Properties of human brain glycine receptors expressed in Xenopus oocytes

Glycine and gamma-aminobutyric acid (GABA) receptors from the foetal human brain were 'transplanted' into the Xenopus oocyte membrane by injecting the oocytes with poly(A)+-mRNA extracted from the cerebral cortex. Activation of both glycine and GABA receptors induced membrane currents carried largely by chloride ions. However, unlike the GABA-activated current, the glycine current was blocked by strychnine, and was not potentiated by barbiturate. At low doses, the glycine current increased with concentration following a 2.7th power relation, suggesting that binding of three molecules of glycine may be required to open a single membrane channel. The current induced by steady application of glycine decreased with hyperpolarization beyond about -60 mV.     

GABA increases both the conductance and mean open time of recombinant GABAA channels co-expressed with GABARAP

The single channel properties of recombinant gamma-aminobutyric acid type A (GABA(A))alphabetagamma receptors co-expressed with the trafficking protein GABARAP were investigated using membrane patches in the outside-out patch clamp configuration from transiently transfected L929 cells. In control cells expressing alphabetagamma receptors alone, GABA activated single channels whose main conductance was 30 picosiemens (pS) with a subconductance state of 20 pS, and increasing the GABA concentration did not alter their conductance. In contrast, when GABA(A) receptors were co-expressed with GABARAP, the GABA-activated single channels displayed multiple, high conductances (> or =40 pS), and GABA (> or =10 microM) was able to increase their conductance, up to a maximum of 60 pS. The mean open time of GABA-activated channels in control cells expressing alphabetagamma receptors alone was 2.3 +/- 0.1 ms for the main 30-pS channel and shorter for the subconductance state (20 pS, 0.8 +/- 0.1 ms). Similar values were measured for the 30- and 20-pS channels active in patches from cells co-expressing GABARAP. However higher conductance channels (> or =40 pS) remained open longer, irrespective of whether GABA or GABA plus diazepam activated them. Plotting mean open times against mean conductances revealed a linear relationship between these two parameters. Since high GABA concentrations increase both the maximum single channel conductance and mean open time of GABA(A) channels co-expressed with GABARAP, trafficking processes must influence ion channel properties. This suggests that the organization of extrasynaptic GABA(A) receptors may provide a range of distinct inhibitory currents in the brain and, further, provide differential drug responses.     

缓激肽抑制大鼠DRG分离神经元的GABA激活电流

本文应用全细胞膜片箝技术在新鲜分离的大鼠背根神经节（ＤＲＧ）神经元上研究缓激肽（ＢＫ）对γ－氨基丁酸（ＧＡＢＡ）反应的调制作用。结果发现：在３４个对ＧＡＢＡ反应的细胞中有３１个细胞对ＢＫ敏感。在对ＢＫ敏感并引起内向电流的２７个细胞中预加ＢＫ,对ＧＡＢＡ－激活电流具有明显的抑制作用,如１０＾－６ｍｏｌ／Ｌ的ＢＫ可抑制ＧＡＢＡ（１０＾－４ｍｏｌ／Ｌ）激活电流３０％。ＢＫ可将ＧＡＢＡ量效曲线明显下移,并     

Insulin reduces neuronal excitability by turning on GABA(A) channels that generate tonic current

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 GABA(A) 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 hippocampal slices that insulin (1 nM) "turns on" new extrasynaptic GABA(A) 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 (EC(50)) in intact CA1 neurons of 17 pM with the maximal current amplitude reached with 1 nM GABA. They are inhibited by GABA(A) 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.     

Properties of GABA-induced transmembrane currents in isolated neurons of the rat spinal ganglia

Using techniques of voltage-clamp in the whole-cell configuration and fast local superfusion, we studied the properties of transmembrane ion currents evoked in freshly isolated neurons of the spinal ganglia of rats by application of γ-aminobutyric acid, GABA, in different concentrations. Increases in the GABA concentration and application time resulted in modification of the amplitude and kinetic parameters of the currents. The dependence between the current amplitude and GABA concentration could be adequately described by the Hill equation. The current rise could be fitted by a sum of two exponential curves with different time constants; the time constant of the second exponent changed with an increase in the GABA concentration, while the first exponent was not sensitive to these changes. The current decay also should be fitted by two exponents. The time constant of the first exponent did not change with increases in the GABA concentration or duration of its application; at the same time the second exponent noticeably depended on the time of GABA application. Our experiments demonstrated that the density of GABA-activated ion channels in the membranes of the studied spinal ganglion cells is relatively high; this finding allows us to suppose possible involvement of these channels in regulation of the transmembrane conductivity in these cells.     

Pre & postsynaptic tuning of action potential timing by spontaneous GABAergic activity

Frequency and timing of action potential discharge are key elements for coding and transfer of information between neurons. The nature and location of the synaptic contacts, the biophysical parameters of the receptor-operated channels and their kinetics of activation are major determinants of the firing behaviour of each individual neuron. Ultimately the intrinsic excitability of each neuron determines the input-output function. Here we evaluate the influence of spontaneous GABAergic synaptic activity on the timing of action potentials in Layer 2/3 pyramidal neurones in acute brain slices from the somatosensory cortex of young rats. Somatic dynamic current injection to mimic synaptic input events was employed, together with a simple computational model that reproduce subthreshold membrane properties. Besides the well-documented control of neuronal excitability, spontaneous background GABAergic activity has a major detrimental effect on spike timing. In fact, GABA(A) receptors tune the relationship between the excitability and fidelity of pyramidal neurons via a postsynaptic (the reversal potential for GABA(A) activity) and a presynaptic (the frequency of spontaneous activity) mechanism. GABAergic activity can decrease or increase the excitability of pyramidal neurones, depending on the difference between the reversal potential for GABA(A) receptors and the threshold for action potential. In contrast, spike time jitter can only be increased proportionally to the difference between these two membrane potentials. Changes in excitability by background GABAergic activity can therefore only be associated with deterioration of the reliability of spike timing.     

The neuroactive steroids alphaxalone and pregnanolone increase the conductance of single GABAA channels in newborn rat hippocampal neurons

The effects of the neuroactive steroids alphaxalone and pregnanolone on single GABA(A) receptor channels were tested in cell-attached and inside-out patches from cultured newborn rat hippocampal neurons. The conductance of these single channels ranged between 10 and 80 pS when exposed to low (0.5-3 microM) GABA concentrations. These GABA concentrations activated low-conducting channels (<40 pS) in 78% of the patches, 22% of patches had channels with a maximum conductance above 40 pS. Alphaxalone at concentrations above 1 microM, and pregnanolone at concentrations above 0.1 microM, significantly increased the conductance of initially low-conducting single channels activated by GABA up to seven-fold and at all concentrations tested, both drugs increased open probability and mean open time and decreased closed probability and mean closed time of channels. Both steroids at higher concentrations could directly activate high conductance (>40 pS) chloride channels. Both the directly activated channels and those channels that had been previously affected by alphaxalone were modulated by diazepam, a benzodiazepine drug that is known to specifically modulate GABA(A) channels. The present study is the first one to show that neurosteroids can significantly increase single GABA(A) channel conductance, thus enlarging our current knowledge on the molecular mechanism of action of these compounds.     

Graded response to GABA by native extrasynaptic GABA receptors

GABA is the main inhibitory neurotransmitter in the mammalian CNS. GABA in the brain is commonly associated with a fast, point-to-point form of signalling called synaptic transmission (phasic inhibition), but there is growing evidence that GABA participates in another, slower and more diffuse form of signalling often referred to as tonic inhibition. Unresolved questions regarding tonic neuronal inhibition concern activation and functional properties of extrasynaptic GABAA receptors (GABARex) present on neurones. Extrasynaptic receptors are exposed to submicromolar GABA concentrations and may modulate the overall excitability of neurones and neuronal networks. Here, we examined GABA-activated single-channel currents in dentate gyrus granule neurones in rat hippocampal slices. We activated three types (I, II, III) of GABARex channels by nanomolar GABA concentrations (EC50 I: 27 +/- 12; II: 4 +/- 3; III: 43 +/- 19 nm). The channels opened after a delay and the single-channel conductance was graded (gammamax I: 61 +/- 3; II: 85 +/- 8, III: 40 +/- 3 pS). The channels were differentially modulated by 1 microm diazepam, 200 nm zolpidem, 1 microm flumazenil and 50 nm THDOC (3alpha, 21-dihydroxy-5alpha-pregnan-20-one), consistent with the following minimal subunit composition of GABARex I alpha1betagamma2, GABARex II alpha4betagamma2 and GABARex III alphabetadelta channels.     

Factors affecting actions of ethanol on GABA-activated chloride channels.

Effects of ethanol in vitro on membrane vesicles (microsacs prepared from mouse cerebral cortex) were evaluated by monitoring 36Cl- influx. Different assay parameters were tested to determine increased or decreased action of ethanol on GABA-activated chloride channels. The ability of 30 mM ethanol to augment 36Cl- flux was seen at 0 degrees C, in the absence of GABA ("direct" action of ethanol), and at 34 degrees C in the presence of GABA, using two different assay procedures. Picrotoxin blocked the direct effects of ethanol (at 0 degrees C) suggesting GABAa involvement. Endogenous GABA in the medium surrounding the microsacs was assayed at different temperatures both in the presence and absence of GABA and ethanol. The direct effect of ethanol did not appear to involve the action of endogenous GABA. In addition to temperature effects on the assay, time of membrane storage also influenced ethanol action. Microsacs stored on ice for 2 hours or more lost their ability to respond to ethanol but not to GABA, pentobarbital or flunitrazepam. When these drugs were tested on membranes from mice that had been sacrificed by cervical dislocation as opposed to decapitation, ethanol did not augment GABA-stimulated chloride flux. The method of sacrifice did not influence the response to GABA, pentobarbital or flunitrazepam.     

Patch-clamp recordings of spiking and nonspiking interneurons from rabbit olfactory bulb slices: GABA- and other transmitter receptors

Summary Transmitter receptor ion channels from previously identified rabbit olfactory bulb neurons were studied by using a thin slice preparation in combination with patch-clamp measurements. PG cells, which closely resembled previously described periglomerular interneurons in their morphology, responded to microapplication of GABA, acetylcholine, norepinephrine and glycine with the activation of distinct ionic currents. JG cells, which belong either to the class of short axon cells or external tufted cells, never showed GABA responses. In mitral cells ionic currents activated by GABA, acetylcholine, norepinephrine and glutamate could be elicited. Further measurements of GABA-activated currents of PG cells were made and indicated that these cells expressed two different types of GABA receptors: one which showed fast desensitization with a decay time constant of about 5 s, and one which slowly desensitized with a decay time constant of about 20–30 s. Both types were completely inhibited by bicuculline methiodide (50 M). GABA receptors were not blocked by Zn²⁺ (0.1 mM). From the dose-response relationship of the peak GABA-activated currents, an apparent dissociation constant of 50 M was derived. From single channel measurements in excised outside-out patches, a single channel conductance of GABA-activated Cl^– currents of 24 pS was obtained during continuous application of the agonist. Single channel events had a mean open time of 1.9 ms.     

非洲爪蟾卵母细胞GABAB和GABAc受体介导的电流反应

实验应用双电极电压箝技术,在具有滤泡膜的非洲爪蟾(Xenopuslaevis)卵母细胞上记录到γ-氨基丁酸(γ-aminobutyricacid,GABA)-激活电流。此GABA-激活电流的特点及有关GABA受体类型的研究和分析如下(1)在35.5%(55/155)的受检细胞外加GABA可引起一慢的浓度依赖性的外向电流。(2)GABAA受体的选择性拮抗剂bicuculline(10     

咖啡因对大鼠背根神经节急性分离神经元GABA-激活电流的抑制作用

应用全细胞膜片钳记录技术,在大鼠新鲜分离背根神经节(dorsal root ganglion,DRG)神经元上,观察预加咖啡因对GABA-激活电流(IGABA)的调制作用。实验中,大部分受检细胞(97．4％,l13／116)对外加GABA敏感。1-1000μmol／L GABA引起一剂量依赖性、有明显上敏感作用的内向电流。在受检的108个DRG细胞中,约有半数(53．7％,58／108)对胞外加咖啡因(0．1-100μmol／L)敏感．产生一幅值很小的内向电流。倾加咖啡因(0．1～100μmol／L)30s后再加GABA能明显抑制GABA(100μmol／L)激活电流的幅值。预加咖啡因后GABA量效曲线明显下移;GABA-激活电流的最人值较之对照下降约57％;而Kd值(30μmol／L)几乎不变,表示此种抑制为非竞争性的。预加安定(diazepam,1μmol／L)对GABA(100μmol／L)激活电流有增强作用,而预加咖啡因(10μmol／L)有拈抗安定增强IGABA的作用。胞内透析H-8后,几乎可以完全消除咖啡因对,IGABA的抑制作用。已知GABA作用于初级感觉神经元能引起初级传入去极化,因而实验结果提示,咖啡因有可能在初级传入末梢产生对抗突触前抑制的效应。     

The GABA postsynaptic membrane receptor-ionophore complex

Summary The function of the inhibitory neurotransmitter, -aminobutyric acid (GABA), has been implicated in the mode of action of many drugs which excite or depress the central nervous system. Many convulsant agents appear to block GABA action whereas anticonvulsants enhance GABA action. Some of these drug effects involve altered GABA-mediated synaptic transmission at the level of GABA biosynthesis, release from nerve endings, uptake into cells, and metabolic degradation. A greater number of agents of diverse classes appear to affect GABA action at the postsynaptic membrane, as determined from both electrophysiological and biochemical studies. The recently developedin vitro radioactive receptor binding assays have led to a wealth of new information about GABA action and its alteration by drugs. GABA inhibitory transmission involves the regulation, by GABA binding to its receptor site, of chloride ion channels. In this GABA receptor-ionophore system, other drug receptor sites, one for benzodiazepines and one for barbiturates/picrotoxinin (and related agents) appear to form a multicomponent complex. In this complex, the drugs binding to any of the three receptor categories are visualized to have an effect on GABA-associated chloride channel regulation. Available evidence suggests that the complex mediates many of the actions of numerous excitatory and depressant drugs showing a variety of pharmacological effects.     

Pharmacological analysis of the activation and receptor properties of the tonic GABA(C)R current in retinal bipolar cell terminals

GABAergic inhibition in the central nervous system (CNS) can occur via rapid, transient postsynaptic currents and via a tonic increase in membrane conductance, mediated by synaptic and extrasynaptic GABA(A) receptors (GABA(A)Rs) respectively. Retinal bipolar cells (BCs) exhibit a tonic current mediated by GABA(C)Rs in their axon terminal, in addition to synaptic GABA(A)R and GABA(C)R currents, which strongly regulate BC output. The tonic GABA(C)R current in BC terminals (BCTs) is not dependent on vesicular GABA release, but properties such as the alternative source of GABA and the identity of the GABA(C)Rs remain unknown. Following a recent report that tonic GABA release from cerebellar glial cells is mediated by Bestrophin 1 anion channels, we have investigated their role in non-vesicular GABA release in the retina. Using patch-clamp recordings from BCTs in goldfish retinal slices, we find that the tonic GABA(C)R current is not reduced by the anion channel inhibitors NPPB or flufenamic acid but is reduced by DIDS, which decreases the tonic current without directly affecting GABA(C)Rs. All three drugs also exhibit non-specific effects including inhibition of GABA transporters. GABA(C)R ρ subunits can form homomeric and heteromeric receptors that differ in their properties, but BC GABA(C)Rs are thought to be ρ1-ρ2 heteromers. To investigate whether GABA(C)Rs mediating tonic and synaptic currents may differ in their subunit composition, as is the case for GABA(A)Rs, we have examined the effects of two antagonists that show partial ρ subunit selectivity: picrotoxin and cyclothiazide. Tonic and synaptic GABA(C)R currents were differentially affected by both drugs, suggesting that a population of homomeric ρ1 receptors contributes to the tonic current. These results extend our understanding of the multiple forms of GABAergic inhibition that exist in the CNS and contribute to visual signal processing in the retina.     

Biochemical pharmacology of the gamma-aminobutyric acid receptor/ionophore protein

The synaptic receptor sites for the neurotransmitter gamma-aminobutyric acid (GABA) can be assayed in vitro with several radiolabeled agonists and one antagonist. Numerous criteria of specificity have been met for these binding sites. All of the ligands show heterogeneity in binding affinities. The subpopulations thus defined have a remarkably similar specificity for GABA analogs, which suggests an intimate relationship and possible interconvertibility. Modulation of GABA receptor binding by barbiturates, anions, and other membrane treatments that affect agonists and antagonists in an opposite manner suggests a three-state model of interconvertible affinities. The complex of GABA receptor and chloride ion channel contains modulatory sites for barbiturates and benzodiazepines, drugs that enhance GABA responses in neurons. The receptor complex can be solubilized in detergent with the three mutually interacting receptor activities intact. The complex has an apparent molecular weight of 355,000 and has been partially purified. GABA agonist function has been assayed at the biochemical level by measuring the activation of 36Cl- efflux from preloaded hippocampal slices by GABA, muscimol, and barbiturates. This response is blocked by the antagonists of the GABA site (bicuculline) and the barbiturate site (picrotoxin). Comparison of binding and function on the same tissue should be useful in analyzing the mechanism of action of GABA.     

SKF38393抑制大鼠DRG分离神经元GABA-激活电流

在大鼠新鲜分离ＤＲＧ神经元标本上应用全细胞膜片箝记录,观察了多巴胺Ｄ１受体的选择性激动剂ＳＫＦ３８３９３ＨＣＩ对ＧＡＢＡ－激活电流的作用。大部分受检细胞对ＧＡＢＡ敏感,１０＾－６－１０＾－３－ｍｏｌ／Ｌ ＧＡＢＡ可于引起呈剂量依赖性的明显去敏感作用的内向电流。     

可乐定对背根神经节神经元GABA激活电流的抑制作用

本实验在新鲜分离大鼠背根神经节（ＤＲＧ）细胞上应用全细胞膜片的箝记录研究贤上腺素α２－受体激动剂可乐定（ｃｌｏｎｉｄｉｎｅ）对ＧＡＢＡ－激活电流的调制作用。发现缘大多数ＤＲＧ细胞对ＧＡＢＡ（１０＾－６￣１０＾－３ｍｏｌ／Ｌ）敏感（７２／７５）,产生浓度依赖性的内向电流;并且可被ｂｉｃｕｃｕｌｉｎｅ（１０＾－５￣１０＾－４ｍｏｌ／Ｌ）所阻断。在多数细胞中（５１／７２）预加可乐定（１０＾－８￣１０＾－     

GABAB-receptor-mediated suppression of sympathetic outflow from the spinal cord of neonatal rats.

Using a splanchnic nerve-spinal cord preparation in vitro that could spontaneously generate sympathetic nerve discharge (SND), we investigated the roles of intraspinal GABA(B) receptors in the regulation of SND. Despite an age-dependent difference in sensitivity, bath applications of baclofen (Bac; GABA(B)-receptor agonist) consistently reduced SND in a concentration-dependent manner. The drug specificity of Bac in activation of GABA(B) receptors was verified by application of its antagonist saclofen (Sac) or CGP-46381 (CGP). Sac or CGP alone did not change SND. However, in the presence of Sac or CGP, the effects of Bac on SND inhibition were reversibly attenuated. The splanchnic sympathetic preganglionic neuron (SPN) was recorded by blind whole cell, patch-clamp techniques. We examined Bac effects on electrical membrane properties of SPNs. Applications of Bac reduced excitatory synaptic events, induced membrane hyperpolarizations, and inhibited SPN firing. In the presence of 12 mM Mg2+ or 0.5 microM TTX to block Ca2+- or action potential-dependent synaptic transmissions, applications of Bac induced an outward baseline current that reversed at -29 +/- 6 mV. Because the K+ equilibrium potential in our experimental conditions was -100 mV, the Bac-induced currents could not simply be attributed to an alteration of K+ conductance. On the other hand, applications of Bac to Cs+-loaded SPNs reduced Cd2+-sensitive and high-voltage-activated inward currents, indicating an inhibition of voltage-gated Ca2+ currents. Our results suggest that the activation of intraspinal GABA(B) receptors suppresses SND via a mixture of ion events that may link to a change in Ca2+ conductance.     

Interaction of barbiturates with dihydropicrotoxinin binding sites related to the GABA receptor-ionophore system.

Barbiturate drugs of diverse chemical structure inhibited the binding of [³H] α-dihydropicrotoxinin to rat brain membranes. This biologically active analoque of picrotoxin labels membrane sites related to the convulsant action of these drugs in inhibiting GABA postsynaptic receptor-ionophore function at a site distinct from the GABA receptor. Depressant barbiturates such as pentobarbital inhibited dihydropicrotoxinin binding competitively at therapeutic concentrations (IC₅₀ = 50 μM) whereas the drug does not alter GABA receptors, uptake, or release at this concentration. Antiepileptics such as phenobarbital (IC₅₀=400 μM), were weaker inhibitors of binding. Convulsant barbiturates, however, such as dimethylbutylbarbiturate (IC₅₀=0.05 μM) and cyclohexylidene-ethyl barbiturate (IC₅₀=0.7 μM), were potent inhibitors. The displacement of radioactive dihydropicrotoxinin binding by the convulsant barbiturates had different slopes and Hill numnbers (0.4) compared to displacement by depressant barbiturates and picrotoxinin itself (Hill numbers = 1.0), indicating heterogeneity of binding sites or negative cooperativity. These potent intractions of barbiturates with dihydropicrotoxinin binding sites are consistent with neurophysiological evidence that depressant or convulsant action of barbiturates may involve modulation of CNS inhibitory synaptic transmission at the level of the postsynaptic GABA receptor-ionophores.     

A conductance decrease after application of GABA to crayfish muscle fibers

Relaxations after voltage steps of membrane current elicited by superfusion with low concentrations of GABA (up to 50 mumol/l) were measured. In many preparations, a conductance decrease due to GABA was observed. The response to GABA was shown to consist of two major components: the well known opening of synaptic chloride channels, and the closing of previously open channels, presumably permeable to K+ ions. The latter component could not be blocked by picrotoxin.