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GABA(A)R, Beta3 antibody

RRID:AB_10673389

Antibody ID

AB_10673389

Target Antigen

GABA(A)R Beta3 null

Proper Citation

(UC Davis/NIH NeuroMab Facility Cat# 73-149, RRID:AB_10673389)

Clonality

monoclonal antibody

Comments

Originating manufacturer of this product. Applications: IB, ICC, IHC, WB. Validation status: IF or IB (Pass), IB in brain (Pass), IHC in brain (Pass), KO (ND).

Clone ID

N87/25

Host Organism

mouse

Vendor

UC Davis/NIH NeuroMab Facility Go To Vendor

Cat Num

73-149

SHANK3 overexpression causes manic-like behaviour with unique pharmacogenetic properties.

  • Han K
  • Nature
  • 2013 Nov 7

Literature context:


Abstract:

Mutations in SHANK3 and large duplications of the region spanning SHANK3 both cause a spectrum of neuropsychiatric disorders, indicating that proper SHANK3 dosage is critical for normal brain function. However, SHANK3 overexpression per se has not been established as a cause of human disorders because 22q13 duplications involve several genes. Here we report that Shank3 transgenic mice modelling a human SHANK3 duplication exhibit manic-like behaviour and seizures consistent with synaptic excitatory/inhibitory imbalance. We also identified two patients with hyperkinetic disorders carrying the smallest SHANK3-spanning duplications reported so far. These findings indicate that SHANK3 overexpression causes a hyperkinetic neuropsychiatric disorder. To probe the mechanism underlying the phenotype, we generated a Shank3 in vivo interactome and found that Shank3 directly interacts with the Arp2/3 complex to increase F-actin levels in Shank3 transgenic mice. The mood-stabilizing drug valproate, but not lithium, rescues the manic-like behaviour of Shank3 transgenic mice raising the possibility that this hyperkinetic disorder has a unique pharmacogenetic profile.

Funding information:
  • Medical Research Council - G0800578(United Kingdom)

Defining the diversity of phenotypic respecification using multiple cell lines and reprogramming regimens.

  • Alicea B
  • Stem Cells Dev.
  • 2013 Oct 1

Literature context:


Abstract:

To better understand the basis of variation in cellular reprogramming, we performed experiments with two primary objectives: first, to determine the degree of difference, if any, in reprogramming efficiency among cells lines of a similar type after accounting for technical variables, and second, to compare the efficiency of conversion of multiple similar cell lines to two separate reprogramming regimens-induced neurons and induced skeletal muscle. Using two reprogramming regimens, it could be determined whether converted cells are likely derived from a distinct subpopulation that is generally susceptible to reprogramming or are derived from cells with an independent capacity for respecification to a given phenotype. Our results indicated that when technical components of the reprogramming regimen were accounted for, reprogramming efficiency was reproducible within a given primary fibroblast line but varied dramatically between lines. The disparity in reprogramming efficiency between lines was of sufficient magnitude to account for some discrepancies in published results. We also found that the efficiency of conversion to one phenotype was not predictive of reprogramming to the alternate phenotype, suggesting that the capacity for reprogramming does not arise from a specific subpopulation with a generally "weak grip" on cellular identity. Our findings suggest that parallel testing of multiple cell lines from several sources may be needed to accurately assess the efficiency of direct reprogramming procedures, and that testing a larger number of fibroblast lines--even lines with similar origins--is likely the most direct means of improving reprogramming efficiency.

Funding information:
  • NHGRI NIH HHS - R01 HG005238(United States)

Altered cortical GABAA receptor composition, physiology, and endocytosis in a mouse model of a human genetic absence epilepsy syndrome.

  • Zhou C
  • J. Biol. Chem.
  • 2013 Jul 19

Literature context:


Abstract:

Patients with generalized epilepsy exhibit cerebral cortical disinhibition. Likewise, mutations in the inhibitory ligand-gated ion channels, GABAA receptors (GABAARs), cause generalized epilepsy syndromes in humans. Recently, we demonstrated that heterozygous knock-out (Hetα1KO) of the human epilepsy gene, the GABAAR α1 subunit, produced absence epilepsy in mice. Here, we determined the effects of Hetα1KO on the expression and physiology of GABAARs in the mouse cortex. We found that Hetα1KO caused modest reductions in the total and surface expression of the β2 subunit but did not alter β1 or β3 subunit expression, results consistent with a small reduction of GABAARs. Cortices partially compensated for Hetα1KO by increasing the fraction of residual α1 subunit on the cell surface and by increasing total and surface expression of α3, but not α2, subunits. Co-immunoprecipitation experiments revealed that Hetα1KO increased the fraction of α1 subunits, and decreased the fraction of α3 subunits, that associated in hybrid α1α3βγ receptors. Patch clamp electrophysiology studies showed that Hetα1KO layer VI cortical neurons exhibited reduced inhibitory postsynaptic current peak amplitudes, prolonged current rise and decay times, and altered responses to benzodiazepine agonists. Finally, application of inhibitors of dynamin-mediated endocytosis revealed that Hetα1KO reduced base-line GABAAR endocytosis, an effect that probably contributes to the observed changes in GABAAR expression. These findings demonstrate that Hetα1KO exerts two principle disinhibitory effects on cortical GABAAR-mediated inhibitory neurotransmission: 1) a modest reduction of GABAAR number and 2) a partial compensation with GABAAR isoforms that possess physiological properties different from those of the otherwise predominant α1βγ GABAARs.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/J010375/1(United Kingdom)

Unique somato-dendritic distribution pattern of Kv4.2 channels on hippocampal CA1 pyramidal cells.

  • Kerti K
  • Eur. J. Neurosci.
  • 2012 May 7

Literature context:


Abstract:

A-type K(+) current (I(A)) plays a critical role in controlling the excitability of pyramidal cell (PC) dendrites. In vitro dendritic patch-pipette recordings have demonstrated a prominent, sixfold increase in I(A) density along the main apical dendrites of rat hippocampal CA1 PCs. In these cells, I(A) is mediated by Kv4.2 subunits, whose precise subcellular distribution and densities in small-diameter oblique dendrites and dendritic spines are still unknown. Here we examined the densities of the Kv4.2 subunit in 13 axo-somato-dendritic compartments of CA1 PCs using a highly sensitive, high-resolution quantitative immunogold localization method (sodium dodecyl sulphate-digested freeze-fracture replica-labelling). Only an approximately 70% increase in Kv4.2 immunogold density was observed along the proximo-distal axis of main apical dendrites in the stratum radiatum with a slight decrease in density in stratum lacunosum-moleculare. A similar pattern was detected for all dendritic compartments, including main apical dendrites, small-diameter oblique dendrites and dendritic spines. The specificity of the somato-dendritic labelling was confirmed in Kv4.2(-/-) tissue. No specific immunolabelling for the Kv4.2 subunit was found in SNAP-25-containing presynaptic axons. Our results demonstrate a novel distribution pattern of a voltage-gated ion channel along the somato-dendritic surface of CA1 PCs, and suggest that the increase in the I(A) along the proximo-distal axis of PC dendrites cannot be solely explained by a corresponding increase in Kv4.2 channel number.

Funding information:
  • NIA NIH HHS - P01 AG009975(United States)

GABRB3 mutation, G32R, associated with childhood absence epilepsy alters α1β3γ2L γ-aminobutyric acid type A (GABAA) receptor expression and channel gating.

  • Gurba KN
  • J. Biol. Chem.
  • 2012 Apr 6

Literature context:


Abstract:

A GABA(A) receptor β3 subunit mutation, G32R, has been associated with childhood absence epilepsy. We evaluated the possibility that this mutation, which is located adjacent to the most N-terminal of three β3 subunit N-glycosylation sites, might reduce GABAergic inhibition by increasing glycosylation of β3 subunits. The mutation had three major effects on GABA(A) receptors. First, coexpression of β3(G32R) subunits with α1 or α3 and γ2L subunits in HEK293T cells reduced surface expression of γ2L subunits and increased surface expression of β3 subunits, suggesting a partial shift from ternary αβ3γ2L receptors to binary αβ3 and homomeric β3 receptors. Second, β3(G32R) subunits were more likely than β3 subunits to be N-glycosylated at Asn-33, but increases in glycosylation were not responsible for changes in subunit surface expression. Rather, both phenomena could be attributed to the presence of a basic residue at position 32. Finally, α1β3(G32R)γ2L receptors had significantly reduced macroscopic current density. This reduction could not be explained fully by changes in subunit expression levels (because γ2L levels decreased only slightly) or glycosylation (because reduction persisted in the absence of glycosylation at Asn-33). Single channel recording revealed that α1β3(G32R)γ2L receptors had impaired gating with shorter mean open time. Homology modeling indicated that the mutation altered salt bridges at subunit interfaces, including regions important for subunit oligomerization. Our results suggest both a mechanism for mutation-induced hyperexcitability and a novel role for the β3 subunit N-terminal α-helix in receptor assembly and gating.

Funding information:
  • NINDS NIH HHS - 5R01NS056307-08(United States)

Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABA(A) receptors in cortical pyramidal cells.

  • Sumegi M
  • J. Physiol. (Lond.)
  • 2012 Apr 1

Literature context:


Abstract:

Recently developed pharmacogenetic and optogenetic approaches, with their own advantages and disadvantages, have become indispensable tools in modern neuroscience. Here, we employed a previously described knock-in mouse line (GABA(A)Rγ2(77I)lox) in which the γ2 subunit of the GABA(A) receptor (GABA(A)R) was mutated to become zolpidem insensitive (γ2(77I)) and used viral vectors to swap γ2(77I) with wild-type, zolpidem-sensitive γ2 subunits (γ2(77F)). The verification of unaltered density and subcellular distribution of the virally introduced γ2 subunits requires their selective labelling. For this we generated six N- and six C-terminal-tagged γ2 subunits, with which cortical cultures of GABA(A)Rγ2(−/−) mice were transduced using lentiviruses. We found that the N-terminal AU1 tag resulted in excellent immunodetection and unimpaired synaptic localization. Unaltered kinetic properties of the AU1-tagged γ2 ((AU1)γ2(77F)) channels were demonstrated with whole-cell patch-clamp recordings of spontaneous IPSCs from cultured cells. Next, we carried out stereotaxic injections of lenti- and adeno-associated viruses containing Cre-recombinase and the (AU1)γ2(77F) subunit (Cre-2A-(AU1)γ2(77F)) into the neocortex of GABA(A)Rγ2(77I)lox mice. Light microscopic immunofluorescence and electron microscopic freeze-fracture replica immunogold labelling demonstrated the efficient immunodetection of the AU1 tag and the normal enrichment of the (AU1)γ2(77F) subunits in perisomatic GABAergic synapses. In line with this,miniature and action potential-evoked IPSCs whole-cell recorded from transduced cells had unaltered amplitudes, kinetics and restored zolpidem sensitivity. Our results obtained with a wide range of structural and functional verification methods reveal unaltered subcellular distributions and functional properties of γ2(77I) and (AU1)γ2(77F) GABA(A)Rs in cortical pyramidal cells. This transgenic–viral pharmacogenetic approach has the advantage that it does not require any extrinsic protein that might endow some unforeseen alterations of the genetically modified cells. In addition, this virus-based approach opens up the possibility of modifying multiple cell types in distinct brain regions and performing alternative recombination-based intersectional genetic manipulations.

Funding information:
  • NIMH NIH HHS - R37 MH063394(United States)

Setting the time course of inhibitory synaptic currents by mixing multiple GABA(A) receptor α subunit isoforms.

  • Eyre MD
  • J. Neurosci.
  • 2012 Apr 25

Literature context:


Abstract:

The kinetics of IPSCs influence many neuronal processes, such as the frequencies of oscillations and the duration of shunting inhibition. The subunit composition of recombinant GABA(A) receptors (GABA(A)Rs) strongly affects the deactivation kinetics of GABA-evoked currents. However, for GABAergic synapses, the relationship between subunit composition and IPSC decay is less clear. Here we addressed this by combining whole-cell recordings of miniature IPSCs (mIPSCs) and quantitative immunolocalization of synaptic GABA(A)R subunits. In cerebellar stellate, thalamic relay, and main olfactory bulb (MOB) deep short-axon cells of Wistar rats, the only synaptic α subunit was α1, and zolpidem-sensitive mIPSCs had weighted decay time constants (τ(w)) of 4-6 ms. Nucleus reticularis thalami neurons expressed only α3 as the synaptic α subunit and exhibited slow (τ(w) = 28 ms), zolpidem-insensitive mIPSCs. By contrast, MOB external tufted cells contained two α subunit types (α1 and α3) at their synapses. Quantitative analysis of multiple immunolabeled images revealed small within-cell, but large between-cell, variability in synaptic α1/α3 ratios. This corresponded to large cell-to-cell variability in the decay (τ(w) = 3-30 ms) and zolpidem sensitivity of mIPSCs. Currents evoked by rapid application of GABA to patches excised from HEK cells expressing different mixtures of α1 and α3 subunits displayed highly variable deactivation times that correlated with the α1/α3 cDNA ratio. Our results demonstrate that diversity in the decay of IPSCs can be generated by varying the expression of different GABA(A)R subunits that alone confer different decay kinetics, allowing the time course of inhibition to be tuned to individual cellular requirements.

Funding information:
  • NHLBI NIH HHS - 1K25HL68704(United States)

Cross-talk between P2X4 and gamma-aminobutyric acid, type A receptors determines synaptic efficacy at a central synapse.

  • Jo YH
  • J. Biol. Chem.
  • 2011 Jun 3

Literature context:


Abstract:

The essence of neuronal function is to generate outputs in response to synaptic potentials. Synaptic integration at postsynaptic sites determines neuronal outputs in the CNS. Using immunohistochemical and electrophysiological approaches, we first reveal that steroidogenic factor 1 (SF-1) green fluorescent protein (GFP)-positive neurons in the ventromedial nucleus of the hypothalamus express P2X4 subunits that are activated by exogenous ATP. Increased membrane expression of P2X4 channels by using a peptide competing with P2X4 intracellular endocytosis motif enhances neuronal excitability of SF-1 GFP-positive neurons. This increased excitability is inhibited by a P2X receptor antagonist. Furthermore, increased surface P2X4 receptor expression significantly decreases the frequency and the amplitude of GABAergic postsynaptic currents of SF-1 GFP-positive neurons. Co-immunopurification and pulldown assays reveal that P2X4 receptors complex with aminobutyric acid, type A (GABA(A)) receptors and demonstrate that two amino acids in the carboxyl tail of the P2X4 subunit are crucial for its physical association with GABA(A) receptors. Mutation of these two residues prevents the physical association, thereby blocking cross-inhibition between P2X4 and GABA(A) receptors. Moreover, disruption of the physical coupling using competitive peptides containing the identified motif abolishes current inhibition between P2X4 and GABA(A) receptors in recombinant system and P2X4 receptor-mediated GABAergic depression in SF-1 GFP-positive neurons. Our present work thus provides evidence for cross-talk between excitatory and inhibitory receptors that appears to be crucial in determining GABAergic synaptic strength at a central synapse.

Funding information:
  • NCRR NIH HHS - P 41 RR08605-06(United States)

Ubiquitin-dependent lysosomal targeting of GABA(A) receptors regulates neuronal inhibition.

  • Arancibia-Cárcamo IL
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2009 Oct 13

Literature context:


Abstract:

The strength of synaptic inhibition depends partly on the number of GABA(A) receptors (GABA(A)Rs) found at synaptic sites. The trafficking of GABA(A)Rs within the endocytic pathway is a key determinant of surface GABA(A)R 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(A)Rs. We demonstrate that the targeting of synaptic GABA(A)Rs into the degradation pathway is facilitated by ubiquitination of a motif within the intracellular domain of the gamma2 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 gamma2 subunit retards the lysosomal targeting of GABA(A)Rs and is sufficient to block the loss of synaptic GABA(A)Rs after anoxic insult. Together, our results establish a previously unknown mechanism for influencing inhibitory transmission under normal and pathological conditions.