Conductance of Recombinant GABA Channels Is Increased in Cells Co-expressing GABAA A Receptor-associated Protein

Abstract

High conductance ␥-aminobutyric acid type A (GABA A ) channels (>40 picosiemens (pS)) have been reported in some studies on GABA A channels in situ but not in others, whereas recombinant GABA A channels do not appear to display conductances above 40 pS. Furthermore, the conductance of some native GABA A channels can be increased by diazepam or pentobarbital, which are effects not reported for expressed GABA A channels. GABARAP, a protein associated with native GABA A channels, has been reported to cause clustering of GABA A receptors and changes in channel kinetics. We have recorded single channel currents activated by GABA in L929 cells expressing ␣ 1 , ␤ 1 , and ␥ 2S subunits of human GABA A receptors. Channel conductance was never higher than 40 pS and was not significantly increased by diazepam or pentobarbital, although open probability was increased. In contrast, in cells expressing the same three subunits together with GABARAP, channel conductance could be significantly higher than 40 pS, and channel conductance was increased by diazepam and pentobarbital. GABARAP caused clustering of receptors in L929 cells, and we suggest that there may be interactions between subunits of clustered GABA A receptors that make them open co-operatively to give high conductance "channels." Recombinant channels may require the influence of GABARAP and perhaps other intracellular proteins to adopt a fuller repertoire of properties of native channels.

GABA Increases both the Conductance and Mean Open Time of Recombinant GABAA Channels Co-expressed with GABARAP

“…At the macroscopic level, changes in the rates of receptor desensitization and deactivation have been reported in addition to an increase in the GABA EC 50 (concentration required to elicit a half-maximal response) (12). At the single channel level we have shown that increased single channel conductances (Ն40 pS) are associated with GABA A receptors co-expressed with GABARAP and that drugs are able to increase the conductance of these channels (9). The aim of the present study was to analyze the single channel properties of GABA A receptors co-expressed with GABARAP and to see if these newly acquired properties correlated with these receptors being organized at a higher density (i.e.…”

“…We have, however, been able to mimic the behavior exhibited by neuronal extrasynaptic GABA A receptors in a recombinant system and change the dispersion of receptors in the membrane simply by co-expressing the trafficking protein GABARAP with GABA A receptors (9). GABARAP (GABA A receptor-associated protein) was originally identified because of its physical association with GABA A receptors following their isolation by immunoprecipitation from solubilized rat brain (10).…”

“…Subsequent immunolocalization data and the biochemical identification of the GABARAP interaction partners has led to the suggestion that it participates in trafficking and membrane fusion events underlying organizational processes at GABAergic synapses but does not remain associated with receptors once they are inserted at the synapse (11). In heterologous expression systems, recombinant GABARAP has been shown to promote clustering of ␥ subunit-containing GABA A receptors in the plasma membrane (9,12). As a consequence of this ordered packing arrangement, the recombinant GABA A receptors function differently from receptors expressed in the absence of GABARAP.…”

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

“…We speculate that functional modulation of GABA A Rs by binding of structurally altered GABARAP is responsible for the RP expression. When GABARAP is expressed with GABA A R composed of ␣1, ␤2/3, and ␥2 subunits in L929 fibroblasts, both single-channel conductance and mean open time of GABA A R are increased (Everitt et al, 2004;Luu et al, 2006). Considering that the majority of GABA A R subunits expressed in a PN are ␣1, ␤2/3, and ␥2, interaction of GABARAP and GABA A R␥2 might upregulate functional properties of GABA A R in a PN.…”

“…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.…”