Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABA
            <sub>A</sub>
            receptor γ2 subunit

Long-term whisker removal alters the balance of excitation and inhibition in rodent barrel cortex, yet little is known about the contributions of individual cells and synapses in this process. We studied synaptic inhibition in four major types of neurons in live tangential slices that isolate layer 4 in the posteromedial barrel subfield. Voltage-clamp recordings of layer 4 neurons reveal that fast decay of synaptic inhibition requires ␣1-containing GABAA receptors. After 7 weeks of deprivation, we found that GABA A-receptor-mediated inhibitory postsynaptic currents (IPSCs) in the inhibitory lowthreshold-spiking (LTS) cell recorded in deprived barrels exhibited faster decay kinetics and larger amplitudes in whisker-deprived barrels than those in nondeprived barrels in age-matched controls. This was not observed in other cell types. Additionally, IPSCs recorded in LTS cells from deprived barrels show a marked increase in zolpidem sensitivity. To determine if the faster IPSC decay in LTS cells from deprived barrels indicates an increase in ␣1 subunit functionality, we deprived ␣1(H101R) mutant mice with zolpidem-insensitive ␣1-containing GABA A receptors. In these mice and matched wild-type controls, IPSC decay kinetics in LTS cells were faster after whisker removal; however, the deprivation-induced sensitivity to zolpidem was reduced in ␣1(H101R) mice. These data illustrate a change of synaptic inhibition in LTS cells via an increase in ␣1-subunit-mediated function. Because ␣1 subunits are commonly associated with circuitspecific plasticity in sensory cortex, this switch in LTS cell synaptic inhibition may signal necessary circuit changes required for plastic adjustments in sensory-deprived cortex.interneurons ͉ networks ͉ plasticity S ynaptic inhibition is involved in sensory processing at the very first stage of cortical integration in layer 4 of the rodent primary somatosensory (barrel) cortex (1). Both feedforward and feedback inhibition from local circuit inhibitory neurons regulates the integration of thalamic and intracortical inputs (2, 3). Inhibitory neurons are distinguished from each other by action potential firing properties, morphology, and biochemical expression (4, 5). Cortical inhibitory neurons are largely separated into two functional circuits that are thought to carry out two intertwined yet separate functions in sensory processing (6). Fast-spiking (FS) cells are either basket cells or chandelier cells and express parvalbumin (4). They are readily activated by thalamic afferents and are the primary mediator of thalamocortical feedforward inhibition, and recurrent synaptic inhibition onto FS cells is very fast (6, 7). Low-threshold-spiking (LTS) cells express varying combinations of somatostatin, vasoactive intestinal peptide, and cholecystokinin (4), are weakly activated by thalamic afferents, and contribute to intracortical inhibitory transmission, and recurrent synaptic inhibition onto LTS cells decays slowly (7,8).In cortex and other regions, an emerging feature in brain networks is the ''G...

Section: Long-term Sensory Deprivation Causes a Switch In Functional contrasting

confidence: 69%

Long-term sensory deprivation selectively rearranges functional inhibitory circuits in mouse barrel cortex

Rudolph

Huntsman

2009

“…Having established that lysosomal degradation of GABA A Rs is an important determinant of the number of surface and synaptic GABA A Rs, we next set out to identify the molecular mechanisms that mediate lysosomal targeting of GABA A Rs. Because the ␥2 subunit is an important determinant for the trafficking of GABA A Rs, and their entry into the endocytic pathway (22)(23)(24)(25), we examined the possible role that this subunit plays in receptor lysosomal targeting. We compared the lysosomal targeting in HEK-293 cells of GABA A Rs composed of ␣␤ and ␣␤␥2 subunits modified with N-terminal 9E10 reporters.…”

Section: Identification Of a Sorting Motif In The Gabaar ␥2 Subunit Thatmentioning

confidence: 99%

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

Arancibia-Cárcamo

Yuen

Muir

et al. 2009

Self Cite

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...

“…Recruitment of GABA A Rs into the endocytic pathway is facilitated via the interactions of the intracellular domains of receptor ␤1-3 and ␥2 subunits with 2-AP2 (7,11). In vitro studies identified the presence of a basic patch 2-AP2 binding motif between residues 401-412 for the ␤3 subunit (7) and showed that phosphorylation of S408/9 sites, the principal sites of phosphorylation for cAMPdependent protein kinase (PKA) and protein kinase C (1), inhibits binding of 2-AP2 to the ␤3 subunit.…”

Section: Discussionmentioning

confidence: 99%

“…3A). mIPSCs were isolated by the inclusion of TTX, DAP5, and CNQX to block voltage-gated sodium channels, and glutamate receptors, respectively (4,11). Under these conditions, the remaining events were blocked by picrotoxin confirming that they arise via the activation of GABA A Rs (Fig.…”

Section: Mutation Of S408/9 Slows Endocytosis Of the ␤3 Subunit In Hementioning

confidence: 91%

GABA _A receptor membrane trafficking regulates spine maturity

Jacob

Wan²,

Vithlani³

et al. 2009

Self Cite

GABAA receptors (GABAARs), the principal sites of synaptic inhibition in the brain, are dynamic entities on the neuronal cell surface, but the role their membrane trafficking plays in shaping neuronal activity remains obscure. Here, we examined this by using mutant receptor ␤3 subunits (␤3S408/9A), which have reduced binding to the clathrin adaptor protein-2, a critical regulator of GABAAR endocytosis. Neurons expressing ␤3S408/9A subunits exhibited increases in the number and size of inhibitory synapses, together with enhanced inhibitory synaptic transmission due to reduced GABAAR endocytosis. Furthermore, neurons expressing ␤3S408/9A subunits had deficits in the number of mature spines and reduced accumulation of postsynaptic density protein-95 at excitatory synapses. This deficit in spine maturity was reversed by pharmacological blockade of GABAARs. Therefore, regulating the efficacy of synaptic inhibition by modulating GABAAR membrane trafficking may play a critical role in regulating spine maturity with significant implications for synaptic plasticity together with behavior.endocytosis ͉ inhibition ͉ phosphorylation F ast neuronal inhibition in the brain is largely mediated by GABA A Rs, which are Cl-selective pentameric ligand-gated ion channels assembled from 7 subunit classes with multiple members; ␣ (1-6), ␤ (1-3), ␥ (1-3), ␦, , , and . Consensus opinion suggests that the majority of synaptic receptor subtypes are assembled from ␣1-3, ␤, and ␥2 subunits. The accumulation of GABA A Rs on the neuronal membrane is a critical factor in determining synaptic inhibition and is largely dependent on rates of receptor exo-and endocytosis (1). After ER assembly, GABA A Rs are inserted in the neuronal plasma membrane primarily at extrasynaptic sites and access the inhibitory postsynaptic specialization via lateral diffusion where they are stabilized via interaction with complements of the subsynaptic cytoskeleton (2, 3). Extrasynaptic receptor populations exhibit short cell surface half-lives and are rapidly removed from the plasma membrane via clathrin dependent-endocytosis (2, 4).The endocytosis of GABA A Rs is facilitated via the direct binding of the intracellular domains of receptor ␤1-3, ␥1-2, and ␦ subunits to the 2 adaptin of the AP2 complex (2-AP2) (5, 6). Subsequent analysis has revealed the presence of a conserved basic patchbinding motif for 2-AP2 within all receptor ␤ subunits (7), which contains the principal sites of phosphorylation within GABA A Rs for cAMP-dependent protein kinase and protein kinase C, residues S408/9 respectively (1). In keeping with this, in vitro studies showed reduced binding of the GST-␤3 intracellular domain (ICD) to 2-AP2 with phosphorylation at S408/9 (7).To determine the significance of regulated GABA A R endocytosis in a neuronal context and the physiological consequence of altered GABA A R cell surface levels, we expressed receptor ␤3 subunits in which S408/9 have been mutated to alanines in neurons. This mutation dramatically increased the synaptic accumulation of GABA ...