Toward Understanding Functional Properties and Subunit Arrangement of α4β2δ γ-Aminobutyric Acid, Type A (GABAA) Receptors.
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ABSTRACT:
SUBMITTER: Wongsamitkul N
PROVIDER: S-EPMC5000092 | biostudies-other | 2016 Aug
REPOSITORIES: biostudies-other
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Project description:PURPOSE:To describe the subunit composition of glutamate and gamma-aminobutyric acid (GABA) receptors in brain tissue from patients with different types of status epilepticus. PATIENTS AND METHODS:The subunit composition of glutamate and GABA receptors was analyzed in: (1) surgical brain samples from three patients with refractory convulsive status epilepticus, three patients with electrical status epilepticus in sleep, and six patients with refractory epilepsy, and (2) brain autopsy samples from four controls who died without neurological disorders. Subunit expression was quantified with Western blotting and messenger ribonucleic acid (mRNA) expression was quantified with reverse polymerase chain reaction. RESULTS:Western blot analysis demonstrated the following patterns (as compared to controls): (1) alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors: elevated GluA1/GluA2 ratio in electrical status epilepticus in sleep (465%±119) and refractory epilepsy (329%±125; p<0.01); (2) N-methyl-d-aspartate (NMDA) receptors: increased GluN2B/GluN2A ratio in electrical status epilepticus in sleep (3682%±1000) and refractory convulsive status epilepticus (3520%±751; p<0.05); (3) GABA receptors: elevated ?2/?1 ratio in refractory epilepsy (321%±138; p<0.05) and refractory convulsive status epilepticus (346%±74; p<0.05); and (4) patients with underlying malformation of cortical development had increased ratios in GluA1/GluA2 (382%±149; p<0.01), GluN2B/GluN2A (3321%±1581; p<0.05) and ?2/?1 (303%±86; p<0.01). Quantification of mRNA demonstrated an elevated GABRA2/GABRA1 ratio in refractory epilepsy (712; p<0.05) as compared to controls. CONCLUSIONS:The subunit composition of glutamate and GABA receptors in patients with status epilepticus mirrors that found in animal models of refractory status epilepticus and may promote self-sustaining seizures. Receptor subunit changes may provide additional targets for improved treatment.
| S-EPMC6294571 | biostudies-literature
Project description:Enhancement of gamma-aminobutyric acid type A receptor (GABA(A)R)-mediated inhibition is a property of most general anesthetics and a candidate for a molecular mechanism of anesthesia. Intravenous anesthetics, including etomidate, propofol, barbiturates, and neuroactive steroids, as well as volatile anesthetics and long-chain alcohols, all enhance GABA(A)R function at anesthetic concentrations. The implied existence of a receptor site for anesthetics on the GABA(A)R protein was supported by identification, using photoaffinity labeling, of a binding site for etomidate within the GABA(A)R transmembrane domain at the beta-alpha subunit interface; the etomidate analog [(3)H]azietomidate photolabeled in a pharmacologically specific manner two amino acids, alpha1Met-236 in the M1 helix and betaMet-286 in the M3 helix (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599-11605). Here, we use [(3)H]azietomidate photolabeling of bovine brain GABA(A)Rs to determine whether other structural classes of anesthetics interact with the etomidate binding site. Photolabeling was inhibited by anesthetic concentrations of propofol, barbiturates, and the volatile agent isoflurane, at low millimolar concentrations, but not by octanol or ethanol. Inhibition by barbiturates, which was pharmacologically specific and stereospecific, and by propofol was only partial, consistent with allosteric interactions, whereas isoflurane inhibition was nearly complete, apparently competitive. Protein sequencing showed that propofol inhibited to the same extent the photolabeling of alpha1Met-236 and betaMet-286. These results indicate that several classes of general anesthetics modulate etomidate binding to the GABA(A)R: isoflurane binds directly to the site with millimolar affinity, whereas propofol and barbiturates inhibit binding but do not bind in a mutually exclusive manner with etomidate.
| S-EPMC2838283 | biostudies-literature
Project description:ObjectiveHuman hypothalamic hamartomas (HHs) are highly associated with treatment-resistant gelastic seizures. HHs are intrinsically epileptogenic, although the basic cellular mechanisms responsible for seizure activity are unknown. Altered gamma-aminobutyric acid (GABA) function can contribute to epileptogenesis in humans and animal models. Recently, functional GABA(A) receptor (GABA(A) R) rundown has been described in surgically resected human temporal lobe epilepsy tissue. We asked whether functional GABA(A) R rundown also occurs in human HH neurons.MethodsGABA(A) R-mediated currents were measured using perforated patch-clamp recordings in single neurons acutely dissociated from surgically resected HH tissue. In addition, functional GABA(A) Rs were expressed in Xenopus oocytes after microinjection with membrane fractions from either HH or control hypothalamus, and were studied with 2-electrode voltage-clamp recordings.ResultsPerforated patch-clamp recordings in dissociated HH neurons showed that repetitive exposure to GABA (5 consecutive exposures to 0.1 mM GABA with 1-second duration and at 20-second intervals) induced a time-dependent rundown of whole-cell currents in small HH neurons, whereas large HH neurons showed much less rundown using the same protocol. Functional rundown was not observed in HH neurons with repetitive exposure to glycine or glutamate. Two-electrode voltage-clamp recordings (6 consecutive exposures to 1 mM GABA with 10-second duration and at 40-second intervals) induced GABA current rundown in Xenopus oocytes microinjected with HH membrane proteins, but not in the oocytes expressing hypothalamic membrane proteins derived from human autopsy controls. Functional rundown of GABA currents was significantly attenuated by intracellular application of adenosine triphosphate or the nonspecific phosphatase inhibitor, okadaic acid.InterpretationNeurons from surgically resected human HH demonstrate functional rundown of GABA(A) R-mediated transmembrane currents in response to GABA agonist exposure. Rundown may be a marker for impaired GABAergic function and a contributing mechanism for seizure genesis within HH tissue.
| S-EPMC3082609 | biostudies-literature
Project description:The pharmacological properties and functional role of native GABA(A) receptors (GABA(A)Rs) were investigated in rat hypothalamic neurons expressing the epsilon-subunit with the help of whole-cell patch-clamp recording and single-cell reverse transcription-PCR. Two cell groups were identified: histaminergic tuberomamillary and orexinergic/hypocretinergic neurons. Approximately 25% of histaminergic and 70% of orexinergic neurons contained mRNA encoding for the epsilon-subunit. Double-immunofluorescence staining revealed a somatic localization of this protein in these two neuronal groups. Constitutive activity, diazepam modulation, fast desensitization of maximal currents, and activation by propofol (6-98 microm) of GABA(A)Rs did not correlate with epsilon-subunit expression. Propofol at 3-12 microm potentiated GABA-mediated currents similarly in all neurons. However, noise variance analysis of GABA-mediated currents enhanced by propofol revealed a significant difference between epsilon-positive and epsilon-negative neurons. The former displayed no difference between control and potentiated responses, and, in the latter, noise was decreased in the presence of propofol. Spontaneous IPSCs recorded in cultured hypothalamic neurons were prolonged in the presence of propofol in all epsilon-negative neurons, whereas propofol-resistant IPSCs were recorded in epsilon-positive cells. The infrequent expression of the epsilon-subunit may be a key factor in the recently discovered central role of the tuberomamillary nucleus in anesthesia.
| S-EPMC6725214 | biostudies-literature
Project description:The accumulation of γ-aminobutyric acid receptors (GABAARs) at the appropriate postsynaptic sites is critical for determining the efficacy of fast inhibitory neurotransmission. Although we know that the majority of synaptic GABAAR subtypes are assembled from α1-3, β, and γ2 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 α2 subunit (pHα2). Using immunoaffinity purification and mass spectroscopy, we identified a stable complex of 174 proteins that were associated with pHα2, including other GABAAR subunits, and previously identified receptor-associated proteins such as gephyrin and collybistin. 149 of these proteins were novel GABAAR 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 GABAARs. 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 GABAARs, validating our proteomic analysis. Thus, our experiments illustrate the complexity of the GABAAR proteome and enhance our understanding of the mechanisms neurons use to construct inhibitory synapses.
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Project description:gamma-Aminobutyric acid type A (GABA-A) receptors are a major mediator of inhibitory neurotransmission in the mammalian central nervous system, and the site of action of a number of clinically important drugs. These receptors exist as a family of subtypes with distinct temporal and spatial patterns of expression and distinct properties that presumably underlie a precise role for each subtype. The newest member of this gene family is the theta subunit. The deduced polypeptide sequence is 627 amino acids long and has highest sequence identity (50.5%) with the beta1 subunit. Within the rat striatum, this subunit coassembles with alpha2, beta1, and gamma1, suggesting that gamma-aminobutyric acid type A receptors consisting of arrangements other than alpha beta + gamma, delta, or epsilon do exist. Expression of alpha2beta1gamma1theta in transfected mammalian cells leads to the formation of receptors with a 4-fold decrease in the affinity for gamma-aminobutyric acid compared with alpha2beta1gamma1. This subunit has a unique distribution, with studies so far suggesting significant expression within monoaminergic neurons of both human and monkey brain.
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Project description:Nineteen GABA(A) receptor (GABA(A)R) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of beta1-subunit-containing GABA(A)Rs is unknown. Here we report the discovery of a new structural class of GABA(A)R positive modulators with unique beta1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed alpha1betaxgamma2L (x-for 1,2,3) GABA(A)R FDD were 6 times more potent at beta1- versus beta2- and beta3-containing receptors. Serine at position 265 was essential for the high sensitivity of the beta1-subunit to FDD and the beta1N286W mutation nearly abolished modulation; vice versa the mutation beta3N265S shifted FDD sensitivity toward the beta1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to beta1-negative cerebellar Purkinje neurons. Immunostaining for the beta1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by beta1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of beta1-containing GABA(A)Rs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA(A)Rs.
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Project description:N-Methyl-d-aspartate (NMDA) receptors, the main mediators of excitatory synaptic transmission, are heterotetrameric receptors. Typically, glycine binding NR1 subunits co-assemble with glutamate binding NR2 subunits to form a functional receptor. Here we have used luminescence resonance energy transfer (LRET) investigations to establish the specific configuration in which these subunits assemble to form the functional tetramer and show that the dimer of dimers structure is formed by the NR1 subunits assembling diagonally to each other. The distances measured by LRET are consistent with the NMDA structure predicted based on cross-linking investigations and on the structure of the full-length alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid (AMPA) receptor structure (1). Additionally, the LRET distances between the NR1 and NR2A subunits within a dimer measured in the desensitized state of the receptor are longer than the distances in the previously published crystal structure of the isolated ligand binding domain of NR1-NR2A. Because the dimer interface in the isolated ligand binding domain crystallizes in the open channel structure, the longer LRET distances would be consistent with the decoupling of the dimer interface in the desensitized state. This is similar to what has been previously observed for the AMPA subtype of the ionotropic glutamate receptors, suggesting a similar mechanism for desensitization in the two subtypes of the glutamate receptor.
| S-EPMC2865314 | biostudies-literature
Project description:It has been recently proposed that ?5-subunit containing GABAA receptors (?5-GABAA receptors) that mediate tonic inhibition might be involved in pain. The purpose of this study was to investigate the contribution of ?5-GABAA receptors in the loss of GABAergic inhibition and in formalin-induced, complete Freund's adjuvant (CFA)-induced and L5 and L6 spinal nerve ligation-induced long-lasting hypersensitivity. Formalin or CFA injection and L5 and L6 spinal nerve ligation produced long-lasting allodynia and hyperalgesia. Moreover, formalin injection impaired the rate-dependent depression of the Hofmann reflex. Peripheral and intrathecal pretreatment or post-treatment with the ?5-GABAA receptor antagonist, L-655,708 (0.15-15 nmol), prevented and reversed, respectively, these long-lasting behaviors. Formalin injection increased ?5-GABAA receptor mRNA expression in the spinal cord and dorsal root ganglia (DRG) mainly at 3 days. The ?5-GABAA receptors were localized in the dorsal spinal cord and DRG colabeling with NeuN, CGRP, and IB4 which suggests their presence in peptidergic and nonpeptidergic neurons. These receptors were found mainly in small and medium sized neurons. Formalin injection enhanced ?5-GABAA receptor fluorescence intensity in spinal cord and DRG at 3 and 6 days. Intrathecal administration of L-655,708 (15 nmol) prevented and reversed formalin-induced impairment of rate-dependent depression. These results suggest that ?5-GABAA receptors play a role in the loss of GABAergic inhibition and contribute to long-lasting secondary allodynia and hyperalgesia.
| S-EPMC4950669 | biostudies-literature
Project description:Ionotropic glutamate receptors (iGluRs), including the NMDA receptor subtype, are ligand-gated ion channels critical to fast signaling in the CNS. NMDA receptors are obligate heterotetramers composed of two GluN1 and typically two GluN2 subunits. However, the arrangement of GluN subunits in functional receptors-whether like subunits are adjacent to (N1/N1/N2/N2) or diagonal to (N1/N2/N1/N2) one another-remains unclear. Recently, a crystal structure of a homomeric AMPA receptor revealed that the four identical subunits adopt two distinct and subunit-specific conformations termed A/C and B/D with subunits of like conformations (e.g., A/C) diagonal to one another. In the structure, the two conformers were notable at the level of the linkers (S1-M1, M3-S2, and S2-M4) that join the ligand-binding domain to the transmembrane ion channel with the M3-S2 linker positioned more proximal to the central axis of the channel pore in the A/C conformation and S2-M4 more proximal in the B/D conformation. Using immunoblots and functional assays, we show that introduced cysteines in the M3/M3-S2 linker of GluN1, but not GluN2, show dimer formation and oxidation-induced changes in current amplitudes predictive of the A/C conformation. Conversely, introduced cysteines in the S2-M4 linker of GluN2, but not GluN1, showed similar functional effects, suggesting that the GluN2 subunit adopts the B/D conformation. Thus, we show that NMDA receptors, like AMPA receptors, possess distinct subunit-specific conformations with GluN1 approximating the A/C and GluN2 the B/D conformation. GluN subunits are therefore positioned in a N1/N2/N1/N2 arrangement in functional NMDA receptors.
| S-EPMC3207322 | biostudies-literature