using-an-bungarotoxin-binding-site-tag-to-study-gaba-receptor

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

University of Pittsburgh School of Medicine

Proneuropeptides are packaged into dense-core vesicles in which they are processed into active peptides by copackaged enzymes. Proprotein convertases (PCs) cleave precursors after dibasic residues, and carboxypeptidases remove basic residues from the C terminals. We show here that the Caenorhabditis elegans egl-21 gene encodes a protein that is very similar to carboxypeptidase E (CPE) and is broadly expressed in the nervous system. Mutants lacking either egl-21 CPE or egl-3, which encodes the C. elegans ortholog of PC type 2 (PC2), were defective for processing endogenously expressed FMRFamide (Phe-Met-Arg-Phe-NH2)-related peptides (FaRPs). Mutants lacking the unc-104 kinesin motor protein were defective for anterograde movement of dense-core vesicle components, including egl-3 PC2, egl-21 CPE, and FaRPs. We provide evidence that egl-3 PC2 and egl-21 CPE mutants have diminished acetylcholine release at neuromuscular junctions (NMJs). Taken together, these results suggest that egl-21 CPE and egl-3 PC2 process endogenous neuropeptides that facilitate acetylcholine release at C. elegans NMJs.

The EGL-21 carboxypeptidase E facilitates acetylcholine release at Caenorhabditis elegans neuromuscular junctions.

The efficacy of fast synaptic inhibition is critically dependent on the accumulation of GABAA receptors at inhibitory synapses, a process that remains poorly understood. Here, we examined the dynamics of cell surface GABAA receptors using receptor subunits modified with N-terminal extracellular ecliptic pHluorin reporters. In hippocampal neurons, GABAA 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 GABAA 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 GABAA 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 GABAA receptors have lower levels of lateral mobility compared with their extrasynaptic counterparts, and suggest a specific role for gephyrin in reducing the diffusion of GABAA receptors, facilitating their accumulation at inhibitory synapses.

Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors.

GABA(A) receptors (GABA(A)Rs) are the major mediators of fast synaptic inhibition in the brain. In neurons, these receptors undergo significant rates of endocytosis and exocytosis, processes that regulate both their accumulation at synaptic sites and the efficacy of synaptic inhibition. Here we have evaluated the role that neuronal activity plays in regulating the residence time of GABA(A)Rs on the plasma membrane and their targeting to synapses. Chronic blockade of neuronal activity dramatically increases the level of the GABA(A)R ubiquitination, decreasing their cell surface stability via a mechanism dependent on the activity of the proteasome. Coincident with this loss of cell surface expression levels, TTX treatment reduced both the amplitude and frequency of miniature inhibitory synaptic currents. Conversely, increasing the level of neuronal activity decreases GABA(A)R ubiquitination enhancing their stability on the plasma membrane. Activity-dependent ubiquitination primarily acts to reduce GABA(A)R stability within the endoplasmic reticulum and, thereby, their insertion into the plasma membrane and subsequent accumulation at synaptic sites. Thus, activity-dependent ubiquitination of GABA(A)Rs and their subsequent proteasomal degradation may represent a potent mechanism to regulate the efficacy of synaptic inhibition and may also contribute to homeostatic synaptic plasticity.

Activity-dependent ubiquitination of GABA(A) receptors regulates their accumulation at synaptic sites.

Classical benzodiazepine sensitive GABA(A) receptor subtypes, the major mediators of fast synaptic inhibition in the brain are heteropentamers that can be assembled from alpha1-3/5, beta1-3, and gamma2 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 alpha2 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 alpha2 subunits to gephyrin in vitro. Our results thus reveal that the specific accumulation of GABA(A) receptor subtypes containing alpha2 subunits at inhibitory synapses is dependent on their ability to bind gephyrin.

The clustering of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor alpha 2 subunits to gephyrin.

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.

GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition.

GABA(A) receptors (GABA(A)Rs), 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 beta3 subunits (beta3S408/9A), which have reduced binding to the clathrin adaptor protein-2, a critical regulator of GABA(A)R endocytosis. Neurons expressing beta3S408/9A subunits exhibited increases in the number and size of inhibitory synapses, together with enhanced inhibitory synaptic transmission due to reduced GABA(A)R endocytosis. Furthermore, neurons expressing beta3S408/9A 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 GABA(A)Rs. Therefore, regulating the efficacy of synaptic inhibition by modulating GABA(A)R membrane trafficking may play a critical role in regulating spine maturity with significant implications for synaptic plasticity together with behavior.

GABA(A) receptor membrane trafficking regulates spine maturity.

Benzodiazepines potentiate γ-aminobutyric acid type A receptor (GABA(A)R) activity and are widely prescribed to treat anxiety, insomnia, and seizure disorders. Unfortunately, clinical use of benzodiazepines (BZs) is severely limited by tolerance. The mechanisms leading to BZ tolerance are unknown. BZs bind at the interface between an α and γ subunit of GABA(A)Rs, preferentially enhancing synaptic receptors largely composed of α(1-3, 5), β3, and γ2 subunits. Using confocal imaging and patch-clamp approaches, we show that treatment with the BZ flurazepam decreases GABA(A)R surface levels and the efficacy of neuronal inhibition in hippocampal neurons. A dramatic decrease in surface and total levels of α2 subunit-containing GABA(A)Rs occurred within 24 h of flurazepam treatment, whereas GABA(A)Rs incorporating α1 subunits showed little alteration. The GABA(A)R surface depletion could be reversed by treatment with the BZ antagonist Ro 15-1788. Coincident with decreased GABA(A)R surface levels, flurazepam treatment reduced miniature inhibitory postsynaptic current amplitude, which returned to control levels with acute Ro 15-1788 treatment. GABA(A)R endocytosis and insertion rates were unchanged by flurazepam treatment. Treatment with leupeptin restored flurazepam lowered receptor surface levels, strongly suggesting that flurazepam increases lysosomal degradation of GABA(A)Rs. Together, these data suggest that flurazepam exposure enhances degradation of α2 subunit-containing GABA(A)Rs after their removal from the plasma membrane, leading to a reduction in inhibitory synapse size and number along with a decrease in the efficacy of synaptic inhibition. These reported subtype-specific changes in GABA(A)R trafficking provide significant mechanistic insight into the initial neuroadaptive responses occurring with BZ treatment.

Article	Year
The EGL-21 carboxypeptidase E facilitates acetylcholine release at Caenorhabditis elegans neuromuscular junctions. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 12657671	2003
Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 16280585	2005
Activity-dependent ubiquitination of GABA(A) receptors regulates their accumulation at synaptic sites. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 18045928	2007
The clustering of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor alpha 2 subunits to gephyrin. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 18256255	2008
GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition. Nature reviews. Neuroscience\| PubMed ID: 18382465	2008
GABA(A) receptor membrane trafficking regulates spine maturity. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19617557	2009
Benzodiazepine treatment induces subtype-specific changes in GABA(A) receptor trafficking and decreases synaptic inhibition. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23091016	2012

Tija C. Jacob

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