Distinct properties of murine α5 γ‐aminobutyric acid type a receptors revealed by biochemical fractionation and mass spectroscopy

Ju, Young Su; Guzzo, Angelina; Chiu, Mary; Taylor, Paul; Moran, Michael F.; Gurd, James W.; MacDonald, John F.; Orser, Beverley A.

doi:10.1002/jnr.21991

Cited by 24 publications

(19 citation statements)

References 55 publications

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“…Although these results are not quantitative and do not discriminate between surface and intracel- lular populations, our results do suggest the existence of multiple receptor subtypes with mixed ␣ and/or ␤ subunits, supporting previous observations of the coexistence of different ␣ subunits in a single receptor complex (42)(43)(44)(45)(46). Consistent with our results, previous studies to identify proteins associated with the GABA A R ␣5 subunit through MS analysis exclusively identified other GABA A R subunits, including ␣1-3, ␣5, ␤1-3, and ␥2 (47). A more recent investigation into the proteins associated with the GABA A R ␣1 subunit isolated 18 associated proteins via MS analysis, more than half of which were other GABA A R subunits (48), further supporting the possibility of a more heterogeneous population of receptors than originally predicted (5,49).…”

Section: Discussionsupporting

confidence: 92%

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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Section: Discussionsupporting

confidence: 92%

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 channel is thought to be composed of two αs, two βs and a third subunit type that is often the δ or the γ2 subunit [24]. Whether the native channels are homo or heteromeric in terms of the αs and βs subunits is not clear [25]. We examined two compounds that are normally inert at α5βγ2 channels: the benzodiazepine flumazenil (1 µM) and zolpidem (100 and 200 nM), a positive modulator at α1, α2 and α3 in αβγ2 channels.…”

Section: Resultsmentioning

confidence: 99%

“…GABA A channels having the α5 subunit in their channel complex are known to be mostly located extrasynaptically in CA1 neurons but are not or minimally activated by the ambient GABA concentration. How the new channels differ from the α5-channels normally in the membrane is not clear but heteromeric α subunits in the channel complex, different intracellular modification or associations with intracellular proteins can all give rise to the differences observed [24], [25]. Interestingly, the induced tonic current is inhibited by flumazenil and zolpidem, indicating a distinct pharmacology of these novel GABA A channels.…”

Section: Discussionmentioning

confidence: 98%

Insulin Reduces Neuronal Excitability by Turning on GABAA Channels that Generate Tonic Current

et al. 2011

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Insulin signaling to the brain is important not only for metabolic homeostasis but also for higher brain functions such as cognition. GABA (γ-aminobutyric acid) decreases neuronal excitability by activating GABAA channels that generate phasic and tonic currents. The level of tonic inhibition in neurons varies. In the hippocampus, interneurons and dentate gyrus granule cells normally have significant tonic currents under basal conditions in contrast to the CA1 pyramidal neurons where it is minimal. Here we show in acute rat hippocamal slices that insulin (1 nM) “turns on” new extrasynaptic GABAA channels in CA1 pyramidal neurons resulting in decreased frequency of action potential firing. The channels are activated by more than million times lower GABA concentrations than synaptic channels, generate tonic currents and show outward rectification. The single-channel current amplitude is related to the GABA concentration resulting in a single-channel GABA affinity (EC50) in intact CA1 neurons of 17 pM with the maximal current amplitude reached with 1 nM GABA. They are inhibited by GABAA antagonists but have novel pharmacology as the benzodiazepine flumazenil and zolpidem are inverse agonists. The results show that tonic rather than synaptic conductances regulate basal neuronal excitability when significant tonic conductance is expressed and demonstrate an unexpected hormonal control of the inhibitory channel subtypes and excitability of hippocampal neurons. The insulin-induced new channels provide a specific target for rescuing cognition in health and disease.

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“…Each averaged value was compared with the baseline value (t test; *p<0.05, **p<0.01, ***p<0.005). Comparison between groups was done with the unpaired Mann-Whitney test most frequently co-assembled with β3 subunits [13]. Taking into account the low detection rate of this subunit in TMN neurons, studies on recombinant GABA A R are warranted in order to determine activation and modulation of α5-containing receptor types by propofol vs FDD.…”

Section: Discussionmentioning

confidence: 99%

GABAA receptors involved in sleep and anaesthesia: β1- versus β3-containing assemblies

Yanovsky

Schubring

Fleischer

et al. 2011

Pflugers Arch - Eur J Physiol

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The histaminergic neurons of the posterior hypothalamus (tuberomamillary nucleus-TMN) control wakefulness, and their silencing through activation of GABA(A) receptors (GABA(A)R) induces sleep and is thought to mediate sedation under propofol anaesthesia. We have previously shown that the β1 subunit preferring fragrant dioxane derivatives (FDD) are highly potent modulators of GABA(A)R in TMN neurons. In recombinant receptors containing the β3N265M subunit, FDD action is abolished and GABA potency is reduced. Using rat, wild-type and β3N265M mice, FDD and propofol, we explored the relative contributions of β1- and β3-containing GABA(A)R to synaptic transmission from the GABAergic sleep-on ventrolateral preoptic area neurons to TMN. In β3N265M mice, GABA potency remained unchanged in TMN neurons, but it was decreased in cultured posterior hypothalamic neurons with impaired modulation of GABA(A)R by propofol. Spontaneous and evoked GABAergic synaptic currents (IPSC) showed β1-type pharmacology, with the same effects achieved by 3 μM propofol and 10 μM PI24513. Propofol and the FDD PI24513 suppressed neuronal firing in the majority of neurons at 5 and 100 μM, and in all cells at 10 and 250 μM, respectively. FDD given systemically in mice induced sedation but not anaesthesia. Propofol-induced currents were abolished (1-6 μM) or significantly reduced (12 μM) in β3N265M mice, whereas gating and modulation of GABA(A)R by PI24513 as well as modulation by propofol were unchanged. In conclusion, β1-containing (FDD-sensitive) GABA(A)R represent the major receptor pool in TMN neurons responding to GABA, while β3-containing (FDD-insensitive) receptors are gated by low micromolar doses of propofol. Thus, sleep and anaesthesia depend on different GABA(A)R types.

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Distinct properties of murine α5 γ‐aminobutyric acid type a receptors revealed by biochemical fractionation and mass spectroscopy

Cited by 24 publications

References 55 publications

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

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

Insulin Reduces Neuronal Excitability by Turning on GABAA Channels that Generate Tonic Current

GABAA receptors involved in sleep and anaesthesia: β1- versus β3-containing assemblies

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