GABA (gamma-aminobutyric acid) type A receptors (GABA(A)Rs) mediate most fast synaptic inhibition in the mammalian brain, controlling activity at both the network and the cellular levels. The diverse functions of GABA in the CNS are matched not just by the heterogeneity of GABA(A)Rs, but also by the complex trafficking mechanisms and protein-protein interactions that generate and maintain an appropriate receptor cell-surface localization. In this Review, we discuss recent progress in our understanding of the dynamic regulation of GABA(A)R composition, trafficking to and from the neuronal surface, and lateral movement of receptors between synaptic and extrasynaptic locations. Finally, we highlight a number of neurological disorders, including epilepsy and schizophrenia, in which alterations in GABA(A)R trafficking occur.
The efficacy of fast synaptic inhibition is critically dependent on the accumulation of GABA A receptors at inhibitory synapses, a process that remains poorly understood. Here, we examined the dynamics of cell surface GABA A receptors using receptor subunits modified with N-terminal extracellular ecliptic pHluorin reporters. In hippocampal neurons, GABA A receptors incorporating pHluorin-tagged subunits were found to be clustered at synaptic sites and also expressed as diffuse extrasynaptic staining. By combining FRAP (fluorescence recovery after photobleaching) measurements with live imaging of FM4-64-labeled active presynaptic terminals, it was evident that clustered synaptic receptors exhibit significantly lower rates of mobility at the cell surface compared with their extrasynaptic counterparts. To examine the basis of this confinement, we used RNAi to inhibit the expression of gephyrin, a protein shown to regulate the accumulation of GABA A receptors at synaptic sites. However, whether gephyrin acts to control the actual formation of receptor clusters, their stability, or is simply a global regulator of receptor cell surface number remains unknown. Inhibiting gephyrin expression did not modify the total number of GABA A receptors expressed on the neuronal cell surface but significantly decreased the number of receptor clusters. Live imaging revealed that clusters that formed in the absence of gephyrin were significantly more mobile compared with those in control neurons. Together, our results demonstrate that synaptic GABA A receptors have lower levels of lateral mobility compared with their extrasynaptic counterparts, and suggest a specific role for gephyrin in reducing the diffusion of GABA A receptors, facilitating their accumulation at inhibitory synapses.
Postsynaptic long-term potentiation of inhibition (iLTP) can rely on increased GABAA receptors (GABAARs) at synapses by promoted exocytosis. However, the molecular mechanisms that enhance the clustering of postsynaptic GABAARs during iLTP remain obscure. Here we demonstrate that during chemically induced iLTP (chem-iLTP), GABAARs are immobilized and confined at synapses, as revealed by single-particle tracking of individual GABAARs in cultured hippocampal neurons. Chem-iLTP expression requires synaptic recruitment of the scaffold protein gephyrin from extrasynaptic areas, which in turn is promoted by CaMKII-dependent phosphorylation of GABAAR-β3-Ser383. Impairment of gephyrin assembly prevents chem-iLTP and, in parallel, blocks the accumulation and immobilization of GABAARs at synapses. Importantly, an increase of gephyrin and GABAAR similar to those observed during chem-iLTP in cultures were found in the rat visual cortex following an experience-dependent plasticity protocol that potentiates inhibitory transmission in vivo. Thus, phospho-GABAAR-β3-dependent accumulation of gephyrin at synapses and receptor immobilization are crucial for iLTP expression and are likely to modulate network excitability.
Classical benzodiazepine sensitive GABA A receptor subtypes, the major mediators of fast synaptic inhibition in the brain are heteropentamers that can be assembled from ␣1-3/5, 1-3, and ␥2 subunits, but how neurons orchestrate their selective accumulation at synapses remains obscure. We have identified a 10 amino acid hydrophobic motif within the intracellular domain of the ␣2 subunit that regulates the accumulation of GABA A receptors at inhibitory synaptic sites on both axon initial segments and dendrites in a mechanism dependent on the inhibitory scaffold protein gephyrin. This motif was sufficient to target CD4 (cluster of differentiation molecule 4) molecules to inhibitory synapses, and was also critical in regulating the direct binding of ␣2 subunits to gephyrin in vitro. Our results thus reveal that the specific accumulation of GABA A receptor subtypes containing ␣2 subunits at inhibitory synapses is dependent on their ability to bind gephyrin.
Neuroactive peptides are packaged as proproteins into dense core vesicles or secretory granules, where they are cleaved at dibasic residues by copackaged proprotein convertases. We show here that the Caenorhabditis elegans egl-3 gene encodes a protein that is 57% identical to mouse proprotein convertase type 2 (PC2), and we provide evidence that this convertase regulates mechanosensory responses. Nose touch sensitivity (mediated by ASH sensory neurons) is defective in mutants lacking GLR-1 glutamate receptors (GluRs); however, mutations eliminating the egl-3 PC2 restored nose touch sensitivity to glr-1 GluR mutants. By contrast, body touch sensitivity (mediated by the touch cells) is greatly diminished in egl-3 PC2 mutants. Taken together, these results suggest that egl-3 PC2-processed peptides normally regulate the responsiveness of C. elegans to mechanical stimuli.
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