Structural determinants and regulation of spontaneous activity in GABAA receptors.
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ABSTRACT:
SUBMITTER: Sexton CA
PROVIDER: S-EPMC8443696 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background and purposeThe δ-subunit-containing GABAA receptors, α4 β1 δ and α4 β3 δ, in dentate gyrus granule cells (DGGCs) are known to exhibit both spontaneous channel openings (i.e. constitutive activity) and agonist-induced current. The functional implications of spontaneous gating are unclear. In this study, we tested the hypothesis that constitutively active α4 β1/3 δ receptors limit agonist efficacy.Experimental approachWhole-cell electrophysiological recordings of adult male rat and mouse hippocampal DGGCs were used to characterize known agonists and antagonists at δ-subunit-containing GABAA receptors. To separate constitutive and agonist-induced currents, different recording conditions were employed.Key resultsRecordings at either 24°C or 34°C, including the PKC autoinhibitory peptide (19-36) intracellularly, removed spontaneous gating by GABAA receptors. In the absence of spontaneous gating, DGGCs responded to the α4 β1/3 δ orthosteric agonist Thio-THIP with a four-fold increased efficacy relative to recording conditions favouring constitutive activity. Surprisingly, the neutral antagonist gabazine was unable to antagonize the current by Thio-THIP. Furthermore, a current was elicited by gabazine alone only when the constitutive current was silenced (EC50 2.1 μM). The gabazine-induced current was inhibited by picrotoxin, potentiated by DS2, completely absent in δ-/- mice and reduced in β1 -/- mice, but could not be replicated in human α4 β1/3 δ receptors expressed heterologously in HEK cells.Conclusion and implicationsKinase activity infers spontaneous gating in α4 β1/3 δ receptors in DGGCs. This significantly limits the efficacy of GABAA agonists and has implications in pathologies involving aberrant excitability caused by phosphorylation (e.g. addiction and epilepsy). In such cases, the efficacy of δ-preferring GABAA ligands may be reduced.
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Project description:γ-Aminobutyric acid type A (GABAA) receptors mediate fast inhibitory signaling in the brain and are targets of numerous drugs and endogenous neurosteroids. A subset of neurosteroids are GABAA receptor positive allosteric modulators; one of these, allopregnanolone, is the only drug approved specifically for treating postpartum depression. There is a consensus emerging from structural, physiological and photolabeling studies as to where positive modulators bind, but how they potentiate GABA activation remains unclear. Other neurosteroids are negative modulators of GABAA receptors, but their binding sites remain debated. Here we present structures of a synaptic GABAA receptor bound to allopregnanolone and two inhibitory sulfated neurosteroids. Allopregnanolone binds at the receptor-bilayer interface, in the consensus potentiator site. In contrast, inhibitory neurosteroids bind in the pore. MD simulations and electrophysiology support a mechanism by which allopregnanolone potentiates channel activity and suggest the dominant mechanism for sulfated neurosteroid inhibition is through pore block.
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Project description:Epilepsy is a serious neurological disease characterized by recurrent unprovoked seizures. The exact etiology of epilepsy is not fully understood. Protrudin is a neural membrane protein and is found to be mutated in hereditary spastic paraplegia that characterized by symptoms like seizures. Here, we reported that the expression of protrudin was downregulated in the temporal neocortex of epileptic patients and in the hippocampus and cortex of pentylenetetrazol and kainic acid-kindled epileptic mouse models. Behavioral and electroencephalogram analyses indicated that overexpression of protrudin in the mouse hippocampus increased the latency of the seizure and decreased the frequency and duration of seizure activity. Using whole-cell patch clamp, overexpression of protrudin in the mouse hippocampus resulted in a reduction in action potential frequency and an increase in gamma-aminobutyric acid (GABA)ergic inhibitory current amplitude. Moreover, western blot analysis showed that the membrane expression of the GABA A receptor β2/3 subunit was also upregulated after protrudin overexpression, and coimmunoprecipitation resulted in a protein-protein interaction between protrudin, GABAARβ2/3 and GABA receptor-associated protein in the hippocampus of epileptic mice. These findings suggest that protrudin probably inhibits the occurrence and development of epilepsy through the regulation of GABAA receptor-mediated synaptic transmission, and protrudin might be a promising target for the treatment of epilepsy.
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Project description:Metabotropic GABAB receptors mediate a significant fraction of inhibitory neurotransmission in the brain. Native GABAB receptor complexes contain the principal subunits GABAB1 and GABAB2, which form an obligate heterodimer, and auxiliary subunits, known as potassium channel tetramerization domain-containing proteins (KCTDs). KCTDs interact with GABAB receptors and modify the kinetics of GABAB receptor signaling. Little is known about the molecular mechanism governing the direct association and functional coupling of GABAB receptors with these auxiliary proteins. Here, we describe the high-resolution structure of the KCTD16 oligomerization domain in complex with part of the GABAB2 receptor. A single GABAB2 C-terminal peptide is bound to the interior of an open pentamer formed by the oligomerization domain of five KCTD16 subunits. Mutation of specific amino acids identified in the structure of the GABAB2-KCTD16 interface disrupted both the biochemical association and functional modulation of GABAB receptors and G protein-activated inwardly rectifying K+ channel (GIRK) channels. These interfacial residues are conserved among KCTDs, suggesting a common mode of KCTD interaction with GABAB receptors. Defining the binding interface of GABAB receptor and KCTD reveals a potential regulatory site for modulating GABAB-receptor function in the brain.
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Project description:Humulus lupulus L. (hops) is a major constituent of beer. It exhibits neuroactive properties that make it useful as a sleeping aid. These effects are hypothesized to be mediated by an increase in GABAA receptor function. In the quest to uncover the constituents responsible for the sedative and hypnotic properties of hops, recent evidence revealed that humulone, a prenylated phloroglucinol derivative comprising 35-70% of hops alpha acids, may act as a positive modulator of GABAA receptors at low micromolar concentrations. This raises the question whether humulone plays a key role in hops pharmacological activity and potentially interacts with other modulators such as ethanol, bringing further enhancement in GABAA receptor-mediated effects of beer. Here we assessed electrophysiologically the positive modulatory activity of humulone on recombinant GABAA receptors expressed in HEK293 cells. We then examined humulone interactions with other active hops compounds and ethanol on GABA-induced displacement of [3H]EBOB binding to native GABAA receptors in rat brain membranes. Using BALB/c mice, we assessed humulone's hypnotic behavior with pentobarbital- and ethanol-induced sleep as well as sedation in spontaneous locomotion with open field test. We demonstrated for the first time that humulone potentiates GABA-induced currents in α1β3γ2 receptors. In radioligand binding to native GABAA receptors, the inclusion of ethanol enhanced humulone modulation of GABA-induced displacement of [3H]EBOB binding in rat forebrain and cerebellum as it produced a leftward shift in [3H]EBOB displacement curves. Moreover, the additive modulatory effects between humulone, isoxanthohumol and 6-prenylnaringenin were evident and corresponded to the sum of [3H]EBOB displacement by each compound individually. In behavioral tests, humulone shortened sleep onset and increased the duration of sleep induced by pentobarbital and decreased the spontaneous locomotion in open field at 20 mg/kg (i.p.). Despite the absence of humulone effects on ethanol-induced sleep onset, sleep duration was increased dose-dependently down to 10 mg/kg (i.p.). Our findings confirmed humulone's positive allosteric modulation of GABAA receptor function and displayed its sedative and hypnotic behavior. Humulone modulation can be potentially enhanced by ethanol and hops modulators suggesting a probable enhancement in the intoxicating effects of ethanol in hops-enriched beer.
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Project description:Metabotropic GABAB receptor is a G protein-coupled receptor (GPCR) that mediates slow and prolonged inhibitory neurotransmission in the brain. It functions as a constitutive heterodimer composed of the GABAB1 and GABAB2 subunits. Each subunit contains three domains; the extracellular Venus flytrap module, seven-helix transmembrane region and cytoplasmic tail. In recent years, the three-dimensional structures of GABAB receptor extracellular and intracellular domains have been elucidated. These structures reveal the molecular basis of ligand recognition, receptor heterodimerization and receptor activation. Here we provide a brief review of the GABAB receptor structures, with an emphasis on describing the different ligand-bound states of the receptor. We will also compare these with the known structures of related GPCRs to shed light on the molecular mechanisms of activation and regulation in the GABAB system, as well as GPCR dimers in general. This article is part of the "Special Issue Dedicated to Norman G. Bowery".
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Project description:Numerous studies recently showed that the inhibitory neurotransmitter, γ-aminobutyric acid (GABA), can stimulate cerebral angiogenesis and promote neurovascular coupling by activating the ionotropic GABAA receptors on cerebrovascular endothelial cells, whereas the endothelial role of the metabotropic GABAB receptors is still unknown. Preliminary evidence showed that GABAA receptor stimulation can induce an increase in endothelial Ca2+ levels, but the underlying signaling pathway remains to be fully unraveled. In the present investigation, we found that GABA evoked a biphasic elevation in [Ca2+]i that was initiated by inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-dependent Ca2+ release from neutral and acidic Ca2+ stores, respectively, and sustained by store-operated Ca2+ entry. GABAA and GABAB receptors were both required to trigger the endothelial Ca2+ response. Unexpectedly, we found that the GABAA receptors signal in a flux-independent manner via the metabotropic GABAB receptors. Likewise, the full Ca2+ response to GABAB receptors requires functional GABAA receptors. This study, therefore, sheds novel light on the molecular mechanisms by which GABA controls endothelial signaling at the neurovascular unit.
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Project description:Acid-sensing ion channels (ASICs) are proton-gated ion channels that mediate nociception in the peripheral nervous system and contribute to fear and learning in the central nervous system. Sevanol was reported previously as a naturally-occurring ASIC inhibitor from thyme with favorable analgesic and anti-inflammatory activity. Using electrophysiological methods, we found that in the high micromolar range, the compound effectively inhibited homomeric ASIC1a and, in sub- and low-micromolar ranges, positively modulated the currents of α1β2γ2 GABAA receptors. Next, we tested the compound in anxiety-related behavior models using a targeted delivery into the hippocampus with parallel electroencephalographic measurements. In the open field, 6 µM sevanol reduced both locomotor and θ-rhythmic activity similar to GABA, suggesting a primary action on the GABAergic system. At 300 μM, sevanol markedly suppressed passive avoidance behavior, implying alterations in conditioned fear memory. The observed effects could be linked to distinct mechanisms involving GABAAR and ASIC1a. These results elaborate the preclinical profile of sevanol as a candidate for drug development and support the role of ASIC channels in fear-related functions of the hippocampus.
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