GABA_AReceptor-Associated Protein Regulates GABA_AReceptor Cell-Surface Number inXenopus laevisOocytes

Abstract

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.

NMDA Receptor Activation Potentiates Inhibitory Transmission through GABA Receptor-Associated Protein-Dependent Exocytosis of GABAAReceptors

“…NSF has been implicated in the exocytosis of many receptor types in the CNS, including GABA A Rs, AMPARs, ␤2-adrenergic receptors, and dopaminergic receptors (for review, see Zhao et al, 2007), so perhaps the specificity of NSF-mediated receptor delivery is conferred by associated proteins such as GABARAP. Several studies have shown that exogenous GABARAP can lead to increased surface GABA A R expression (Leil et al, 2004;Chen et al, 2005), but our data provide the first indication of a similar role for the endogenous protein. These results at first seem at odds with data from a GABARAP knock-out mouse that showed no alteration in GABA A R expression (O'Sullivan et al, 2005).…”

C-Terminal Modification Is Required for GABARAP-Mediated GABAA Receptor Trafficking

“…Furthermore, the single channel conductance was significantly higher in L929 cells coexpressing ␣1␤2␥2S subunits with GABARAP than without (Everitt et al, 2004), showing high conductance states similar to those observed in native receptors (Fatima-Shad and Barry, 1992;Curmi et al, 1993;Birnir et al, 1994Birnir et al, , 2001. Recently, we and others reported that overexpression of GABARAP increased the cell-surface number of GABA A receptors (Leil et al, 2004;Boileau et al, 2005;Chen et al, 2005). Immunostaining microscopy showed that GABARAP colocalized with intracellular vesicular GABA A receptors and to some extent with plasma membrane GABA A receptors but not with synaptically localized GABA A receptors and gephyrin (Kneussel et al, 2000;Kittler et al, 2001;Leil et al, 2004).…”

“…Capped mRNA (cRNA) was synthesized by in vitro transcription from Apa I-linearized cDNA constructs using the mMessage Machine kit (Ambion, Austin, TX) as described previously (Chang et al, 2003;Chen et al, 2005). Oocytes were injected with a total volume of 50 nl of cRNA mixed in a ratio of 1:1:2:4 (␣1:␤2:␥2: H 2 O, GABARAP or mutant GABARAP).…”

“…Mutation in GABARAP G116A blocks stimulation of GABA receptor channel expression in X. laevis oocytes We have reported previously that GABARAP increased both GABA currents and cell-surface content of GABA A receptor polypeptides in X. laevis oocytes (Chen et al, 2005). The effects of the Gly116 mutation of GABARAP were studied in this system.…”

Sustained Structural Change of GABAA Receptor-Associated Protein Underlies Long-Term Potentiation at Inhibitory Synapses on a Cerebellar Purkinje Neuron

“…Among them, we attended to GABARAP, a linker protein between GABA A R and tubulin (Wang et al, 1999). The number and/or function of surface GABA A R can be regulated by GABARAP in both heterologous expression systems and neurons (Chen et al, 2000(Chen et al, , 2005Nymann-Andersen et al, 2002;Everitt et al, 2004;Leil et al, 2004;Luu et al, 2006). However, implication of GABARAP in synaptic plasticity has not been explored.…”