce qualitatively similar effects on GABA-induced currents, but differ in their potency (which correlates with lipid solubility) (Nakahiro et al., 1991). Furthermore, recent single-channel patch clamp experiments have clearly shown that ethanol and n-octanol have identical effects to modulate GABA receptor channels in DRG neurons (Tatebayashi et al., 1998).

In the present study, alcohol actions on the 122S receptor were examined during a brief period of coapplication and after prolonged pretreatment with either GABA or n-octanol. n-Octanol exerted multiple actions on the receptor depending on the experimental condition. We have attempted to explain various effects of n-octanol on the 122S receptor using two models. One is based on the classical allosteric model in which n-octanol binds to a site different from the GABA binding site to modulate the GABA-induced response. The other is based on a partial agonist model in which n-octanol acts as a weak partial agonist to bind to the same site as GABA does. The binding to the agonist site manifests multiple actions as has been demonstrated with nicotinic receptors (Cachelin and Rust, 1994; Steinbach and Chen, 1995; Fletcher and Steinbach, 1996). The latter model is unique in that many features of n-octanol action can be satisfactorily accounted for, whereas the allosteric model can only explain limited observations.

	Materials and Methods

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Preparation. HEK293 cells were transfected with 1, 2, and 2S subunit cDNAs derived from rat brain GABA_A receptors. The techniques for the stable expression of these GABA_A receptors were detailed in a previous report (Hamilton et al., 1993). All cells were grown in modified Eagle's minimum essential medium supplemented with 10% fetal calf serum in the presence of humidified air containing 5% CO₂. These cells exhibited GABA dose-dependent responses similar to those reported by other groups for this subunit combination, and were sensitive to benzodiazepines requiring the presence of 1, 2, and 2S subunits (Kurata et al., 1993).

Electrical Recording. GABA-induced currents were recorded with the whole-cell configuration of the patch clamp technique. Patch electrodes were made from 1.0-mm (o.d.) borosilicate glass capillary tubes using a vertical pipette puller (Narishige PP-83, Tokyo, Japan). Electrode resistance ranged from 2 to 5 M when filled with internal solution. All currents were recorded with an Axopatch-1C amplifier (Axon Instrument Co., Foster City, CA) and were stored on an PDP 11/73 computer (Digital Equipment, Pittsburgh, PA).

Solutions. Cells were dialyzed with an internal (electrode) solution of the following composition: 140 mM KCl, 1 mM MgCl₂, 5 mM HEPES, 5 mM EGTA, and 5 mM Mg-ATP. The normal external solution contained: 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 5 mM HEPES, and 10 mM D-glucose. The pH of both solutions was adjusted to 7.3 with NaOH.

Drug Application. External solutions were perfused into the recording chamber at a normal rate of 1 to 2 ml/min and were increased to 10 ml/min when solutions were changed. GABA-containing solutions with or without n-octanol were directly applied to the cell by a variant of the U-tube method (Marszalec et al., 1994). This allowed localized solution exchanges within a range of 20 to 30 ms. For prolonged application of GABA or n-octanol, bath application was used.

Data Analysis. The dose-response relationship for agonistic action was analyzed by the following logistic equation:

(1)

where y represents the percentage of maximal response induced by GABA or n-octanol at a concentration of C, with 100% being the maximal obtainable response. EC₅₀ and n_H represent the half-maximal effective concentration of GABA and the Hill coefficient, respectively.

(2)

(3)

(4)

(5)

(6)

	Results

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Characteristics of GABA Responses under Control Conditions. The dose-response relationship for GABA to induce inward currents was fitted by two methods as described under Materials and Methods. Figure 1A depicts the fit of data with a logistic eq. 1 yielding an EC₅₀ of 13.3 µM and a Hill coefficient (n_H) of 1.40. Figure 1B illustrates the fit of data with eq. 4. The open probability was set to 80% as a scaling factor for the original dose-response data (Weiss and Magleby, 1989; Newland et al., 1991). A least-squares fit to the data by the velocity equation based on the partial agonist model yielded the following parameters: a cooperating factor, a, of 0.8, an intrinsic dissociation constant, K_g, of 22 µM and an equilibrium channel open ratio, X_G, was assumed to be 4 based on the assumed maximal open probability of doubly liganded GABA_A receptor. These parameters will be used later for fitting the data obtained in the presence of GABA and n-octanol.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   GABA dose-response relationship observed in HEK293 cells expressing 122S subunit combination. Two models were used to fit data: an allosteric model (A) and a partial agonist model (B). A, in the allosteric model, a logistic eq. 1 was used to fit data giving an EC50 of 13.3 µM and a Hill coefficient (nH) of 1.40. Current amplitude was normalized to its maximum value. B, Percentage of response in A was divided by 125 to convert to open probability, assuming that maximal open probability was 0.8. Data was fitted by eq. 4 yielding the following parameters: cooperative factor, a, of 0.8 ± 0.4, intrinsic dissociation constant, KG, of 22.2 ± 8.0 µM, and an equilibrium opening ratio, XG, of 4.

n-Octanol Generates Currents by Itself. In the absence of GABA, n-octanol was capable of generating inward currents in a dose-dependent manner when the membrane potential was held at 60 mV (Fig. 2). The n-octanol-induced currents were small compared with GABA-induced currents. On the average, the current generated by 1000 µM n-octanol was about 35% of that generated by 3 µM GABA and 3.3 ± 0.7% (n = 6) of the maximal current generated by 300 µM GABA.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   n-Octanol generates currents in HEK293 cells expressing the 122S GABAA receptors. A-D, Currents induced by 100 to 1000 µM n-octanol applied for 5 s. E, Current induced by 3 µM GABA. Current induced by 1000 µM n-octanol was about 50% of that produced by 3 µM GABA.

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Dose-response relationship for currents generated by n-octanol. A, amplitude of octanol-induced currents is given as percentage of control current evoked by 300 µM GABA. A, logistic eq. 1 was used to fit data yielding an EC50 of 1127 µM and a Hill coefficient (nH) of 2.0. B, data were fitted with a partial agonist model (eq. 5) yielding the following parameters: cooperative factor, b, of 0.33, intrinsic dissociation constant, KO, of 2821 µM, and an equilibrium opening ratio, XO, of 0.15.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effects of bicuculline (Bic) on GABA- and n-octanol-induced currents. Bicuculline (10 µM) suppressed GABA-induced currents to 1.37 ± 0.45% (n = 6) of control when it was coapplied with 3 µM GABA (A and B) and inhibition was reversible after washing with bicuculline-free solution (C). Same concentration of bicuculline reduced current only to 80.0 ± 5.0% (n = 4) of control when coapplied with 300 µM GABA (D and E). Block was enhanced by bath application and coapplication of 10 µM bicuculline (F). Coapplication of 10 µM bicuculline with n-octanol reversibly suppressed n-octanol-induced current to 3.44 ± 2.0% (n = 5) of control (G, H, and I). Time interval between each application was 5 min.

n-Octanol Potentiates GABA-Induced Peak Current. When n-octanol was coapplied with GABA, the GABA-induced current was increased. The potentiating action of n-octanol on GABA-induced current was examined with respect to its dose dependence. As shown in Fig. 5, 30 to 1000 µM n-octanol increased the peak current evoked by 3 µM GABA in a dose-dependent manner. The potentiating action was reversible upon washing out n-octanol (Fig. 5G).

View larger version (8K): [in this window] [in a new window]   Fig. 5.   n-Octanol reversibly potentiates GABA-induced currents in a dose-dependent manner. GABA was applied through the U-tube with or without n-octanol at 2-min intervals. B-F, n-octanol (30-1000 µM) was coapplied with 3 µM GABA for 3 s. Because there was pronounced decay in GABA-induced current in the presence of 1000 µM n-octanol, the maximum current was obtained by extrapolation to time zero (F). Potentiation and acceleration of decay phase were reversible upon washing out n-octanol (G).

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Potentiation of GABA-induced currents by n-octanol and its simulation by two models. Potentiation by n-octanol was more pronounced in the presence of 3 µM GABA (open symbols) than in the presence of 10 µM GABA (filled symbols). Two models were used to fit data: an allosteric model (A) and a partial agonist model (B). In allosteric model, a logistic eq. 2 was used to fit data giving an EC50 of 191 ± 39 µM and an nH of 1.49 ± 0.30 with A being 6.85 ± 0.06 and 2 ± 0.3 for 3 µM GABA and 10 µM GABA, respectively. Data for potentiation of currents induced by 3 µM and 10 µM GABA were also fitted by using partial agonist model (eq. 6): parameters used were determined from the dose-response relationship for GABA and n-octanol, i.e., a = 0.8, KG = 22.2 µM, XG = 4, b = 0.33, KO = 2821 µM, and XO = 0.15, and the parameter XGO was assumed to be 12. Ordinate in B represents ratios of responses in the presence of GABA and n-octanol over control response, either in the presence of 3 µM GABA or 10 µM GABA alone. Ordinate in Fig. 6A is ratio determined in Fig. 6B  1, representing degree of potentiation.

Potentiating Action of n-Octanol Depends on GABA Concentrations. Figure 7 summarizes the augmentation of GABA-induced peak currents by 100 µM n-octanol as a function of GABA concentration. The n-octanol-induced potentiation of GABA currents decreased as the GABA concentration increased. Peak currents evoked by GABA at 1 and 3 µM were greatly increased but those at 100 to 300 µM GABA were not changed by 100 µM n-octanol. According to the allosteric model, this alcohol-induced peak current potentiation is related to a shift of the GABA dose-response relationship in the direction of lower agonist concentrations. That is, 100 µM n-octanol reduced EC₅₀ from 13 µM to 5.5 µM for activating the GABA_A receptor.

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Potentiation of GABA-induced currents by n-octanol depends on GABA concentration. Potentiation by 100 µM n-octanol was more pronounced at lower GABA concentrations than at higher concentrations. Two models were used to fit data: an allosteric model (A) and a partial agonist model (B). In allosteric model, 100 µM n-octanol reduced EC50 for GABA activation from 13 µM to 5.5 µM without altering nH value and maximal response. Control parameters and those in the presence of 100 µM n-octanol were fitted into eq. 1. In partial agonist model, GABA-induced currents were fitted by eq. 6 with or without 100 µM n-octanol by using parameters used in Fig. 6. In both cases, solid line represents ratio of calculated responses in presence of n-octanol over control values without n-octanol.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   n-Octanol reduces slope of GABA dose-response relationship. Dose-response relationship for GABA () was fitted to eq. 1 to give an EC50 of 13 µM and an nH of 1.4. n-Octanol reduced EC50 to 5.9 µM and nH to 0.99 when it was coapplied with GABA at 100 µM and reduced the EC50 to 1.4 µM and the nH to 0.95 when coapplied with GABA at 300 µM.

Lack of Effects of n-Octanol on Maximal GABA Response. Both the allosteric model and the partial agonist model make a prediction that at high GABA concentrations n-octanol would lose its potentiating action. This prediction proved to be the case as illustrated in Fig. 9. When the receptor was activated by 1000 µM GABA, coapplication of 100 to 1000 µM n-octanol with GABA did not affect the maximal GABA-activated current. GABA-induced currents in the presence of n-octanol relative to the control GABA-current were 101 ± 4.0%, 105 ± 7.0%, 100 ± 4.0%, and 97 ± 4.0% in 100, 300, 500, and 1000 µM n-octanol, respectively.

View larger version (11K): [in this window] [in a new window]   Fig. 9.   Lack of effect of n-octanol on 1000 µM GABA-induced currents. GABA was applied through a U-tube with or without n-octanol at 5-min intervals. n-Octanol was coapplied with 1000 µM GABA for 1 s at concentrations of 100 to 1000 µM (B-E). There was pronounced decay in GABA-induced currents in the presence of 500 and 1000 µM n-octanol, and maximum current was obtained by extrapolation to time zero. Acceleration of decay phase was reversible upon washing out n-octanol (F).

Effects of Prolonged Application of n-Octanol on GABA-Activated Currents. Figure 10A shows that when 1000 µM n-octanol was applied to the bath for 1 to 5 min before a coapplication of 3 µM GABA and 1000 µM n-octanol, n-octanol had little potentiating effect on GABA-induced peak currents. On the average, after a 1-min pretreatment of 1000 µM n-octanol, the current in the presence of both n-octanol and 3 µM GABA was 1.26 ± 0.37-fold (n = 5) the current in the presence of 3 µM GABA alone.

View larger version (13K): [in this window] [in a new window]   Fig. 10.   Effects of prolonged application of n-octanol on GABA-induced currents. A, coapplication of 1000 µM n-octanol and 3 µM GABA to a cell has little or no potentiating effect on GABA-induced currents. GABA was coapplied with n-octanol 1, 3, and 5 min after beginning of bath application of 1000 µM n-octanol (horizontal line). B, coapplication of 1000 µM n-octanol and 300 µM GABA reduces GABA-induced current to 20% of control. Protocol was same as A, but with a higher GABA concentration (300 µM) and coapplication after 1 min of exposure to n-octanol. Time scale calibrations refer to periods of coapplication.

	Discussion

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The present study showed that n-octanol exerted multiple actions on the 122S GABA_A receptor expressed in HEK293 cells. In the absence of GABA, n-octanol was capable of inducing inward currents, and in the presence of GABA, it exerted a dual action on GABA-induced currents depending on the GABA concentration and on how long the receptor was exposed to GABA or n-octanol. For a brief exposure when GABA and n-octanol were coapplied to the cell and when GABA concentrations were lower than its EC₅₀ value, n-octanol caused a great increase in GABA-induced currents. The potentiation diminished with increasing concentration of GABA and eventually disappeared as the GABA concentration was well above EC₅₀ value. When the receptor was exposed to n-octanol for a prolonged period of time, a coapplication of GABA and n-octanol resulted in reduction of GABA-induced currents.

Mechanisms of Action of n-Octanol in Modulation of GABA-Induced Currents

The discussion in the following two sections will focus on the question of whether the multiple actions of n-octanol on the GABA_A receptors involve multiple sites of action, or whether the direct and potentiation actions can be explained by the partial agonistic action of n-octanol as has been previously shown for other receptors (Steinbach and Chen, 1995; Fletcher and Steinbach, 1996). The multiple sites for multiple actions assume that the binding of n-octanol to each site is responsible for each type of n-octanol action, whereas, in the partial agonist model, n-octanol binds to the site identical with the GABA site manifesting its multiple actions.

Multiple Sites of n-Octanol Action. According to the multiple-site model for n-octanol action, the affinities of n-octanol for its binding site on the GABA_A receptor were estimated by fitting the logistic Hill equation to the dose-response relationship for each type of n-octanol action. The EC₅₀ values were 1127 µM (Fig. 3A) and 191 µM (Fig. 6A), respectively, for its direct action and potentiating action.

Partial Agonist Model: One Binding Site Manifesting Multiple Actions. Several observations suggest that n-octanol acts at the GABA site to induce inward currents. n-Octanol-induced currents have a reversal potential similar to that of GABA-induced currents. Both currents are inhibited by bicuculline and picrotoxin. The degree of bicuculline inhibition of the current induced by 3 µM GABA was similar to that induced by 1000 µM n-octanol, suggesting that the GABA_A receptors are bound by GABA and n-octanol to the similar extent at their respective concentrations. Taking these results together, it is concluded that n-octanol acts on the GABA binding site as a weak partial agonist. The kinetic scheme for GABA to activate the receptor depicted in Scheme 1 is applicable to n-octanol.

n-Octanol Potentiation of GABA-Induced Currents. One might wonder how a weak partial agonist is able to exert the potentiating action on the receptor activated by a full agonist as seen in Fig. 6. The interpretation is rather straightforward according to the partial agonist model outlined in Scheme 2. At low GABA concentrations, not many GABA_A receptors are doubly bound by GABA, and with an increasing concentration of n-octanol, more receptors will be bound by one GABA and one n-octanol molecule. Because the mixed ligand-bound receptor has a high probability of opening, the whole-cell current becomes larger as the concentration of n-octanol is increased (Figs. 6B and 8). This may account for the observation that the partial agonist model is better than the allosteric model to account for the potentiating action of n-octanol on the response induced by 1 µM GABA as well (Fig. 7).

Inhibition of GABA-Induced Currents by Alcohol Pretreatment. Consistent with the previous report by Arakawa et al. (1992), high concentrations of n-octanol (300-1000 µM) preperfused before GABA application inhibited peak currents induced by high concentrations of GABA. This implies that the alcohol-induced current can undergo desensitization, which is consistent with the notion that alcohol acts as a partial agonist. After 1 min of application of 1000 µM n-octanol, about 84% of the GABA_A receptors have undergone desensitization (Fig. 10B). The remaining 16% of receptors can still respond to the potentiating action of n-octanol, with a 7-fold increase in GABA-induced currents when GABA was applied at 3 µM. The overall amplitude of GABA-induced current under this condition would be only slightly greater than the current induced by 3 µM GABA alone when all receptors are available.

Comparison with Previous Studies. Many agents are known to exert multiple actions on transmitter-gated receptors. For examples, barbiturates (Schulz and Macdonald, 1981; Parker et al., 1986; Akaike et al., 1987, 1990; Amin and Weiss, 1993), propofol (Hales and Lambert, 1991; Hara et al., 1993, 1994; Orser et al., 1994; Jones et al., 1995; Sanna et al., 1995), and halothane (Yang et al., 1992; Sincoff et al., 1996) have been found to have the direct action on the GABA_A receptor in the absence of GABA. Some of them exert a biphasic potentiating action in the presence of GABA at low concentrations relative to its EC₅₀ value (Parker et al., 1986; Akaike et al., 1990) and inhibit the sustained residual current following exposure to a high desensitizing concentration of GABA (Nakahiro et al., 1989; Hall et al., 1994).