Monoclonal antibodies reveal structural homogeneity of gamma-aminobutyric acid/benzodiazepine receptors in different brain areas.

Häring, P.; Stähli, C.; Schoch, Peter; Takács, B.; Staehelin, Theophil; Möhler, Hanns

doi:10.1073/pnas.82.14.4837

Cited by 164 publications

(73 citation statements)

References 19 publications

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“…They were obtained from Chemicon (Temecula, CA) or were kind gifts of Dr. Robert J. Wenthold (National Institutes of Health, Bethesda, MD). For GABA A receptor immunocytochemistry, we used monoclonal antibody bd-17, which recognizes the ␤2 and ␤3 subunits (Haring et al, 1985). This antibody was a kind gift of Dr. Jean-Marc Fritschy (Institute of Pharmacology, University of Zurich, Zurich, Switzerland).…”

Section: Methodsmentioning

confidence: 99%

Organization of Ionotropic Glutamate Receptors at Dendrodendritic Synapses in the Rat Olfactory Bulb

Sassoè‐Pognetto

Ottersen

2000

J. Neurosci.

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Dendrodendritic synapses between mitral (or tufted) and granule cells of the olfactory bulb play a major role in the processes of odor discrimination and olfactory learning. Release of glutamate at these synapses activates postsynaptic receptors on the dendritic spines of granule cells, as well as presynaptic NMDA receptors in the mitral cell membrane. However, immunocytochemical studies have failed to demonstrate the presence of ionotropic glutamate receptors in granule cell dendrites. By using a postembedding immunogold procedure, we describe here the precise organization of neurotransmitter receptors at dendrodendritic synapses. We show that there is a selective localization of glutamate and GABA receptors at asymmetric and symmetric synaptic junctions, respectively. In addition, we demonstrate that NMDA and AMPA receptors are clustered at postsynaptic specializations on granule cell spines and that they are extensively colocalized. Conversely, glutamate receptors do not appear to be concentrated in clusters on mitral cell dendrites, suggesting that the presynaptic effects of glutamate are mediated by a small complement of extrasynaptic receptors. By analyzing the subsynaptic distribution of the NR1 and GluR2/3 subunits, we show that they are distributed along the entire extent of the postsynaptic specialization, indicating that both NMDA and AMPA receptors are available for dendrodendritic signaling between mitral and granule cells. These results indicate that the principles recently found to underlie the organization of glutamate receptors at axospinous synapses also apply to dendrodendritic synapses.

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Section: Methodsmentioning

confidence: 99%

Organization of Ionotropic Glutamate Receptors at Dendrodendritic Synapses in the Rat Olfactory Bulb

Sassoè‐Pognetto

Ottersen

2000

J. Neurosci.

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“…After 8% SDS-PAGE, proteins were transferred to nitrocellulose sheets (Hybond-ECL; Amersham Pharmacia Biotech) and immunodecorated in a manner similar to that described previously (32). The following primary antibodies were used: bd24 (2 g/ml), GABA A receptor ␤2/3-subunit-specific monoclonal antibody bd17 (33,34), GABA A receptor ␤1-subunit-specific antibody ␤1-(350 -404) (5 g/ml; Refs. 37 and 38), GABA A receptor ␤2-subunit-specific antibody ␤2-(351-405) (2 g/ml; Refs.…”

Section: Methodsmentioning

confidence: 99%

“…For precipitation, the monoclonal antibody bd24 (33,34) covalently coupled to protein A-Sepharose (bd24-beads) was used. With 10 l of packed bd24-beads, GABA A receptor containing approximately 1 g of ␣1-subunit was precipitated.…”

Section: Methodsmentioning

confidence: 99%

A Novel Serine Kinase with Specificity for β3-Subunits Is Tightly Associated with GABAA Receptors

Kannenberg

Schaerer

Fuchs³

et al. 1999

Journal of Biological Chemistry

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Tuning of ␥-aminobutyric acid type A (GABA A ) receptor function via phosphorylation of the receptor potentially allows neurons to modulate their inhibitory input. Several kinases, both of the serine-threonine kinase and the tyrosine kinase families, have been proposed as candidates for such a modulatory role in vivo. However, no GABA A receptor-phosphorylating kinase physically associated with the receptor has been identified so far on a molecular level. In this study, we demonstrate a GABA A receptor-associated protein serine kinase phosphorylating specifically ␤3-subunits of native GABA A receptors. The characteristics of this novel kinase clearly distinguish it from enzymatic activities that have been shown so far to phosphorylate the GABA A receptor. We putatively identify this protein kinase as the previously described GTAP34 (GABA A receptor-tubulin complexassociated protein of molecular mass 34 kDa). Using expressed recombinant fusion proteins, we identify serine 408 as a major target of the phosphorylation reaction, whereas serine 407 is not phosphorylated. This demonstrates the high specificity of the kinase. Phosphorylation of serine 408 is known to result in a decreased receptor function. The direct association of this kinase with the receptor indicates an important physiological role.The ionotropic GABA A 1 receptor is abundantly expressed in the mammalian brain, and its major function is to mediate fast synaptic inhibition (for reviews, see Refs. 1-3). Binding of GABA to the extracellular domain of the receptor leads to the opening of an intrinsic ion channel followed by chloride influx, counteracting depolarization of the neuronal resting potential. Also, excitatory actions of the receptor depending on an altered chloride equilibrium potential have been reported (4, 5). A hallmark of the GABA A receptor is the many clinically important ways of modulation (3, 6).Biochemical purification of the GABA A receptor (7) and cloning of the first two subunits (8) were followed by the description of so far 14 different mammalian subunits named ␣1-6, ␤1-3, ␥1-3, ␦, and ⑀. From these subunits, diverse heterooligomeric receptor subtypes are generated neuronally (9), bearing most likely a pentameric structure (10, 11) equivalent to that of the related nicotinic acetylcholine receptor (12).As also demonstrated for other ion channels (13), GABA A receptor function can be regulated by phosphorylation. This provides a mean for fine tuning inhibitory neuronal inputs. A variety of studies has given evidence for the phosphorylation of the GABA A receptor by serine/threonine kinases such as protein kinase C (14 -19, 23), cAMP-dependent protein kinase (17, 20 -25), cGMP-dependent protein kinase (26, 27), Ca 2ϩ /calmodulindependent kinase II (27), and also by the tyrosine kinase Src (28, 29). These reports point to the intracellular loops of GABA A receptor ␤-and ␥-subunits as major phosphorylation targets. The functional consequences of phosphorylation are either inhibition or enhancement of GABA A receptormediated chlo...

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“…The modified rat ␣ subunit differs from the rat ␣ by one amino acid residue, which confers the subunit-specific bd24 antibody recognition (32,33). This property has previously been used to exclude proteolysis of the linked constructs (31).…”

Section: Construction Of Tandem and Triple Subunit Cdnas-for Simplicitymentioning

confidence: 99%

Forced Subunit Assembly in α1β2γ2 GABAAReceptors

Baumann

Baur

Sigel

2002

Journal of Biological Chemistry

237

137

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The major isoform of the ␥-aminobutyric acid type A (GABA A ) receptor is thought to be composed of 2␣ 1 , 2␤ 2 , and 1␥ 2 subunit(s), which surround the ion pore. Definite evidence for the subunit arrangement is lacking. We show here that GABA A receptor subunits can be concatenated to a trimer that can be functionally expressed upon combination with a dimer. Many combinations did not result in the functional expression. In contrast, four different combinations of triple subunits with dual subunit constructs, all resulting in the identical pentameric receptor ␥ 2 ␤ 2 ␣ 1 ␤ 2 ␣ 1 , could be successfully expressed in Xenopus oocytes. We characterized the functional properties of these receptors in respect to agonist, competitive antagonist, and diazepam sensitivity. All properties were similar to those of wild type ␣ 1 ␤ 2 ␥ 2 GABA A receptors. Thus, together with information on the crystal structure of the homologous acetylcholine-binding protein (Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van Der Oost, J., Smit, A. B., and Sixma, T. K., (2001) Nature 411, 269 -276, we provide evidence for an arrangement ␥ 2 ␤ 2 ␣ 1 ␤ 2 ␣ 1 , counterclockwise when viewed from the synaptic cleft. Forced subunit assembly will also allow receptors containing different subunit isoforms or mutant subunits to be expressed, each in a desired position. The methods established here should be applicable to the entire ion channel family comprising nicotinic acetylcholine, glycine, and 5HT 3 receptors.

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Monoclonal antibodies reveal structural homogeneity of gamma-aminobutyric acid/benzodiazepine receptors in different brain areas.

Cited by 164 publications

References 19 publications

Organization of Ionotropic Glutamate Receptors at Dendrodendritic Synapses in the Rat Olfactory Bulb

Organization of Ionotropic Glutamate Receptors at Dendrodendritic Synapses in the Rat Olfactory Bulb

A Novel Serine Kinase with Specificity for β3-Subunits Is Tightly Associated with GABAA Receptors

Forced Subunit Assembly in α1β2γ2 GABAAReceptors

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