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. Author manuscript; available in PMC: 2008 Apr 30.
Published in final edited form as: Alcohol. 2007 May 23;41(3):155–162. doi: 10.1016/j.alcohol.2007.03.006

Studies of ethanol actions on recombinant δ-containing γ-aminobutyric acid type A (GABAA) receptors yield contradictory results

Cecilia M Borghese 1, R Adron Harris 1
PMCID: PMC2040030  NIHMSID: NIHMS27061  PMID: 17521845

Abstract

The γ-aminobutyric acid type A receptors (GABAA-Rs) display a wide variety of subunit combinations. Drugs such as benzodiazepines have shown differential effects based on GABAA-R subunit composition. Actions of alcohols and volatile anesthetics generally do not vary markedly with subunits composition, with low concentrations of ethanol being poor modulators of these receptors. Recent studies showed α4/6- and δ-containing GABAA-Rs (located extrasynaptically, and responsible for tonic currents in selective brain regions) presenting high sensitivity to low concentrations of ethanol, but these results have not been obtained in other laboratories. We carried out additional experiments varying the receptor level of expression, and GABA and ethanol concentration, but no sensitivity to low concentrations of ethanol was detected. We will discuss these results and attempt an analysis of the possible causes for the discrepancies.

Keywords: Ion channel, inhibitory neurotransmission, delta subunit, allosteric modulation, Xenopus oocyte, electrophysiology

Introduction

Gifted with a long list of subunits (6 α, 3 β, 3 γ, δ, ε, π and θ, several of which possess isoforms), the pentameric γ-aminobutyric acid type A receptor (GABAA-R) has an incredible number of potential subunit combinations, at least in theory. In practice, a comparatively reduced number of combinations have been observed, with the most abundant being α1β2γ2S (Sieghart and Sperk, 2002; Whiting et al., 1999). Two combinations that have attracted attention lately are α4β2/3δ and α6β2/3δ: even though they are not very abundant, they present a more restricted expression, both with respect to regional and neuronal localization, and a very distinctive pharmacology. Numerous studies have provided evidence supporting the coexpression of α4 and δ subunits in brain, mainly in hippocampal dentate granule cells, thalamocortical relay neurons and outer layers of the cerebral cortex (Korpi et al., 2002; Mihalek et al., 1999; Nusser et al., 1999; Peng et al., 2002; Pirker et al., 2000; Sur et al., 1999). In cerebellum, δ is coexpressed with α6 subunits in the granule cells (Jechlinger et al., 1998; Jones et al., 1997; Pirker et al., 2000; Tretter et al., 2001). The localization of δ-containing GABAA-Rs is non-synaptic (Nusser et al., 1998; Sun et al., 2004; Wei et al., 2003). Their higher GABA affinity makes them exquisitely sensitive to the low GABA concentrations in the perisynapsis and extrasynaptic space, which produces a small tonic current, in contrast with the phasic current observed in the synapsis, where GABAA-Rs contain γ subunits (Farrant and Nusser, 2005).

Expression in heterologous systems

While studies of brain slices or homogenates can render valuable information, the characterization of the properties of a particular subunit combination is usually better achieved by expressing the subunits of interest in heterologous systems, therefore controlling the subunit composition of the receptor. Attractive as the approach is, it is not without caveats: the host cell may influence the receptor properties, and it may be difficult to achieve a level of expression that allows the study of the receptor of interest, among other problems. The difficulty in expressing α4/6β2/3δ in oocytes (widely used for the study of other subunit combinations) probably delayed their full characterization. In our experience, expression of α4- and δ-containing receptors in Xenopus oocytes was only possible when three condition were met: subcloning of the coding regions in a modified, high expression vector, injection of low amounts of cRNA, and recording a week or more after injection, as described in Wallner et al. (2003). Both α4- and α6-containing GABAA-Rs were more sensitive to GABA than α1-containing GABAA-Rs, and insensitive to most ligands to the benzodiazepine site (Wafford et al., 1996; Whittemore et al., 1996). For instance, diazepam potentiation was not observed in α4/6-containing GABAA-Rs, but it was pronounced in α1-containing GABAA-Rs; flumazenil did not affect α1-containing GABAA-Rs, but it increased GABA currents through α4/6-containing GABAA-Rs; Ro15-4513 had no effect or was a partial inverse agonist at α1-containing GABAA-Rs, but was a positive modulator of α4/6-containing GABAA-Rs (Wafford et al., 1996; Whittemore et al., 1996). GABAA-Rs composed of α6β3γ2L or α6β3δ (Saxena and Macdonald, 1996), and α1β3, α1β3γ2L or α1β3δ (Fisher and Macdonald, 1997; Haas and Macdonald, 1999) were transiently expressed and their GABA responses characterized in mouse L929 fibroblasts. Later, human α4β3δ subunits were stably expressed in a L(tk-) cell line and pharmacologically characterized, compared with α4β3γ2 (Brown et al., 2002). The α4β3δ were more sensitive to GABA than α4β3γ2 GABAA-Rs, but GABA acted as a partial agonist at α4β3δ, and gaboxadol (4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol, or THIP) showed more efficacy than GABA at this subunit combination. Desensitization following a short application of a maximally effective concentration of GABA was faster in α4β3γ2 than in α4β3δ GABAA-Rs. The sensitivity of these GABAA-Rs to zinc was very similar, but lanthanum inhibition was more pronounced in α4β3γ2 than in α4β3δ. As for GABA modulators, flunitrazepam was inactive in both combinations, but α4β3δ showed Ro15-4513 and bretazenil potentiation, and DMCM inhibition, while α4β3γ2 was unaltered by these drugs. Several neuroactive steroids (5α-pregnane-3α-ol-20-one, alphaxalone, and 5α-pregnane-3α,21-diol-20-one or THDOC) were more potent modulators of GABA in α4β3δ than in α4β3γ2 GABAA-Rs. More recent studies of human and rat α4β3δ expressed in oocytes replicated many of these findings (Borghese et al., 2006; Stórustovu and Ebert, 2006).

Behavioral experiments with δ-knockout mice

The generation and subsequent testing of δ-knockout mice (Mihalek et al., 1999) revealed reduced effects of neuroactive steroids; specifically, decreased sedative, anxiolytic and pro-absence seizure effects. The precise molecular basis for these differences is not clear yet. For instance, THDOC modulation of evoked and spontaneous IPSCs in thalamocortical relay neurons was not modified in δ-knockout mice (Porcello et al., 2003), but both THDOC (Vicini et al., 2002) and alphaxalone (Spigelman et al., 2003) failed to prolong spontaneous IPSCs in cerebellar and dentate granule cells, respectively, from δ-knockout mice. When the effect of THDOC was studied in dentate and cerebellar granule cells, the neuroactive steroid was much more potent in tonic currents mediated by δ-containing GABAA-Rs than in phasic currents (Stell et al., 2003). The tonic conductance in both dentate and cerebellar granule neurons was greatly decreased in δ-knockout mice, and its modulation by THDOC eliminated (Stell et al., 2003).

As has been observed in other GABAA-R subunit knockout mice, the elimination of the δ subunit led to changes in the composition, and sometimes number, of the GABAA-Rs. In the δ-knockout mice, the missing δ subunit was replaced by γ in both cerebellum granule cells (Tretter et al., 2001) and in forebrain regions (Korpi et al., 2002; Peng et al., 2002), where δ normally coexpresses with α6 and α4 subunits, respectively.

Compared with wild type mice, δ-knockout mice presented altered ethanol responses in several tests (reduced ethanol consumption, attenuated withdrawal from chronic ethanol exposure and reduced anticonvulsant effects of ethanol), while other tests were normal (anxiolytic response, and development of acute and chronic tolerance) (Mihalek et al., 2001). Another behavior that was not modified in δ-knockout mice was ethanol-induced sleep time, and Ro15-4513 reduced sleep in a similar way in both wild-type and δ-knockout mice (Mihalek et al., 2001). The δ-knockout mice were also characterized in an ethanol-discrimination procedure, and the authors concluded that “the delta subunit is not necessary in the mediation of ethanol-like effects of any of the GABAA ligands tested, including sensitivity to ethanol, barbiturate, benzodiazepine, and neurosteroid discriminative stimulus effects” (Shannon et al., 2004). Delta-containing GABAA-Rs are particularly sensitive to gaboxadol (see Section 2). Consistent with this finding, gaboxadol-induced sleep time was decreased in δ-knockout mice (Boehm et al., 2006).

Ethanol on α4/6- and δ-containing GABAA-Rs

The results obtained in brain slice preparations is covered in this volume by Valenzuela and others, and will not be reviewed in this article.

Sensitivity to low concentrations of ethanol

Two labs reported that δ-containing receptors expressed in oocytes were sensitive to low concentrations of ethanol, but the results diverged in several points. Sundstrom-Poromaa et al. (2002) showed that GABA-induced responses in human α4β2δ were potentiated by 1-3 mM ethanol (50%), with 10 mM ethanol presenting a decreased potentiation (25%). Other combinations tested (α1β2δ, α1β2γ2S, α4β2γ2S) were not sensitive to these low ethanol concentrations, and other relevant combinations (α4β2, α4β3δ, α4β3, α6β3δ, α6β3) were either not tested or did not express (Sundstrom-Poromaa et al., 2002).

Wallner et al. (2003) published results obtained with GABAA-Rs constituted by rat α4/6 and β2/3, with or without δ or γ2S. The α and β subunits did not affect the GABA affinity, but including δ increased GABA affinity (EC50 αβ ~ αβγ > αβδ). Positive modulators (THDOC in α4β2δ and etomidate in α6β3δ) were capable of potentiating even maximally effective GABA concentrations, suggesting that GABA is a partial agonist at these combinations. Low concentrations of ethanol potentiated EC20 GABA responses when the combination included either α4 or α6, β3 and δ subunits, and 30 mM ethanol produced 75% potentiation in these combinations, but only 21% in α4/6β2δ and none in α4/6β2/3γ2S. This differential sensitivity was maintained at 100 mM ethanol, with all these combinations being potentiated at similar levels by 300 mM ethanol. The combinations including only α and β subunits (α4/6β2/3) did not show any potentiation by ethanol at any concentration.

Recent papers show that ethanol acted as a competitive inhibitor of Ro15-4513 binding on α4/6β3δ (Hanchar et al., 2006), and that Ro15-4513 blocked ethanol potentiation of GABA-mediated currents in α4β3δ GABAA-Rs at ethanol concentrations up to 30 mM (Wallner et al., 2006). In this last paper, flunitrazepam lacked any effect on Ro15-4513 inhibition of ethanol potentiation of GABA-mediated responses, while flumazenil reversed Ro15-4513 inhibition; neither flunitrazepam nor flumazenil blocked ethanol potentiation on its own. The authors concluded that Ro15-4513 is acting on a site different from the classical benzodiazepine binding site, where only certain benzodiazepine-like ligands can bind, but not all. Higher concentrations of ethanol seem to act on transmembrane sites of the GABAA-R subunits, as Ro15-4513 was unable to block the ethanol potentiation, and this high alcohol potentiation was eliminated by a mutation in β3 transmembrane domain (Wallner et al., 2006).

Lack of sensitivity to low concentrations of ethanol

Four labs tested ethanol sensitivity of α4- and δ-containing GABAA-Rs in different systems, and did not observe any sensitivity to low concentrations of ethanol (Borghese et al., 2006). Our laboratory’s setting most closely resembled that used by Olsen’s group, since we used the same rat cDNAs, and followed their protocols. However, we did not observe in α4β3δ GABAA-Rs the sensitivity to low concentrations of ethanol. We took special care in verifying the expression of δ subunit along with α4 and β3: α4β3δ GABAA-Rs presented larger GABA-induced maximal currents, and lower sensitivity to zinc. GABA affinity was not a defining characteristic: EC50 GABA was similar for α4β3 and α4β3δ, while α4β3γ2S GABAA-Rs presented a lower affinity for GABA.

The main discrepancy with previous results was in the ethanol modulation of EC20 GABA responses: 30 mM ethanol produced a reliable but small potentiation (~10%) in all subunit combinations (α4β3, α4β3δ, and α4β3γ2S), 100 mM induced a more pronounced, but not differential, potentiation (25-50%), and 300 mM presented a larger and distinctive pattern of potentiation: α4β3 > α4β3δ > α4β3γ2S. Figures 1 and 2A through C show representative tracings, while pooled results of short applications appear in figure 2D. Interestingly, isoflurane modulation of GABA responses was similar in α4β3δ and α4β3γ2S.

Figure 1.

Figure 1

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Tracings showing the ethanol effect on EC20 GABA responses (long application) in rat α4β3δ expressed in oocytes. The faster desensitization of the GABA current in rat α4β3γ2S is shown for comparison. Reproduced by permission of The American Society for Pharmacology and Experimental Therapeutics, adapted from Borghese et al. (2006).

Figure 2.

Figure 2

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Tracings and graph showing the ethanol effect on EC20 GABA responses (short application) in rat α4β3 (A), α4β3γ2S (B) and α4β3δ (C) expressed in oocytes. (D) Pooled results; values are mean ± S.E.M., n= 6-8, * p< 0.05 versus other subunit combinations at 300 mM ethanol. Reproduced by permission of The American Society for Pharmacology and Experimental Therapeutics, adapted from Borghese et al. (2006).

Experiments expressing human cRNAs in oocytes yielded similar results with respect to ethanol modulation of α4β3δ GABAA-Rs; the human subunits presented a pharmacological profile consistent with the literature, explored in more detail in a companion paper (Stórustovu and Ebert, 2006). The L(tk-) cell line stably expressing human α4β3δ and α4β3γ2S showed lower overall sensitivity to ethanol potentiation, but again no differences between α4β3δ and α4β3γ2S GABAA-Rs.

Our paper also included experiments conducted in mouse hippocampal slices. Dentate granule neurons showed a tonic current blocked by bicuculline, which was not affected by 30 mM ethanol.

A more recent paper studied cerebellar granule cells and Chinese hamster ovary (CHO) cells recombinantly expressing GABAA-Rs (Yamashita et al., 2006). In cerebellar granule cells, GABA-induced currents mediated by α6- and δ-containing GABAA-Rs were decreased by 30% in the presence of 30 mM ethanol. Similarly, in α4β2δ, α6β2δ and α6β3δ GABAA-Rs expressed in CHO cells, 30 mM ethanol had either no effect or inhibitory actions on GABA-mediated responses, while 100 mM ethanol was clearly inhibitory. Several other drugs were tested in α6β2δ GABAA-Rs expressed in CHO cells that agreed with the pharmacological profile of this combination in other systems. Another study of ethanol effect on GABA-induced currents in cerebellar granule cells in culture (Casagrande et al., 2007) found that low ethanol concentrations did not affect the slow (tail) component of GABA currents.

Further experiments in our laboratory

The methodology used was the same as described in Borghese et al. (2006). Essentially, Xenopus laevis oocytes were isolated and injected with cRNAs encoding the GABAA subunits α4, β3, δ and/or γ2S in the ratios 1:1:3 for α4:β3:γ2S and 1:1:10 for α4:β3:δ, unless otherwise indicated (the injected amounts of α4 and β3 were always 0.4 ng each per oocyte). Six to nine days after injection, recordings were carried out using the two-electrode voltage clamp technique.

Delta Subunit Expression Level

Several papers were published concerning the GABAA-R ε subunit: one describing receptors α1β3ε and α2β1ε expressed in HEK293 cells as insensitive to pentobarbital (Davies et al., 1997), and two others describing receptors α1β1ε expressed in Xenopus oocytes showing modulation by pentobarbital, as well as direct effects by this barbiturate (Thompson et al., 1998; Whiting et al., 1997). This controversy would be eventually resolved (Thompson et al., 2002): using α1β1ε expressed in Xenopus oocytes, it was shown that the differences between the ε subunits were not due to amino acid sequences nor to untranslated regions, but to levels of expression determined by the vector in which the DNA encoding the ε subunit was inserted. Overexpression of the subunit eliminated sensitivity to pentobarbital.

This led us to pose the following question: was the δ subunit being overexpressed (leading maybe to an unusual stoichiometry in the receptor), and did this affect the ethanol modulation?

To answer this, we injected different amounts of cRNA encoding the δ subunit, keeping the amounts of α4 and β3 constant (0.4 ng each per oocyte). Our hypothesis was that the level of expression of δ subunit would decrease if the amount of cRNA encoding δ is reduced, and that would allow us to test ethanol sensitivity, and determine if there was any difference.

In our previous paper (Borghese et al., 2006), GABA affinity for α4β3δ and α4β3 GABAA-Rs was very similar (1.29 versus 0.62 μM GABA, respectively), and we did not see any differences in the receptors that contained a lower ratio of δ subunit (Fig. 3A). However, we had previously observed a decreased GABA-induced maximal current in α4β3 compared with α4β3δ GABAA-Rs (12-fold), and we obtained a significant decrease in maximal currents (3.7-fold) when the ratio of cRNA encoding for α:β:δ was reduced to 1:1:0.01 (Fig. 3B). Further proof of a reduced level of δ expression was the zinc sensitivity. GABAA-Rs lacking a δ or γ subunit are very sensitive to zinc inhibition, and we had observed 90% inhibition in α4β3 GABAA-Rs with 1 μM zinc, while α4β3δ GABAA-Rs were inhibited by about 20% (Borghese et al., 2006). When the δ-encoding cRNA ratio was reduced to 1:1:0.01, zinc inhibition was enhanced (Fig. 4A). However, the ethanol sensitivity remained the same, independently of the expression level of δ subunit (Fig. 4B).

Figure 3.

Figure 3

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GABA responses of rat subunits injected in different ratios in oocytes. GABA concentration-response curves (A) and GABA maximal responses 7-9 days after injection (B) when α4β3γ2S (subunit ratio 1:1:3) and α4β3δ (subunit ratio ranging from 1:1:10 to 1:1:0.01) were expressed. In A, n=3-12; in B, *p<0.01 versus α4β3δ 1:1:10 (Dunnett’s Multiple Comparison Test, ANOVA), n=4-25.

Figure 4.

Figure 4

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GABA modulation by zinc and ethanol of rat subunits injected in different ratios in oocytes. Zinc (1 μM) inhibition (A) and ethanol (30 and 100 mM) potentiation (B) of EC20 GABA responses when α4β3γ2S (subunit ratio 1:1:3) and α4β3δ (subunit ratio ranging from 1:1:10 to 1:1:0.01) were expressed. *p< 0.01 (Dunnett’s Multiple Comparison Test, ANOVA), n= 3-8.

Reduced GABA concentration

For many subunit combinations, as GABA concentration increases, ethanol potentiation decreases (Mihic et al., 1994). The difficulty of obtaining large currents in heterologous systems expressing α4β3δ GABAA-Rs has probably resulted in most of the literature reporting ethanol effects on currents elicited by EC20 GABA. We decided to check if the inverse relationship between GABA concentration and ethanol potentiation would hold true, and if sensitivity to low concentrations of ethanol would increase if GABA concentration were diminished. We observed no significant changes in ethanol potentiation when EC5 GABA was used (Figure 5), compared with EC20 GABA (Figure 4), suggesting that there is no inverse relationship for these combinations, at least at the lower range of GABA concentrations.

Figure 5.

Figure 5

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Modulation by ethanol of a lower concentration of GABA (EC5) applied to rat α4β3δ and α4β3γ2S expressed in oocytes. n= 4.

Ethanol-Zinc Interaction

Zinc is accumulated by specific neurons in the CNS into synaptic vesicles, and modulates both excitatory (glutamatergic) and inhibitory (GABAergic and glycinergic) transmission (Hirzel et al., 2006; Smart et al., 2004). Discrete binding sites in α1β2 and α1β2γ2S GABAA-Rs have been described, and the combination αβ is more sensitive to zinc inhibition than αβγ/δ (Borghese et al., 2006; Hosie et al., 2003; Stórustovu and Ebert, 2006). Given its role as endogenous modulator, and the possibility of contamination of buffers through standard laboratory procedures (some latex and nitrile gloves, as well as plastic pipettes contain zinc; Rachel Phelan and John Mihic, personal communication), we decided to study if the presence of zinc could modify ethanol actions on α4β3δ GABAA-Rs. An inverse relationship between neurotransmitter concentration and zinc effect has been described, therefore we used an EC5 GABA for our experiments.

Low concentrations of zinc (10 and 100 nM) had minimal effects on both α4β3δ and α4β3γ2S GABAA-Rs, while 1 μM zinc produced a definite inhibition on the GABA response (Figure 6A). These same concentrations of zinc were tested in the same oocytes in the presence of 30 mM ethanol, which by itself produced a small but reliable potentiation of the GABA response (Figure 6B). The presence of zinc did not modify the ethanol effect (Figure 6B): for α4β3γ2S GABAA-Rs, both the ethanol and zinc effects were significant, but the interaction was not (F3,18= 0.53, p= 0.67), and the same was true for α4β3δ GABAA-Rs (interaction F3,24= 1.67, p= 0.20). Therefore, the presence of zinc did not affect the ethanol modulation of GABA responses.

Figure 6.

Figure 6

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GABA modulation by zinc and ethanol of rat α4β3δ and α4β3γ2S expressed in oocytes. (A) Zinc (0.01-1 μM) effect on EC5 GABA responses. (B) Difference between the percentage of change in the EC5 GABA response when coapplied with different concentrations of zinc (0-1 μM) and 30 mM ethanol, and when coapplied with zinc alone at those same concentrations. N= 4-5.

Possible basis for the discrepancy

We tried several experimental approaches to identify the factors that control sensitivity to low concentrations of ethanol in α4β3δ GABAA-Rs, but none of them produced a different result. So far, there is no ready and easy explanation for the discrepancy in results between eight research groups, a situation not entirely new in the ethanol field.

Differences in system used [transient expression in oocytes and CHO cells, stable cell line L(tk-)], clones (rat versus human) and genetic backgrounds in the studies conducted with brain slices could possibly account for the lack of reproducibility, but in one case the clones and the system used were the same, and the protocol followed closely the one employed in the first place. We will concentrate on this particular case.

Besides the lack of sensitivity to low concentrations of ethanol, other discrepancies that were apparent were differences in a) maximal GABA responses, b) affinities for subunit combinations, and c) desensitization rate. Point a: in Wallner et al. (2006), the authors made a reference to our maximal GABA responses being five times higher than the currents they obtained. We were not able to do that comparison, because the authors did not publish their maximal currents. However, the EC20 GABA tracings shown in their papers (Wallner et al., 2003; Wallner et al., 2006) were approximately of the same magnitude of ours (Figure 1 and 2C), and the EC20 GABA responses in our experiments involving ethanol modulation ranged from 300 to 1800 nA (mean ± SEM= 1060 ± 100 nA), and there was no correlation between the magnitude of the GABA response and the ethanol effect. Therefore, the differences observed in ethanol sensitivity were not related to the magnitude of the current itself, and we are not sure if the maximal currents could be so divergent. As for the GABA-mediated currents in oocytes injected with human cRNAs, they were considerable smaller than with the rat counterparts (Borghese et al., 2006), but the human α4β3 and α4β3δ expressed in oocytes have been pharmacologically characterized, and all the subunits were present (Stórustovu and Ebert, 2006). Point b: in our paper, the relative order for EC50 GABA was α4β3 ~ α4β3δ < α4β3γ2S, which is the order observed for these and similar combinations (Brown et al., 2002; Fisher and Macdonald, 1997; Stórustovu and Ebert, 2006; Thompson et al., 1997). Therefore, even though there was a shift to the right for α4β3δ compared with Wallner et al. (2003), we feel that the overall results were consistent with the literature. Point c: the desensitization rate for α4β3δ was higher in our preparation than in Wallner et al. (2003, 2006), but α4β3δ always presented less desensitization than α4β3γ2L (Figure 1).

The δ-knockout mice could provide interesting insight into the situation. If ethanol actions at low concentrations are mediated by α4/6β3δ GABAA-Rs, which ethanol behaviors are affected when these receptors are absent? At 1.5 g/kg i.p. (the lowest dose used), ethanol hypothermic and anxiolytic effects were similar in wild-type and δ-knockout mice (Mihalek et al., 2001). Moreover, deletion of the δ subunit did not modify the acquisition of an ethanol/saline discrimination (Shannon et al., 2004). Ethanol consumption was decreased in δ-knockout mice, albeit the decrease was less in females (Mihalek et al., 2001). More testing is necessary, but so far, the behaviors mediated by δ-containing GABAA-Rs seem to be limited. Even though it has been proposed that ethanol anesthetic effects are mediated by different binding sites in the GABAA-R transmembrane domains, it is surprising that ethanol-induced sleeping time was not altered in δ-knockout mice, and was unaltered by Ro15-4513 (Mihalek et al., 2001), when gaboxadol (selective agonist for δ-containing GABAA-Rs) showed a decreased sleeping time in δ-knockout mice (Boehm et al., 2006).

Post-translational modifications can greatly affect the behavior of GABAA-Rs by altering channel currents and/or trafficking (Brandon et al., 2002; Luscher and Keller, 2004). It has been suggested that protein kinase A-induced phosphorylation of β3 subunit in α4β3δ and α4β3γ2L could modify currents through these GABAA-Rs, by respectively decreasing and increasing fast desensitization (Tang and Macdonald, 2005). Differences in the phosphorylation state of the GABAA-R could in principle account for differences in the ethanol sensitivity, although it is difficult to understand how phosphorylation state could vary between laboratories for very similar preparations.

Ten laboratories have published results of low concentrations of ethanol effects on δ-containing receptors in heterologous systems, cultured neurons and slice preparations. Three have observed potentiation: Smith’s and Olsen’s for recombinant receptors (although for different combinations), and Mody’s for slice preparations. Laboratories that have seen no effect or even inhibition include Wafford’s, Ebert’s, Narahashi’s, Robello’s and Harris’ for recombinant receptors and cultured neurons, and Lambert’s and Valenzuela’s for slice preparations. We conclude that there are factor/s still unaccounted for that determine the sensitivity of δ-containing receptors to low concentrations of ethanol.

Acknowledgments

This study was supported by National Institutes of Health Grants AA06399 and GM47818, and by the Waggoner Center for Alcohol and Addiction Research.

Footnotes

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