NMDA receptors regulate GABA _A receptor lateral mobility and clustering at inhibitory synapses through serine 327 on the γ2 subunit

Abstract

Modification of the number of GABA A receptors (GABA A Rs) clustered at inhibitory synapses can regulate inhibitory synapse strength with important implications for information processing and nervous system plasticity and pathology. Currently, however, the mechanisms that regulate the number of GABA A Rs at synapses remain poorly understood. By imaging superecliptic pHluorin tagged GABA A R subunits we show that synaptic GABA A R clusters are normally stable, but that increased neuronal activity upon glutamate receptor (GluR) activation results in their rapid and reversible dispersal. This dispersal correlates with increases in the mobility of single GABA A Rs within the clusters as determined using singleparticle tracking of GABA A Rs labeled with quantum dots. GluRdependent dispersal of GABA A R clusters requires Ca 2+ influx via NMDA receptors (NMDARs) and activation of the phosphatase calcineurin. Moreover, the dispersal of GABA A R clusters and increased mobility of individual GABA A Rs are dependent on serine 327 within the intracellular loop of the GABA A R γ2 subunit. Thus, NMDAR signaling, via calcineurin and a key GABA A R phosphorylation site, controls the stability of synaptic GABA A Rs, with important implications for activitydependent control of synaptic inhibition and neuronal plasticity.ion channels | plasticity | trafficking | diffusion | calcineurin S ynaptic inhibition plays a critical role in regulating neuronal excitability and information processing in the brain. The number of GABA A receptors (GABA A Rs) in the surface membrane and at synaptic sites is an important determinant of inhibitory synapse strength (1), but the mechanisms that rapidly control synaptic GABA A R number and stability remain poorly understood. Activation of Ca 2+ -permeable ionotropic glutamate receptors (GluRs) during plasticity and in pathology can result in down-modulation of inhibitory synapse strength and GABA A R function (2-5) but the molecular and cellular mechanisms underlying GluR-dependent changes in the strength of GABAergic inhibition remain unclear.A major mechanism for modulating GABA A R activity is the direct phosphorylation of residues within the intracellular loops of GABA A R subunits, which can regulate synaptic inhibition, GABA A R channel kinetics, and trafficking (6-9). The rapid movement of neurotransmitter receptors (including GABA A Rs) (10-12) into and out of synapses has also recently emerged as an important mechanism for regulating synaptic strength (13). However, whether GABA A R phosphorylation can directly regulate the synaptic stability of GABA A Rs and their lateral diffusion and movement into and out of synapses is unknown.Here, by live cell imaging of surface GABA A R clusters with pHsensitive superecliptic pHluorin (SEP) and single GABA A Rs with quantum dots (QDs), we investigate the mechanisms that regulate activity-dependent control of the lateral diffusion, clustering, and stability of GABA A Rs at inhibitory synapses. We find that Ca 2+ entry through NMDA receptors (NMDARs) l...

Stabilization of GABAAReceptors at Endocytic Zones Is Mediated by an AP2 Binding Motif within the GABAAReceptor β3 Subunit

“…We found that QD-labeled GABA A Rs could be reversibly recruited to these clathrin-rich structures in which they displayed a reduced lateral mobility compared with receptors outside the EZ, suggesting that the receptors are being confined to these subdomains, before their endocytosis. This is similar to the confined behavior of GABA A Rs observed at the inhibitory postsynaptic site, with similar diffusion coefficient values for the mobility of the receptors previously recorded at the synapse to those observed at EZs (Bannai et al, 2009;Muir et al, 2010). This suggests that both inhibitory postsynaptic sites and EZs are specialized dendritic subdomains that share similar GABA A R confinement mechanisms via reversible interactions with submembranous synaptic or EZ proteins, such as gephyrin and AP2, respectively.…”

“…SEP ␥2 (Superecliptic pHluorin) has been described previously (Muir et al, 2010). Untagged ␣1 was in pRK5 and was described previously (Twelvetrees et al, 2010).…”

“…As a prelude to investigating the role of AP2 adaptor interactions in stabilizing GABA A Rs to EZs, we initially characterized the membrane dynamics of ␤3-subunitcontaining receptors in dendrites with respect to EZs. To test this, we visualized single GABA A R-containing myc-epitope-tagged wildtype GABA A R ( myc ␤3 WT ) subunits, using QD tracking (Muir et al, 2010), a well-established method for following the behavior of single receptors in the surface membrane (Triller and Choquet, 2008). To examine GABA A R mobility using this method in relation to regions of endocytosis in the neuronal plasma membrane, we simultaneously labeled EZs by cotransfecting neurons with YFP-tagged clathrin light chain, which has been used previously to label these subdomains in dendrites (Blanpied et al, 2002;Petrini et al, 2009).…”

“…GABA A Rs are dynamic entities, in terms of both their lateral mobility at the plasma membrane (Bannai et al, 2009;Muir et al, 2010) and their trafficking to and from the cell surface . Clathrin-mediated endocytosis of GABA A Rs is dependent on the clathrin-adaptor AP2 and rapidly modulates synaptic GABA A R number, inhibitory synaptic strength, neuronal excitability, and, ultimately, animal behavior (Kittler et al, 2000(Kittler et al, , 2005Chen et al, 2006;Kurotani et al, 2008;Tretter et al, 2009).…”

Chronic treatment with serotonin reuptake inhibitor antidepressant (SSRI) combined with an antipsychotic regulates GABA-A receptor in rat prefrontal cortex

“…HALO-FLU may regulate GABA-A receptor activity by altering the number of GABA-A receptors available to GABAergic ligands on the cell membrane, resulting in increased GABAergic transmission, GABA release, and compensatory downregulation of GABA-A receptors (Kumar et al 2005). There is evidence that GABAergic deficit in PFC of schizophrenia patients is accompanied by compensatory upregulation of GABA-A receptors (Wassef et al 2003), so downregulation of GABA-A receptors, induced by chronic HALO-FLU, might follow an increase in GABAergic activity, consistent with evidence of dynamic changes in GABA receptor localization (Muir et al 2010). …”