Functional properties of recombinant rat GABAA receptors depend upon subunit composition

Verdoorn, Todd A.; Draguhn, Andreas; Ymer, Sanie; Seeburg, Peter H.; Sakmann, Bert

doi:10.1016/0896-6273(90)90145-6

Cited by 534 publications

(312 citation statements)

References 32 publications

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“…That is, precedents exist, with the nicotinic ACh receptor and the GABA, receptor, indicating that singlechannel conductance is determined by the specific subunits constituting the receptor (Mishina et al, 1986;Verdoom et al, 1990). Although phosphorylation (Swope et al, 1992) or other posttranslational processes may modify channel kinetics, no such processes have been shown to date to alter single-channel conductance.…”

Section: Discussionmentioning

confidence: 99%

Glutamate-activated currents in outside-out patches from spiny versus aspiny hilar neurons of rat hippocampal slices

1993

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The desensitization rate of non-NMDA glutamate receptors was investigated in outside-out membrane patches obtained from morphologically identified spiny “mossy cells” (SMCs) and aspiny hilar interneurons (AHIs) in young rat hippocampal slices. The fast application of a 1 mM step of L-glutamate for 50–100 msec in the presence of TTX and dizolcipine (MK-801) onto patches excised from these neurons produced large glutamate-activated currents (GACs) that decayed with a single or double exponential time course despite the continued presence of agonist. These desensitization rates of alpha- amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate- sensitive receptors differed markedly between patches obtained from the two cell types. The fast time constant of desensitization in AHIs (n = 34) averaged 3.3 +/- 0.93 msec (mean +/- SD), while that of SMCs (n = 57) averaged 6.8 +/- 2.0 msec. Current-voltage relationships of the GACs did not differ between SMCs and AHIs, with comparable reversal potentials and no evidence of inward rectification. We also failed to observe significant Ca2+ permeability in either cell type. However, brief (< 1 msec) pulses of 1 mM glutamate produced rapidly decaying GACs with distinct kinetics in the two neuronal classes. Furthermore, analysis of the single glutamate-activated channel currents in outside- out patches from hilar neurons revealed a larger predominant single- channel current in AHIs versus SMCs. Lastly, we observed a greater sensitivity to cyclothiazide in SMCs versus AHIs, with half-maximal removal of desensitization being 90 mM and 200 mM, respectively. Taken together, these differences in GACs between SMCs and AHIs might indicate a functional correlate to the substantial heterogeneity in the molecular structure of glutamate receptor subunits or might be related to posttranslational modifications of these subunits, perhaps provided by the unique microenvironment in the spines covering SMCs.

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Section: Discussionmentioning

confidence: 99%

Glutamate-activated currents in outside-out patches from spiny versus aspiny hilar neurons of rat hippocampal slices

1993

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“…After 3 d of growth in uncoated conditions, PSA-NCAM ϩ cells generated spheres and were shown to express multiple GABA A R subunit genes (i.e., ␣ 2,4,5 , ␤ 1,3 , and ␥ 1-3 ) that are known to be necessary for forming heteromeric functional receptors (Levitan et al, 1988;Malherbe et al, 1990;Sigel et al, 1990;Verdoorn et al, 1990). It is acknowledged that at least ␣ 4 , ␤ 1 , and ␥ 1 , which are detected in rodent progenitors from the neocortical proliferative zone Barker, 1995, 1998;, may compose GABA A Rs that have an extrasynaptic function in neural development.…”

Section: Discussionmentioning

confidence: 99%

Autocrine/Paracrine Activation of the GABA_AReceptor Inhibits the Proliferation of Neurogenic Polysialylated Neural Cell Adhesion Molecule-Positive (PSA-NCAM⁺) Precursor Cells from Postnatal Striatum

et al. 2003

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GABA and its type A receptor (GABA A R) are present in the immature CNS and may function as growth-regulatory signals during the development of embryonic neural precursor cells. In the present study, on the basis of their isopycnic properties in a buoyant density gradient, we developed an isolation procedure that allowed us to purify proliferative neural precursor cells from early postnatal rat striatum, which expressed the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). These postnatal striatal PSA-NCAM ϩ cells were shown to proliferate in the presence of epidermal growth factor (EGF) and formed spheres that preferentially generated neurons in vitro. We demonstrated that PSA-NCAM ϩ neuronal precursors from postnatal striatum expressed GABA A R subunits in vitro and in situ. GABA elicited chloride currents in PSA-NCAM ϩ cells by activation of functional GABA A R that displayed a typical pharmacological profile. GABA A R activation in PSA-NCAM ϩ cells triggered a complex intracellular signaling combining a tonic inhibition of the mitogen-activated protein kinase cascade and an increase of intracellular calcium concentration by opening of voltagegated calcium channels. We observed that the activation of GABA A R in PSA-NCAM ϩ neuronal precursors from postnatal striatum inhibited cell cycle progression both in neurospheres and in organotypic slices. Furthermore, postnatal PSA-NCAM ϩ striatal cells synthesized and released GABA, thus creating an autocrine/paracrine mechanism that controls their proliferation. We showed that EGF modulated this autocrine/paracrine loop by decreasing GABA production in PSA-NCAM ϩ cells. This demonstration of GABA synthesis and GABA A R function in striatal PSA-NCAM ϩ cells may shed new light on the understanding of key extrinsic cues that regulate the developmental potential of postnatal neuronal precursors in the CNS.

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“…GABA A Rs containing ␣1-3 and ␥ are enriched at inhibitory synapses and mediate phasic inhibition, whereas those containing ␣4 -6 and ␦ are found at extrasynaptic locales and mediate tonic inhibition (1,2). Notably, subunit composition impacts the pharmacological and physiological properties of these varying receptor subtypes (1,7,8). Moreover, GABA A Rs containing unique subunit combinations are selectively targeted to distinct types of inhibitory synapses.…”

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confidence: 99%

Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse

Nakamura

Morrow

Modgil

et al. 2016

Journal of Biological Chemistry

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The accumulation of ␥-aminobutyric acid receptors (GABA A Rs) at the appropriate postsynaptic sites is critical for determining the efficacy of fast inhibitory neurotransmission. Although we know that the majority of synaptic GABA A R subtypes are assembled from ␣1-3, ␤, and ␥2 subunits, our understanding of how neurons facilitate their targeting to and stabilization at inhibitory synapses is rudimentary. To address these issues, we have created knock-in mice in which the pH-sensitive green fluorescent protein (GFP) and the Myc epitope were introduced to the extracellular domain of the mature receptor ␣2 subunit (pH␣2). Using immunoaffinity purification and mass spectroscopy, we identified a stable complex of 174 proteins that were associated with pH␣2, including other GABA A R subunits, and previously identified receptor-associated proteins such as gephyrin and collybistin. 149 of these proteins were novel GABA A R binding partners and included G-protein-coupled receptors and ion channel subunits, proteins that regulate trafficking and degradation, regulators of protein phosphorylation, GTPases, and a number of proteins that regulate their activity. Notably, members of the postsynaptic density family of proteins that are critical components of excitatory synapses were not associated with GABA A Rs. Crucially, we demonstrated for a subset of these novel proteins (including cullin1, ephexin, potassium channel tetramerization domain containing protein 12, mitofusin2, metabotropic glutamate receptor 5, p21-activated kinase 7, and Ras-related protein 5A) bind directly to the intracellular domains of GABA A Rs, validating our proteomic analysis. Thus, our experiments illustrate the complexity of the GABA A R proteome and enhance our understanding of the mechanisms neurons use to construct inhibitory synapses.

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Functional properties of recombinant rat GABAA receptors depend upon subunit composition

Cited by 534 publications

References 32 publications

Glutamate-activated currents in outside-out patches from spiny versus aspiny hilar neurons of rat hippocampal slices

Glutamate-activated currents in outside-out patches from spiny versus aspiny hilar neurons of rat hippocampal slices

Autocrine/Paracrine Activation of the GABA_AReceptor Inhibits the Proliferation of Neurogenic Polysialylated Neural Cell Adhesion Molecule-Positive (PSA-NCAM⁺) Precursor Cells from Postnatal Striatum

Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse

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Functional properties of recombinant rat GABAA receptors depend upon subunit composition

Cited by 534 publications

References 32 publications

Glutamate-activated currents in outside-out patches from spiny versus aspiny hilar neurons of rat hippocampal slices

Glutamate-activated currents in outside-out patches from spiny versus aspiny hilar neurons of rat hippocampal slices

Autocrine/Paracrine Activation of the GABAAReceptor Inhibits the Proliferation of Neurogenic Polysialylated Neural Cell Adhesion Molecule-Positive (PSA-NCAM+) Precursor Cells from Postnatal Striatum

Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse

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Autocrine/Paracrine Activation of the GABA_AReceptor Inhibits the Proliferation of Neurogenic Polysialylated Neural Cell Adhesion Molecule-Positive (PSA-NCAM⁺) Precursor Cells from Postnatal Striatum