GABAAReceptor-Associated Protein Traffics GABAAReceptors to the Plasma Membrane in Neurons

Abstract

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 γ2 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 γ2 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 γ2-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 γ2 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.