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Type-A receptors for the neurotransmitter GABA (gamma-aminobutyric acid) are ligand-gated chloride channels that mediate inhibitory neurotransmission. Each subunit of the pentameric receptor protein has ligand-binding sites in the amino-terminal extracellular domain and four membrane-spanning regions, one of which forms a wall of the ion channel. Each subunit also has a large intracellular loop that may be a target for protein kinases and be required for subcellular targeting and membrane clustering of the receptor, perhaps by anchoring the receptor to the cytoskeleton. Neurotransmitter receptors need to be positioned in high density in the cell membrane at sites postsynaptic to nerve terminals releasing that neurotransmitter. Other members of the superfamily of ligand-gated ion-channel receptors associate in postsynaptic-membrane clusters by binding to the proteins rapsyn or gephyrin. Here we identify a new cellular protein, GABA(A)-receptor-associated protein (GABARAP), which can interact with the gamma2 subunit of GABA(A) receptors. GABARAP binds to GABA(A) receptors both in vitro and in vivo, and co-localizes with the punctate staining of GABA(A) receptors on cultured cortical neurons. Sequence analysis shows similarity between GABARAP and light chain-3 of microtubule-associated proteins 1A and 1B. Moreover, the N terminus of GABARAP is highly positively charged and features a putative tubulin-binding motif. The interactions among GABA(A) receptors, GABARAP and tubulin suggest a mechanism for the targeting and clustering of GABA(A) receptors.

␥-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...