The Subunit of -Aminobutyric Acid Type A Receptors Does Not Confer Sensitivity to Low Concentrations of Ethanol

GABAA receptors (GABAARs) are usually formed by , , and or subunits. Recently, -containing GABAARs expressed in Xenopus oocytes were found to be sensitive to low concentrations of ethanol (1–3 mM). Our objective was to replicate and extend the study of the effect of ethanol on the function of 4 3 GABAARs. We independently conducted three studies in two systems: rat and human GABAARs expressed in Xenopus oocytes, studied through two-electrode voltage clamp; and human GABAARs stably expressed in the fibroblast L(tk ) cell line, studied through patch-clamp electrophysiology. In all cases, 4 3 GABAARs were only sensitive to high concentrations of ethanol (100 mM in oocytes, 300 mM in the cell line). Expression of the subunit in oocytes was assessed through the magnitude of the maximal GABA currents and sensitivity to zinc. Of the three rat combinations studied, 4 3 was the most sensitive to ethanol, isoflurane, and 5 -pregnan-3 ,21-diol-20one (THDOC); 4 3 and 4 3 2S were very similar in most aspects, but 4 3 was more sensitive to GABA, THDOC, and lanthanum than 4 3 2S GABAARs. Ethanol at 30 mM did not affect tonic GABA-mediated currents in dentate gyrus reported to be mediated by GABAARs incorporating 4 and subunits. We have not been able to replicate the sensitivity of 4 3 GABAARs to low concentrations of ethanol in four different laboratories in independent studies. This suggests that as yet unidentified factors may play a critical role in the ethanol effects on -containing GABAARs. The number of possible molecular targets for ethanol action in brain continues to increase, but the number affected by low ethanol concentrations ( 20 mM) remains remarkably small (Harris, 1999; Walter and Messing, 1999; Narahashi et al., 2001; Mailliard and Diamond, 2004). One of the main candidates as a site of ethanol action is the -aminobutyric acid type A receptor (GABAAR). Five subunits form a chloride ion channel that can be modulated by benzodiazepines, neurosteroids, barbiturates, intravenous and volatile anesthetics, and alcohols (Mehta and Ticku, 1999; Korpi et al., 2002a). The binding site for benzodiazepines is located in the subunit interface (Sigel, 2002), and a putative binding site for alcohols and volatile anesthetics is in the extracellular side of the transmembrane domains (Harris et al., 1997; Ueno et al., 1999; Mascia et al., 2000). The most common subunit combination in brain is 1 2 2 (Fritschy et al., 1992). -Containing GABAARs are not nearly as abundant as -containing GABAARs, but they are gradually emerging as unique and fundamental players in GABAergic inhibition. In the case of 4 and subunits, their immunoreactivities often presented patterns of codistribution in discreet areas (Pirker et al., 2000). In rat thalamus, immunoprecipitation studies determined that two thirds of the 4-containing GABAARs also contained subunits, and in hippocampus, GABAARs containing both 4 and represented half of each subunit’s population (Sur et al., 1999). -Deficient mice (Mihalek et al., 1999) showed decreased 4 subunit expression in forebrain, whereas 2S subunit levels were increased (Peng et al., 2002). These studies suggested that 4 and subunits coassemble and that the absence of subunits allowed 2 subunits to replace them (Korpi et al., 2002b). This study was supported by National Institutes of Health Grants AA06399 and GM47818 and by the Waggoner Center for Alcohol and Addiction Research (R.A.H.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.092452. ABBREVIATIONS: GABAAR, -aminobutyric acid receptor type A; DGC, dentate granule cell; CRC, concentration-response curve; THDOC, 5 -pregnan-3 ,21-diol-20-one; ANOVA, analysis of variance; aCSF, artificial cerebrospinal fluid; mIPSC, miniature inhibitory postsynaptic current; RMS, root mean square. 0022-3565/06/3163-1360–1368$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 316, No. 3 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 92452/3079349 JPET 316:1360–1368, 2006 Printed in U.S.A. 1360 at A PE T Jornals on A ril 1, 2017 jpet.asjournals.org D ow nladed from Later studies showed that, in dentate granule cells (DGCs), immunogold-labeled -containing GABAARs were located in perisynaptic sites (Wei et al., 2003) and 2% of 4 and subunits colocalized with GABAergic synaptic markers (GAD65 and gephyrin) (Sun et al., 2004). In DGCs, the tonic inhibitory current was apparently mediated by -containing receptors, most likely 4 3 , because zolpidem did not affect the tonic current, which was highly sensitive to the hypnotic gaboxadol; the phasic current mediated by -containing receptors present in synaptic sites was enhanced by zolpidem (Nusser and Mody, 2002; Maguire et al., 2005). The -knockout mice presented alterations in some ethanol-induced behaviors compared with wild-type mice (reduced ethanol consumption, withdrawal from chronic ethanol exposure, and the anticonvulsant effect of ethanol); however, the anxiolytic and hypothermic responses and the development of chronic and acute tolerance were normal (Mihalek et al., 2001). In addition, the discriminatory stimulus effect of ethanol was similar in -knockout and wild-type mice (Shannon et al., 2004). Recently, two studies showed that low ethanol concentrations enhanced currents mediated by GABAARs expressed in Xenopus oocytes (Sundstrom-Poromaa et al., 2002; Wallner et al., 2003). In the first study, 4 2 was enhanced by 1 to 3 mM ethanol, but 10 mM ethanol had no effect. In the study of Wallner et al., the receptors most sensitive to ethanol were formed by 4 or 6 subunits in conjunction with 3 and subunits. They found that ethanol enhanced submaximal GABA-induced currents in 4 3 and 6 3 receptors at concentrations as low as 3 mM, with 30 mM producing 75% potentiation. The 4 2 and 6 2 GABAARs were not affected by 3 mM ethanol but were potentiated by 30 mM ethanol, albeit to a lesser extent than the equivalent 3containing GABAARs (21%). The combinations 4 3 2S and 6 3 2S failed to show any enhancement until the ethanol concentration reached 100 mM, and the combinations studied ( 4 2, 6 2, 4 3, and 6 3) did not show any enhancement up to 300 mM ethanol. In DGCs, the tonic inhibition (mediated by -containing GABAARs) was enhanced by low concentrations of ethanol (30 mM), whereas the tonic conductance present in CA1 pyramidal cells (mediated by 5 2-containing GABAARs) was not (Wei et al., 2004). In an attempt to replicate and extend the studies discussed above, we expressed the same clones used by Wallner et al. (2003) (rat 4, 3, and ) in X. laevis oocytes, following their procedures. We compared the effects of several drugs, including ethanol, on the 4 3 , 4 3, and 4 3 2S combinations. Similar studies were performed with equivalent human clones expressed in X. laevis oocytes. To compare results in a different expression system, a stable cell line previously shown to express human 4 3 (Brown et al., 2002) was also investigated using patch-clamp analysis. Finally, we determined the effects of ethanol on native extrasynaptic GABAARs of DGCs, which are reported to contain 4 and subunits. Materials and Methods Rat GABAA Subunits Expressed in X. laevis Oocytes Materials. Adult female X. laevis frogs were obtained from Xenopus Express (Plant City, FL). GABA, ethanol, zinc chloride, and lanthanum chloride were purchased from Sigma Chemical (St. Louis, MO), 5 -pregnan-3 ,21-diol-20-one (THDOC) was purchased from Steraloids (Newport, RI), isoflurane was purchased from Marsam Pharmaceuticals Inc. (Cherry Hill, NJ), and etomidate was purchased from Tocris Cookson Inc. (Ellisville, MO). All other reagents were of reagent grade. GABA, zinc chloride, and lanthanum stocks were prepared in water; etomidate and THDOC were dissolved in dimethyl sulfoxide. The drug stocks were then dissolved in buffer; the final dimethyl sulfoxide concentration was 0.1% v/v or less, which does not affect GABAA-mediated current. Clones, Transcription, and Oocyte Injection. The cDNAs encoding the GABAA subunits were generously provided by Dr. R. W. Olsen (rat 4 and , in a modified pGEM vector), Dr. L. Mahan (rat 3, in a modified pGEM vector), and Dr. M. H. Akabas (rat 2S, in a pGEMHE vector). After linearization, the cDNAs encoding the subunits were used as a template for the synthesis in vitro of 5 -capped RNA (mCAP RNA Capping Kit, Stratagene, La Jolla, CA). X. laevis oocytes were manually isolated from a surgically removed portion of ovary. Oocytes were treated with collagenase (type IA, 0.5 mg/ml) for 10 min, and then placed in incubation medium (composition: 100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5 mM HEPES, adjusted to pH 7.5), supplemented with 1000 units of penicillin and 1 mg of streptomycin per liter. Oocytes were then injected into the cytoplasm with 30 nl of diethyl pyrocarbonate-treated water containing cRNA encoding GABAA subunits. The injected amounts were 0.4:0.4 / , 0.4:0.4:1.2 / / , and 0.4:0.4:4 / / in nanograms per oocyte. The injected oocytes were kept at 18°C in incubation media for 7 to 10 days. Electrophysiological Recordings. Recordings were carried out 7 to 10 days after injection. The oocytes were placed in a rectangular chamber (approximately 100 l) and continuously perfused with ND96 buffer (2 ml/min) at room temperature (23°C). The perfusion buffer composition was 96 mM NaCl, 1 mM CaCl2, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.5. The whole-cell voltage clamp at 80 mV was achieved through two glass electrodes (1.5–8 M ) filled with 3 M KCl, using a Warner Instruments (Hamden, CT) oocyte clamp