Altering the relative abundance of GABA A receptor subunits changes GABA- and ethanol-responses in Xenopus oocytes.
- Authors
- Hurley, Joyce H; Ballard, Carrie J; Edenberg, Howard J
- Year
- 2009
- Journal
- Alcoholism, clinical and experimental research
- PMID
- 19382902
- DOI
- 10.1111/j.1530-0277.2009.00930.x
- PMCID
- PMC4136686
BACKGROUND: Variations in GABRA2 and GABRG3, genes encoding the alpha2 and gamma3 subunits of the pentameric GABA(A) receptor, are associated with the risk of developing alcoholism in adults, conduct disorder at younger ages, and with differences in electroencephalographic power in the beta frequency range. The SNPs associated with alcoholism did not alter the coding of these genes, and extensive DNA sequencing of GABRA2 did not find coding changes in the high-risk haplotypes. Therefore, we hypothesize that the associations arise from differences in gene expression. METHODS: Here we report studies in Xenopus oocytes to examine the functional effects of altering the relative abundance of these 2 receptor subunits on GABA current and response to ethanol, as a model of potential effects of regulatory differences. RESULTS: When human alpha2beta2gamma3 subunits are co-expressed, increasing the amount of the alpha2 subunit mRNA increased GABA current; in contrast, increasing the amount of the gamma3 subunit decreased GABA currents. Acute ethanol treatment of oocytes injected with a 1:1:1 or 2:2:1 ratio of alpha2:beta2:gamma3 subunit mRNAs resulted in significant potentiation of GABA currents, whereas ethanol inhibited GABA currents in cells injected with a 6:2:1 ratio. Overnight treatment with ethanol significantly reduced GABA currents in a manner dependent on the ratio of subunits. CONCLUSIONS: These studies demonstrate that changes in relative expression of GABA(A) receptor subunits alter the response of the resulting channels to GABA and to ethanol.
GABA currents as a function of the ratio of α2, β2 and γ3 subunit mRNAs A. Peak current amplitudes in response to 1 or 10 μM GABA in oocytes injected with a 1:1 ratio of α2:β2 subunit mRNAs or a 1:1:1 ratio of α2:β2:γ3 subunit mRNAs. Inset depicts representative inward GABA currents in response to 10 μM GABA. The solid line above the traces indicates GABA application. B. Peak current amplitudes in response to 1 or 10 μM GABA in oocytes injected with either 1:1:3 or 1:1:1 or 3:1:1 ratios of α2:β2:γ3 subunit mRNAs. The data are shown as the mean ± SEM. * indicates a significant difference between groups (p < 0.05).
GABA currents as a function of the ratio of α2, β2 and γ3 subunit mRNAs. A. Peak current amplitudes in response to 10 μM GABA in oocytes injected with either 2:2:1 or 6:2:1 or 0.5:2:1 ratios of α2:β2:γ3 subunit mRNAs. B–C. GABA concentration-response curves in oocytes injected with either 2:2:1 (B) or 6:2:1 (C). Peak currents at each GABA concentration were normalized to the current at 30 μM GABA for each oocyte and averaged within groups. Data are presented as mean ± SE. The solid line through the data indicates the non-linear regression fit with the correlation coefficient (R2) and calculated EC50 values below. Dotted lines indicate the location of EC50 and EC20 on each graph.
Effects of acute ethanol treatment on GABA current amplitude. A. Peak current amplitudes in response to 1 μM GABA before and after 44 mM ethanol pretreatment in oocytes expressing either 1:1:1 or 2:2:1 or 6:2:1 ratios of α2:β2:γ3 subunit mRNAs. Data are normalized to peak current amplitudes in response to GABA from each oocyte before ethanol treatment. Ethanol was applied for 60 seconds before and concurrently with GABA administration. * indicates a significant difference between groups (p < 0.01) using paired t-tests.
Effects of chronic ethanol treatment on GABA current amplitude. A. Peak current amplitudes in response to 10 μM GABA in oocytes treated for 16 hours with 44 mM ethanol after injection with 2:2:1 or 6:2:1 or 0.5:2:1 ratios of α2:β2:γ3 subunit mRNAs. B. Peak current amplitudes in response to 10 μM GABA in oocytes treated for 16 hours with 100 mM ethanol after injection with 2:2:1 or 6:2:1 or 0.5:2:1 ratios of α2:β2:γ3 subunit mRNAs. Data are normalized to peak current amplitudes of control oocytes; symbols as in Figure 1.
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| A polymorphism in GABRA2 is associated with the medial frontal response to alcohol cues in an fMRI study. | Kareken DA et al. | — | 2010 | → |