Altering the Relative Abundance of GABA A Receptor Subunits Changes GABA- and Ethanol-Responses in Xenopus Oocytes

Alcoholism and alcohol abuse are prevalent conditions which present a significant burden to society. Alcoholism is a complex multi-factorial disease which has a strong genetic component. Linkage and association analyses have led to the identification of a number of genes in which variations increase risk for alcoholism (for reviews, see (Dick and Foroud 2003; Edenberg and Foroud 2006; Kohnke 2008). Family-based studies demonstrated that GABRA2, encoding the α2 subunit of the GABAA receptor, is associated with alcohol dependence and with electroencephalographic differences in the β frequency range of 13–28 Hz (Edenberg et al. 2004). Alcoholics and their offspring differ from controls by having increased power in that frequency band (Bauer and Hesselbrock 1993; Costa and Bauer 1997; Rangaswamy et al. 2002; Rangaswamy et al. 2003). Bauer and Hesselbrock (Bauer and Hesselbrock 1993) reported that enhanced high frequency beta activity, originating from deep anterior regions of the frontal brain, was the best predictor of relapse in substance-dependent patients, and may also be related to initial risk for dependence. The association between GABRA2 and alcoholism has been replicated in several other populations, with the same haplotypes conferring higher risk (Covault et al. 2004; Edenberg and Foroud 2006; Enoch et al. 2006; Fehr et al. 2006; Lappalainen et al. 2005; Soyka et al. 2008). Further analyses demonstrated that at younger ages, the higher-risk alleles of GABRA2 are associated with conduct disorder (Dick et al. 2006a). GABRG3, encoding the γ3 subunit, has also been associated with alcohol dependence (Dick et al. 2004), as has GABRA1, encoding the α1 subunit (Dick et al. 2006b). Our previous study identified multiple SNPs associated with alcoholism, none of which were within coding regions (Edenberg et al. 2004). In addition, sequencing over 40 individuals with high-risk and low-risk haplotypes revealed no nucleotide changes within the coding sequence of GABRA2. Detailed analysis of mRNA from human brain demonstrated alternative splicing and alternative promoter use in the human α2 gene (Tian et al. 2005). Promoter activity of naturally occurring haplotypes also differed (Tian et al. 2005). These observations led us to hypothesize that changes in gene regulation may underlie the alcohol-related phenotypes. Some evidence suggests that there are innate differences in GABA receptor function between lines of rats selectively bred for alcohol preference (P and NP) (Lumeng et al. 1977). Microarray analysis has identified genes expressed differentially in the hippocampus of the P and NP lines including many genes involved in synaptic transmission (Edenberg et al. 2005). For example, the GABRB1 gene was expressed at a 1.6-fold higher level in the hippocampus of the P rat vs. the NP rat and GABRA1 gene expression was higher as well. An additional microarray study corroborated the observation that the GABRB1 gene was more highly expressed in multiple brain regions in the P vs. NP rats (Kimpel et al. 2007). Our hypothesis is that changes in expression levels of the receptor subunits alter GABA signaling. γ-Aminobutyric acidA (GABAA) receptors are ligand-gated ion channels that mediate the majority of fast inhibitory neurotransmission in the central nervous system (Barnard et al. 1998; Mehta and Ticku 1999; Whiting et al. 1999). They are pentameric integral membrane proteins comprised of different classes of subunits. At least 6 α, 3 β, 3 γ, δ, e, θ and π subunit genes and multiple splice variants have been identified. The majority of GABAA receptors are thought to be composed of two α, two β and one γ subunits (Chang et al. 1996; Farrar et al. 1999) with δ sometimes substituting for the γ subunit (Quirk et al. 1995). GABAA receptors are targets of many important clinical agents including benzodiazepines, barbiturates, steroids, anesthetics and anti-convulsants (Barnard et al. 1998; Korpi, Grunder, and Luddens 2002; Sieghart 2006). GABAA receptors are important in many behavioral effects of ethanol consumption including motor in-coordination, sedation, anxiolysis, tolerance, preference, hypothermia and withdrawal (Buck 1996; Grobin et al. 1998; Korpi, Grunder, and Luddens 2002). For example, ethanol mimics some of the effects of GABAA receptor modulators such as benzodiazepines, neurosteroids and barbiturates. Reports about the acute effects of ethanol on GABAA receptors differ. The sensitivity of GABAA receptors to ethanol may vary with subunit composition. Ethanol potently (at low mM concentrations) facilitates extrasynaptic GABAA receptors containing α4 or α6 subunits in combination with δ subunits (Sundstrom-Poromaa et al. 2002; Wallner, Hanchar, and Olsen 2003). In contrast the potency of ethanol is much lower in the more abundant synaptic GABAA receptors which contain γ subunits instead of δ subunits. However other investigators have been unable to replicate these findings and the inconsistency may be due to species differences, differences in expression levels or GABA concentrations tested, or unknown variables (Borghese and Harris 2007; Harris and Mihic 2004). In general, long-term ethanol treatment reduces GABAA receptor function by several mechanisms including changes in gene expression, posttranslational modification and subcellular localization of specific subunits (for reviews, see (Kumar, Fleming, and Morrow 2004) and (Krystal et al. 2006)). For example, chronic ethanol administration in rats reduces the level of mRNA and peptides for the α1, α2 and α3 GABAA receptor subunits in the cerebral cortex (Mhatre and Ticku 1992) but these effects differ across brain regions. Indirect evidence suggests that phosphorylation, particularly by PKC, may differentially alter GABAA receptor function in response to ethanol (Kumar, Fleming, and Morrow 2004; Kumar, Sieghart, and Morrow 2002; Morrow et al. 2004). Chronic ethanol treatment selectively increases α1 subunit protein internalization in rat cerebral cortex (Kumar et al. 2003). In the functional studies presented here, we have mimicked changes in relative gene expression by introducing different ratios of the α2, β2 and γ3 GABAA receptor subunit mRNAs into Xenopus oocytes and have measured GABAA receptor channel function. In addition we have examined the effect of acute and chronic ethanol treatment on the electrophysiological characteristics of the channels that result from expression of different subunit ratios.