A prominent epitope on GABAA receptors is recognized by two different monoclonal antibodies

Ewert, Markus; Blas, Angel L. De; Möhler, Hanns; Seeburg, Peter H.

doi:10.1016/0006-8993(92)90368-j

Cited by 80 publications

(57 citation statements)

References 31 publications

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“…The monoclonal mouse anti-␤ 2/3 (62-3G1) was raised in our laboratory to affinity-purified GABA A R Vitorica et al, 1988). This antibody recognizes an extracellular N-terminus epitope that is common to ␤ 2 and ␤ 3 subunits but is not present in ␤ 1 (Ewert et al, 1992). Antibodies to f usion proteins of the intracellular loops of ␤ 1 , ␤ 2 , and ␤ 3 were also raised in rabbits in our laboratory and affinity purified with purified intracellular loop of the respective isoform (Moreno et al, 1994;Li and De Blas, 1997).…”

Section: Methodsmentioning

confidence: 99%

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABA_AReceptor Clustering in Cultured Hippocampal Neurons

2002

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We have studied the effects of GABAergic innervation on the clustering of GABA A receptors (GABA A Rs) in cultured hippocampal neurons. In the absence of GABAergic innervation, pyramidal cells form small (0.36 Ϯ 0.01 m diameter) GABA A R clusters at their surface in the dendrites and soma. When receiving GABAergic innervation from glutamic acid decarboxylase-containing interneurons, pyramidal cells form large (1.62 Ϯ 0.08 m breadth) GABA A R clusters at GABAergic synapses. This is accompanied by a disappearance of the small GABA A R clusters in the local area surrounding each GABAergic synapse. Although the large synaptic GABA A R clusters of any neuron contained all GABA A R subunits and isoforms expressed by that neuron, the small clusters not localized at GABAergic synapses showed significant heterogeneity in subunit and isoform composition. Another difference between large GABAergic and small non-GABAergic GABA A R clusters was that a significant proportion of the latter was juxtaposed to postsynaptic markers of glutamatergic synapses such as PSD-95 and AMPA receptor GluR1 subunit. The densities of both the glutamate receptor-associated and non-associated small GABA A R clusters were decreased in areas surrounding GABAergic synapses. However, no effect on the density or distribution of glutamate receptor clusters was observed. The results suggest that there are local signals generated at GABAergic synapses that induce both assembly of large synaptic GABA A R clusters at the synapse and disappearance of the small GABA A R clusters in the surrounding area. In the absence of GABAergic innervation, weaker GABA A R-clustering signals, generated at glutamatergic synapses, induce the formation of small postsynaptic GABA A R clusters that remain juxtaposed to glutamate receptors at glutamatergic synapses.

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Section: Methodsmentioning

confidence: 99%

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABA_AReceptor Clustering in Cultured Hippocampal Neurons

2002

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“…The following GABA A R antibodies raised in our laboratory were used: the rabbit and guinea pig anti-rat α1 GABA A R subunit antibodies were to amino acids 1-15 (QPSQDELKDNTTVFT); the rabbit anti-rat α2 GABA A R subunit antibody was to amino acids 417-423 (PVLGVSP); the mouse monoclonal (mAb) anti β2/3 GABA A R subunit antibody (clone 62-3G1) was raised to the affinity purified GABA A receptor Vitorica et al1988), which recognizes an N-terminal epitope present in both β2 and β3 subunits but not in β1 subunit (Ewert et al, 1992); rabbit and guinea pig anti-rat γ2 GABA A R subunit antibodies were to amino acids 1-15 (QKSDDDYEDYASNKT). These anti-GABA A R antibodies have been thoroughly characterized and their specificities determined previously by ELISA, immunoblotting, displacement by antigenic peptide, rat brain immunochemistry at the light microcopy and electron microscopy levels, immunofluorescence of hippocampal cultures and of HEK293 cells transfected with various GABA A R subunits and some knockout and knockdown mutant mice Vitorica et al,1988;Homanics et al, 1999;Miralles et al,1999;Christie et al, 2002a, b;Riquelme et al, 2002;Christie and De Blas 2003;Charych et al, 2004a, b;Chandra et al, 2005;Li et al, 2005a, b;Christie et al, 2006;Serwanski et al, 2006).…”

Section: Antibodiesmentioning

confidence: 99%

Gephyrin clustering is required for the stability of GABAergic synapses

Jiang

Miralles

et al. 2007

Molecular and Cellular Neuroscience

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115

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Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. We report here that knocking down gephyrin expression with small hairpin RNAs (shRNAs) in cultured hippocampal pyramidal cells decreased both the number of gephyrin and GABA(A) receptor clusters. Similar results were obtained by disrupting the clustering of endogenous gephyrin by overexpressing a gephyrin-EGFP fusion protein that formed aggregates with the endogenous gephyrin. Disrupting postsynaptic gephyrin clusters also had transynaptic effects leading to a significant reduction of GABAergic presynaptic boutons contacting the transfected pyramidal cells. Consistent with the morphological decrease of GABAergic synapses, electrophysiological analysis revealed a significant reduction in both the amplitude and frequency of the spontaneous inhibitory postsynaptic currents (sIPSCs). However, no change in the whole-cell GABA currents was detected, suggesting a selective effect of gephyrin on GABA(A) receptor clustering at postsynaptic sites. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses.

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“…The anti-GABA A receptor antibody is specific for the ␤ 2 /␤ 3 -subunits (11,12). Both an avidin-biotin complex (ABC)-horseradish peroxidase technique and fluorescent labeling were used for immunocytochemical detection in rat cerebellum.…”

Section: Methodsmentioning

confidence: 99%

The Neuron-specific K-Cl Cotransporter, KCC2

Williams¹,

Sharp²,

Kumari³

et al. 1999

Journal of Biological Chemistry

211

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The neuron-specific K-Cl cotransporter (KCC2) is hypothesized to function as an active Cl ؊ extrusion pathway important in postsynaptic inhibition mediated by ligand-gated anion channels, like ␥-aminobutyric acid type A (GABA A ) and glycine receptors. To understand better the functional role of KCC2 in the nervous system, we developed polyclonal antibodies to a KCC2 fusion protein and used these antibodies to characterize and localize KCC2 in the rat cerebellum. The antibodies specifically recognized the KCC2 protein which is an ϳ140-kDa glycoprotein detectable only within the central nervous system. The KCC2 protein displayed a robust and punctate distribution in primary cultured retinal amacrine cells known to form exclusively GABA A ergic synapses in culture. In immunolocalization studies, KCC2 was absent from axons and glia but was highly expressed at neuronal somata and dendrites, indicating a specific postsynaptic distribution of the protein. In the granule cell layer, KCC2 exhibited a distinct colocalization with the ␤ 2 /␤ 3 -subunits of the GABA A receptor at the plasma membrane of granule cell somata and at cerebellar glomeruli. KCC2 lightly labeled the plasma membrane of Purkinje cell somata. Within the molecular layer, KCC2 exhibited a distinctly punctate distribution along dendrites, indicating it may be highly localized at inhibitory synapses along these processes. The distinct postsynaptic localization of KCC2 and its colocalization with GABA A receptor in the cerebellum are consistent with the putative role of KCC2 in neuronal Cl ؊ extrusion and postsynaptic inhibition.

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A prominent epitope on GABAA receptors is recognized by two different monoclonal antibodies

Cited by 80 publications

References 31 publications

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABA_AReceptor Clustering in Cultured Hippocampal Neurons

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABA_AReceptor Clustering in Cultured Hippocampal Neurons

Gephyrin clustering is required for the stability of GABAergic synapses

The Neuron-specific K-Cl Cotransporter, KCC2

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A prominent epitope on GABAA receptors is recognized by two different monoclonal antibodies

Cited by 80 publications

References 31 publications

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABAAReceptor Clustering in Cultured Hippocampal Neurons

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABAAReceptor Clustering in Cultured Hippocampal Neurons

Gephyrin clustering is required for the stability of GABAergic synapses

The Neuron-specific K-Cl Cotransporter, KCC2

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Product

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GABAergic Innervation Organizes Synaptic and Extrasynaptic GABA_AReceptor Clustering in Cultured Hippocampal Neurons

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABA_AReceptor Clustering in Cultured Hippocampal Neurons