Ubiquitin-dependent lysosomal targeting of GABA _A receptors regulates neuronal inhibition

Abstract

The strength of synaptic inhibition depends partly on the number of GABA A receptors (GABAARs) found at synaptic sites. The trafficking of GABA ARs within the endocytic pathway is a key determinant of surface GABA AR number and is altered in neuropathologies, such as cerebral ischemia. However, the molecular mechanisms and signaling pathways that regulate this trafficking are poorly understood. Here, we report the subunit specific lysosomal targeting of synaptic GABA ARs. We demonstrate that the targeting of synaptic GABAARs into the degradation pathway is facilitated by ubiquitination of a motif within the intracellular domain of the ␥2 subunit. Blockade of lysosomal activity or disruption of the trafficking of ubiquitinated cargo to lysosomes specifically increases the efficacy of synaptic inhibition without altering excitatory currents. Moreover, mutation of the ubiquitination site within the ␥2 subunit retards the lysosomal targeting of GABA ARs and is sufficient to block the loss of synaptic GABA ARs after anoxic insult. Together, our results establish a previously unknown mechanism for influencing inhibitory transmission under normal and pathological conditions. endocytosis ͉ trafficking ͉ ischemia ͉ ion channels ͉ synapse T he strength of inhibitory synapses is a major determinant of neuronal processing and excitability and can be determined by the number of ␥-amino butyric acid type A receptors (GABA A Rs) found at synaptic sites (1-3). Moreover, in a number of neuropathologies, including status epilepticus and in vitro and in vivo models of cerebral ischemia, the rapid downmodulation of synaptic GABA A Rs has been reported to contribute to pathological disinhibition, excitotoxicity, and cell death (4-8). However, the molecular mechanisms and signaling pathways that regulate GABA A R trafficking, under normal or pathological conditions, remain unclear.GABA A Rs undergo significant rates of endocytosis, a process that can rapidly modify receptor number at synapses (9). Upon internalization, receptors are either reinserted into the plasma membrane or targeted for degradation (10). The balance between these two processes is critical for determining the number of receptors expressed at synapses and hence neuronal excitability (11). However, the regulatory mechanisms that control this endocytic sorting of synaptic GABA A Rs remain unknown.The trafficking of membrane proteins can be regulated via the covalent attachment of the 76 amino acid (aa) peptide, ubiquitin, or ubiquitin-like proteins to lysine residues within target proteins (12,13). A number of studies on the role of ubiquitination in regulating synaptic function under normal conditions and in pathological conditions such as ischemia have focused on the ubiquitin-proteasome system (14-18). However, within the endocytic pathway, mono-ubiquitination (or the addition of short ubiquitin chains) is a key signal that results in the specific targeting of cargo to late endosomes for subsequent lysosomal degradation (12,19,20). The significance of ubiquitinati...

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