The trafficking of GABA A receptors is an important component of the pathway that regulates plasticity of inhibitory synapses. The 17 kDa GABA A receptor-associated protein (GABARAP) has been implicated in the trafficking of GABA A receptors because of its ability to interact not only with the ␥2 subunit of the receptor but also with microtubules and the N-ethylmaleimide-sensitive factor (NSF). To elucidate the role of GABARAP in the trafficking of GABA A receptors, we have constructed a yellow fluorescent protein (YFP) fusion protein of GABARAP and expressed it in neurons using adenovirus, so that its function may be examined. YFP-GABARAP colocalized with ␥2 subunit-containing GABA A receptors and NSF to the perinuclear cytoplasm in cultured hippocampal neurons and to the proximal regions of dendrites that are making synaptic contact. Expression of YFP-GABARAP in Cos7 cells and cultured hippocampal neurons was able to increase the level of GABA A receptors detected at the plasma membrane, even at low levels of YFP-GABARAP expression. This effect is specific to the function of GABARAP on GABA A receptor trafficking, because point mutations in the ␥2-binding domain of YFP-GABARAP interfered with the ability of YFP-GABARAP to increase GABA A receptor surface levels. These mutations also disrupted the colocalization of YFP-GABARAP with the ␥2 subunit and with NSF in hippocampal neurons. The results of this study show for the first time that GABARAP has a functional effect on the trafficking of GABA A receptors and provide decisive evidence for the role of GABARAP in transporting GABA A receptors to the plasma membrane in neurons.

General anesthetics allosterically modulate the activity of neuronal ␥-aminobutyric acid, type A (GABA A ), receptors. Previous mutational studies from our laboratory and others have shown that the regions in transmembrane domain 1 (M1) and pre-M1 of ␣ and ␤ subunits in GABA receptors are essential for positive modulation of GABA binding and function by the intravenous (IV) general anesthetics. Mutation of ␤2Gly-219 to Phe corresponded in nearly eliminated the modulatory effect of IV anesthetics in ␣1/␤2/␥2S combination. However, the general anesthetics retained the ability to directly open the channel of mutant G219F, and the apparent affinity for GABA was increased, and desensitization rate was reduced. In this study, we made additional single mutations such as 219 Ser, Cys, Ile, Asp, Arg, Tyr, and Trp. The larger side chains of the replacement residues produced the greatest reduction in enhancement of GABA currents by IV anesthetics at clinical concentrations (Trp > Tyr ‫؍‬ Phe > Arg > Asp > Ile > Cys > Ser ‫؍‬ wild type). Compared with a 2-3-fold response in wild type, pentobarbital and propofol enhanced less than 0.5-fold; etomidate and alphaxalone modulation was reduced from more than 4-to 1-fold in G219F, G219Y, and G219W. A linear correlation was observed between the volume of the residue at position 219 and the loss of modulation. An identical correlation was found for the effect of modulation on left-shift in the GABA EC 50 value; furthermore, the same rank order of residues, related to size, was found for reduction in the maximal direct channel-gating by pentobarbital (1 mM) and etomidate (100 M) and for increased apparent affinity for direct gating by the IV anesthetics.Considerable evidence supports the hypothesis that general anesthesia can be produced, at least in part, by enhancing neuronal inhibition mediated by GABA A receptors (GABAR). 1 GABA and most volatile and general intravenous (IV) anesthetics bind to GABAR. These are ligand-gated channels that enhance the flow of chloride ions (Cl Ϫ ) into the postsynaptic neuron, thus increasing its resting potential and making it less likely to fire, i.e. the inhibitory effect in GABA synapses. GABAR are modulated positively by a wide variety of structurally diverse general anesthetics (1). In particular, volatile anesthetics (2, 3) and IV anesthetic agents such as the barbiturates (4, 5), propofol (6), etomidate (7), and neuroactive steroid anesthetics, like alphaxalone (8 -10), all enhance the function of GABAR at clinically relevant concentrations. In addition, IV and volatile anesthetics can activate GABAR directly in the absence of GABA (11). Anesthetics appear to allosterically modulate ligand-gated ion channel (LGIC) receptor function by binding to the receptor itself, but not at the agonist-binding site, and to affect the conformation of the membrane protein in a functional manner. Exactly how such modulatory sites affect agonist-regulated channel opening is a major contemporary question in pharmacology and neurobiology. Obviously, the struct...

GABA A receptor-associated protein (GABARAP) was isolated previously in a yeast two-hybrid screen using the intracellular loop of the ␥2 subunit of the GABA A receptor as bait. GABARAP has been shown to participate in the membraneclustering and intracellular-trafficking of GABA A receptors, including a stimulation of the surface expression of GABA A receptors. To assess this quantitatively, we used Xenopus laevis oocytes expressing ␣1␤2␥2S-containing GABA A receptors to demonstrate that coexpression of GABARAP increased net surface levels of GABA A receptors as shown by both increased GABA currents and surface-expressed protein. This GABARAP stimulation of GABA currents required the receptor ␥2 subunit and full-length GABARAP: deletion of the microtubule-binding domain (amino acids 1-22) or disrupting the polymerization of microtubules abolished the enhancement, indicating that the effect of GABARAP was derived from the interaction with microtubules. GABARAP coexpression did not alter the general properties of GABA A receptors such as sensitivity to GABA or benzodiazepines, but it increased surface levels of receptor protein in oocytes. Rather, it seems to supplement inadequate amounts of endogenous GABARAP to support optimum trafficking and/or stabilization of surface GABA A receptors.

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We investigated the ubiquitin-like modification of GABA A receptor-associated protein (GABARAP) and its function. A fusion protein of GABARAP with v5 in the N terminus and myc in the C terminus was expressed in rat cultured hippocampal neurons and PC12 cells. Western blotting with antibodies to v5 and myc revealed that the C terminus of GABARAP was cleaved off. Cleavage was blocked by mutating the C-terminal Gly116 to Ala, suggesting that G116 is required for the processing. Unlike ubiquitin, GABARAP was not incorporated covalently into higher-molecular-weight protein complexes. Nor was GABARAP degraded by ubiquitinylation through the proteasome, although GABARAP formed noncovalent dimers. Immunofluorescent confocal microscopy demonstrated that recombinantly expressed GABARAP was diffusely localized in PC12 cells. However, prevention of C-terminal processing in the mutant GABARAP G116A resulted in redistribution to the Golgi. In neurons, punctate cytoplasmic staining of GABARAP was seen in soma and processes, whereas GABARAP G116A was limited to soma. Compared with wild-type GABARAP, the colocalization and interaction of GABARAP G116A with GABA A receptors were significantly reduced, resulting in a reduction in expression of receptors in the plasma membrane. When ␣1␤2␥2S-containing GABA A receptors were expressed in oocytes, the increased surface expression of GABA A receptors, as shown by increased GABA currents and surface-accessible GABA A receptor subunit polypeptides resulting from GABARAP coexpression, was prevented by mutation G116A. In addition, the distribution of NSF (N-ethylmaleimide-sensitive factor) was affected in GABARAP G116A -expressing neurons. These results suggest that glycine 116 is required for C-terminal processing of GABARAP and that processing is essential for the localization of GABARAP and its functions as a trafficking protein.