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

Abstract

GABA A receptors (GABA A Rs) are usually formed by ␣, ␤, and ␥ or ␦ subunits. Recently, ␦-containing GABA A Rs 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 ␦ GABA A Rs. We independently conducted three studies in two systems: rat and human GABA A Rs expressed in Xenopus oocytes, studied through two-electrode voltage clamp; and human GABA A Rs stably expressed in the fibroblast L(tk Ϫ ) cell line, studied through patch-clamp electrophysiology. In all cases, ␣ 4 ␤ 3 ␦ GABA A Rs 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-20-one (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 GABA A Rs. Ethanol at 30 mM did not affect tonic GABA-mediated currents in dentate gyrus reported to be mediated by GABA A Rs incorporating ␣ 4 and ␦ subunits. We have not been able to replicate the sensitivity of ␣ 4 ␤ 3 ␦ GABA A Rs 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 GABA A Rs.

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

“…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 α 4 β 3 δ than in α 4 β 3 γ 2 GABA A -Rs. More recent studies of human and rat α 4 β 3 δ expressed in oocytes replicated many of these findings (Borghese et al, 2006;Stórustovu and Ebert, 2006).…”

“…Four labs tested ethanol sensitivity of α 4 -and δ-containing GABA A -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.…”

Deleterious Effects of a Low Amount of Ethanol on LTP-Like Plasticity in Human Cortex

“…However, these statements deserve caution, given that the systems level approach of our experiments does not permit direct insights into the cellular mechanisms. Furthermore, the high sensitivity of extrasynaptic GABAARs to low doses of EtOH has not been unanimously replicated (Borghese et al, 2006;Yamashita et al, 2006).…”

“…Cellular studies showed that low-dose EtOH (p30 mM) produces mainly two effects: potentiation of extrasynaptic gamma-aminobutyric acid type A receptor (GABAAR)-mediated tonic inhibition (Sundstrom-Poromaa et al, 2002;Wallner et al, 2003;Wei et al, 2004) (for review, (Olsen et al, 2007)), although this has not been unanimously supported (Borghese et al, 2006;Yamashita et al, 2006), and N-methyl-D-aspartate receptor (NMDAR) inhibition (Lovinger et al, 1989(Lovinger et al, , 1990Weitlauf and Woodward, 2008).…”

Alcohol Modulation of Extra-synaptic Gamma-aminobutyric Acid Type A Receptors

“…On the other hand, these results generated some controversy in the field as the findings regarding the high alcohol affinity of the extra-synaptic receptors have not been replicated by some groups and presynaptic mechanisms have been proposed for the alcohol potentiation of GABAergic system (Carta et al, 2004;Borghese et al, 2006;Botta et al, 2007a, b;Korpi et al, 2007;Baur 2009). Several experimental errors or methodological issues may cause this contradiction.…”