␥-Aminobutyric acid type A receptors (GABARs) have long been implicated in mediating ethanol (EtOH) actions, but so far most of the reported recombinant GABAR combinations have shown EtOH responses only at fairly high concentrations (>60 mM). We show that GABARs containing the ␦-subunit, which are highly sensitive to ␥-aminobutyric acid, slowly inactivating, and thought to be located outside of synapses, are enhanced by EtOH at concentrations that are reached with moderate, social EtOH consumption. Reproducible ethanol enhancements occur at 3 mM, a concentration six times lower than the legal blood-alcohol intoxication (driving) limit in most states (0.08% wt͞vol or 17.4 mM). GABARs responsive to these low EtOH concentrations require the GABAR ␦-subunit, which is thought to be associated exclusively with ␣4-and ␣6-subunits in vivo, and the ␤3-subunit, which has recently been shown to be essential for the in vivo anesthetic actions of etomidate and propofol. GABARs containing ␤2-instead of ␤3-subunits in ␣4␤␦-and ␣6␤␦-receptor combinations are almost 10 times less sensitive to EtOH, with threshold enhancement at 30 mM. GABARs containing ␥2-instead of ␦-subunits with ␣4␤ and ␣6␤ are three times less sensitive to EtOH, with threshold responses at 100 mM, a concentration not usually reached with social EtOH consumption. These combined findings suggest that ''extrasynaptic'' ␦-subunit-containing GABARs, but not their ''synaptic'' ␥-subunit-containing counterparts, are primary targets for EtOH. Despite the fact that ethanol (EtOH) is the most widely used psychoactive agent, its actions on brain functions are poorly understood. Several types of receptors and channels have been shown to be functionally altered by EtOH, which include Nmethyl-D-aspartate (1) and non-N-methyl-D-aspartate glutamate receptors (2, 3), serotonin (4), glycine (5, 6), and GABARs (7,8), and G protein-coupled inwardly rectifying K ϩ channels (9, 10). With a few exceptions (3,(8)(9)(10)(11)(12), EtOH effects on these targets are seen only at fairly high concentrations (Ն60 mM).The GABAR, the major inhibitory neurotransmitter receptor, has been a long-time focus for studies on EtOH and anesthetic actions. For example, it has been shown that EtOH at low intoxicating concentrations was able to enhance Cl Ϫ flux in synaptoneurosomes (13,14) and cultured neurons (15). However, electrophysiological studies of GABARs in single neurons and recombinant receptors showed current enhancement only at fairly high concentrations (Ͼ50 mM) of EtOH (5,7,16), which now appears to be due to the fact that these studies focused on synaptic and͞or ␥-subunit-containing receptors. It is thought that replacement of the ␥-subunit in the GABAR 2␣-2␤-1␥ pentameric complex by the ␦-subunit changes not only the localization of the receptor from mainly postsynaptic to extrasynaptic, but also leads to up to a 50-fold increase in ␥-aminobutyric acid (GABA) affinity and slower desensitization (17)(18)(19). These functional properties are consistent with ␣␤␦ GABARs, which are activat...

Neuronal mechanisms underlying alcohol intoxication are unclear. We find that alcohol impairs motor coordination by enhancing tonic inhibition mediated by a specific subtype of extrasynaptic GABA A receptor (GABAR), α6β3δ, expressed exclusively in cerebellar granule cells. In recombinant studies, we characterize a naturally occurring single-nucleotide polymorphism that causes a single amino acid change (R100Q) in α6 (encoded in rats by the Gabra6 gene). We show that this change selectively increases alcohol sensitivity of α6β3δ GABARs. Behavioral and electrophysiological comparisons of Gabra6 100R/100R and Gabra6 100Q/100Q rats strongly suggest that alcohol impairs motor coordination by enhancing granule cell tonic inhibition. These findings identify extrasynaptic GABARs as critical targets underlying low-dose alcohol intoxication and demonstrate that subtle changes in tonic inhibition in one class of neurons can alter behavior.Humans have been consuming alcohol for thousands of years, and the use of alcoholic beverages pervades human culture and society and can have substantial health effects 1 . Different mechanisms by which ethanol might depress brain function have been proposed based on ethanol's ability to modulate a wide variety of ion channels 2-4 , neurotransmitter receptors 5-10 and transporters 11 . Among these diverse targets, however, GABARs are arguably the most attractive candidates. This is in part because other classes of known GABAR modulators such as benzodiazepines, barbiturates and certain anesthetics lead to behavioral effects that closely resemble ethanol intoxication. Yet despite strong evidence implicating GABARs in ethanol's action, critical details remain unclear. For instance, although it is known that native GABARs are heteropentamers assembled from 19 possible subunits 12,13 , it has not been possible to link the activity of particular GABAR subunits to changes in behavioral sensitivity to ethanol.Recent studies suggest that specific combinations of GABAR subunits (those containing α4β3δ and α6β3δ) are uniquely sensitive to ethanol, showing dose dependencies that mirror blood alcohol levels associated with intoxication in humans 9,10 . GABARs containing α4 and δ subunits are expressed in many brain regions 14,15 , but α6 is found in only two types of neurons (cerebellar granule cells and granule cells in the cochlear nucleus) and is expressed together with δ only in cerebellar granule cells 14,16,17 . In granule cells α6 and δ combine with β subunits Correspondence should be addressed to M.W. (mwallner@mednet.ucla.edu) or T.S.O. (otist@ucla.edu). 3 These authors contributed equally to this work. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. We set out to examine whether such extrasynaptic GABARs containing α6 and δ subunits account for behavioral effects of ethanol at moderately intoxicating doses. To link these particular GABARs to behavioral sensitivity, we first characterized a naturally occurring single-nucleotide polymo...

This review discusses evidence for and apparent controversy about, GABA A receptor subtypes that mediate alcohol effects experienced during social drinking. GABA A receptors that contain the β3 and δ subunits were shown to be enhanced by alcohol concentrations that mirror the concentrationdependence of alcohol responses in humans. A mutation (α6R100Q) previously found in alcohol non-tolerant (ANT) rats in the cerebellar GABA A receptor α6 subunit is sufficient for increased alcohol-induced ataxia in rats homozygous for this mutation (α6-100QQ) and further increases alcohol-sensitivity of tonic GABA currents (mediated by α6βδ receptors) in cerebellar granule cells of α6-100QQ rats and in recombinant α6R100Qβ3δ receptors. This provided the first direct evidence that these types of receptors mediate behavioral effects of ethanol. Furthermore the behavioral alcohol antagonist Ro15-4513 specifically reverses ethanol enhancement on α4/6β3δ receptors. Unexpectedly, native and recombinant α4/6β3δ receptors bind the behavioral alcohol antagonist Ro15-4513 with high affinity and this binding is competitive with EtOH, suggesting a specific and mutually exclusive (competitive) ethanol/Ro15-4513 site which explains the puzzling activity of Ro15-4513 as a behavioral alcohol antagonist.Our conclusion from these findings is that alcohol/Ro15-4513-sensitive GABA A receptor subtypes are important alcohol targets and that alcohol at relevant concentrations is more specific than previously thought. In this review we discuss technical difficulties in expressing recombinant δ subunit-containing receptors in oocytes and mammalian cells, that may have contributed to negative results and confusion. Not only because we have reproduced detailed positive results numerous times, and we and many others have built extensively on basic findings, but also because we explain and combine many previously puzzling results into a coherent and highly plausible paradigm on how alcohol exerts an important part of its action in the brain, we are confident about our findings and conclusions. However, many important open questions remain to be answered.

Although it is now more than two decades since it was first reported that the imidazobenzodiazepine Ro15-4513 reverses behavioral alcohol effects, the molecular target(s) of Ro15-4513 and the mechanism of alcohol antagonism remain elusive. Here, we show that Ro15-4513 blocks the alcohol enhancement on recombinant ''extrasynaptic'' ␣4͞6␤3␦ GABAA receptors at doses that do not reduce the GABA-induced Cl ؊ current. At low ethanol concentrations (<30 mM), the Ro15-4513 antagonism is complete. However, at higher ethanol concentrations (>100 mM), there is a Ro15-4513-insensitive ethanol enhancement that is abolished in receptors containing a point mutation in the second transmembrane region of the ␤3 subunit (␤3N265M). Therefore, ␣4͞6␤3␦ GABA receptors have two distinct alcohol modulation sites: (i) a low-dose ethanol site present in ␣4͞6␤3␦ receptors that is antagonized by the behavioral alcohol antagonist Ro15-4513 and (ii) a site activated at high (anesthetic) alcohol doses, defined by mutations in membrane-spanning regions. Receptors composed of ␣4␤3N265M␦ subunits that lack the high-dose alcohol site show a saturable ethanol dose-response curve with a half-maximal enhancement at 16 mM, close to the legal blood alcohol driving limit in most U.S. states (17.4 mM). Like in behavioral experiments, the alcohol antagonist effect of Ro15-4513 on recombinant ␣4␤3␦ receptors is blocked by flumazenil and ␤-carboline-ethyl ester (␤-CCE). Our findings suggest that ethanol͞Ro15-4513-sensitive GABA A receptors are important mediators of behavioral alcohol effects.alcohol intoxication ͉ alcohol receptor ͉ anesthetics A lthough alcohol is one of the most widely used and abused drugs, the molecular targets that mediate alcohol effects at concentrations relevant for mild social intoxication are only beginning to be revealed. Neurotransmitter receptors for GABA, NMDA and glycine, and G protein-gated K ϩ channels have been identified as potential alcohol targets that are sensitive to intoxicating alcohol concentrations (1-4). GABA A receptors (GABA A Rs) have long been suspected to be important mediators of alcohol effects (5, 6) because benzodiazepines (BZs) and barbiturates, classic GABA A R agonists, share common pharmacological properties with ethanol, such as sedativehypnotic, anti-anxiety, and motor in-coordinating and anticonvulsant effects and have additive, possibly even synergistic effects, when taken together with ethanol (7). In addition, BZs, barbiturates, and ethanol produce tolerance and cross-tolerance to each other (8), consistent with GABA A Rs as targets of action.We have recently identified subtypes of GABA A Rs, those containing the ␦ and the ␤3 subunit, that are uniquely sensitive to low alcohol concentrations (9). Consistent with the view that ␦ subunit-containing receptors are important mediators of alcohol actions is the finding that GABA A R ␦-subunit knockout mice show multiple defects in behavioral responses to ethanol (10). Receptors containing the ␦ subunit have an exclusively nonsynaptic distribution, ...

Many spikes in amperometric records of exocytosis events initially exhibit a prespike feature, or foot, which represents a steady-state flux of neurotransmitter through a stable fusion pore spanning both the vesicle and plasma membranes and connecting the vesicle lumen to the extracellular fluid. Here, we present the first evidence indicating that vesicular volume before secretion is strongly correlated with the characteristics of amperometric foot events. L-3,4-Dihydroxyphenylalanine and reserpine have been used to increase and decrease, respectively, the volume of single pheochromocytoma cell vesicles. Amperometry and transmission electron microscopy have been used to determine that as vesicle size is decreased the frequency with which foot events are observed increases, the amount and duration of neurotransmitter released in the foot portion of the event decreases, and vesicles release a greater percentage of their total contents in the foot portion of the event. This previously unidentified correlation provides new insight into how vesicle volume can modulate the activity of the exocytotic fusion pore.