Confocal microscopic study of GABA(A) receptors in Xenopus oocytes after rat brain mRNA injection: modulation by tyrosine kinase activity

The expression of GABA(A) receptors in Xenopus oocytes injected with rat brain mRNA was studied by immunocytochemistry and evaluation of the distribution of fluorescent probes at the confocal microscope. The beta(2/3) subunit distributed exclusively on the membrane at the animal pole of the oocytes. Treatment of oocytes for 20 min with the protein tyrosine kinase inhibitor genistein, 200 microM, resulted in a lower presence of GABA(A) receptors on the membrane. The inactive genistein analogue daidzein, 200 microM, had no effect even with a 30 min treatment. Alkaline phosphatase but not a protein tyrosine phosphatase, when injected into oocytes, reduced GABA(A) receptor membrane expression. The data indicate that protein tyrosine phosphorylation modulates the expression on the plasma membrane of presynthesized GABA(A) receptors.     

Responses to GABA, glycine and beta-alanine induced in Xenopus oocytes by messenger RNA from chick and rat brain

Poly (A)+ messenger RNA (mRNA) was extracted from rat and chick brains, and injected into oocytes of Xenopus laevis. This led to the expression of receptors that evoked membrane currents in response to gamma-aminobutyric acid (GABA), glycine and beta-alanine. These currents all inverted at about the chloride equilibrium potential in the oocyte, and showed a marked rectification at negative potentials. Oocytes injected with mRNA from chick optic lobe gave large responses to GABA and beta-alanine, but small responses to glycine. In contrast, one fraction of mRNA from rat cerebral cortex (obtained by sucrose density gradient centrifugation) caused oocytes to develop sensitivity to GABA, glycine and beta-alanine, but very little to GABA. The pharmacological properties of the three amino acid responses also differed. Barbiturate and benzodiazepines potentiated the responses to GABA and beta-alanine, but not to glycine. Strychnine reduced the responses to glycine and beta-alanine, but not to GABA, whereas bicuculline reduced the responses to GABA and beta-alanine, but not to glycine. We conclude that different species of mRNA code for receptors to GABA and glycine, and possibly also for separate beta-alanine receptors.     

Expression of Native GABAA Receptors in Xenopus Oocytes Injected With Rat Brain Synaptosomes

Abstract: Oocytes from the frog Xenopus laevis were shown recently to express native nicotinic acetylcholine receptors after injection with purified Torpedo electroplaque membrane vesicles. Injection of Xenopus oocytes with rat cortical or nigral synaptosomes has now been shown to result in the expression of γ-aminobutyric acid type A (GABA_A) receptor-mediated Cl⁻ currents. Electrophysiological characterization of the responses of these receptors to GABA and other agents revealed that they were incorporated into the oocyte membrane and that they retained their original pharmacological properties, such as sensitivity to Cl⁻ channel blockers, benzodiazepines, and general anesthetics. These results suggest that this approach to the expression of heterologous proteins in Xenopus oocytes may facilitate the study of native synaptic proteins derived from brain tissue.     

红藻氨酸和GABA受体在中华大蟾蜍卵母细胞的表达

本文报道了利用中华大蟾蜍卵母细胞作为外源性膜蛋白的表达及其功能特性研究的模式系统。将大鼠脑的mRNA微量注入蟾蜍卵母细胞(每个卵母细胞注射50ng),在19℃下经48h以上培养后,由外源mRNA表达的大鼠脑的红藻氨酸和γ-氨基丁酸受体被整合到了卵母细胞膜上。红藻氨酸(5×10~(-5)mol/L)和γ-氨基丁酸(10~(-4)mol/L)所诱导的膜电流分别达到294.0±6.4nA(n=5)和309.5±4.9nA(n=4)。红藻氨酸浓度在10~(-3)mol/L时,其诱导的膜电流达最大值。进而,注射mRNA的卵母细胞,~(36)Cl~-流入速度比对照组高一倍多。这些结果表明,中华大蟾蜍卵母细胞,如同爪蟾卵母细胞一样,能表达具有功能的外源膜蛋白(受体蛋白和离子运输蛋白)。     

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.     

Minimal model to account for the membrane conductance increase and desensitization of gamma-aminobutyric acid receptors synthesized in the Xenopus oocytes injected with rat brain mRNA

gamma-Aminobutyric acid (GABA) receptors, which translocate chloride anion with binding GABA, were synthesized in Xenopus oocytes by injecting rat brain mRNA. GABA-elicited responses in the oocytes were measured electrophysiologically by the current-clamped method. Five different measurements were made to establish the relationship between GABA concentration and the electrical responses: (1) the GABA-elicited conductance increase before desensitization; (2) the rate of desensitization of GABA receptors; (3) the rate of recovery of desensitized receptors upon removal of GABA; (4) the GABA-elicited conductance increase after desensitization equilibrium; (5) the fraction of the active form of GABA receptors after desensitization equilibrium. These results were interpreted on the basis of the minimal model proposed for nicotinic acetylcholine receptor in Electrophorus electricus electroplax [Hess, G. P., Cash, D. J., & Aoshima, H. (1983) Annu. Rev. Biophys. Bioeng. 12, 443-473]. Estimated equilibrium and rate constants in the model for GABA receptors could successfully explain the results of the five above measurements.     

Effect of lipid hydroperoxide on Xenopus oocytes and on neurotransmitter receptors synthesized in Xenopus oocytes injected with exogenous mRNA

The effect of 13-L-hydroperoxylinoleic acid (LOOH) on both Xenopus oocytes and neurotransmitter receptors synthesized in the oocytes was studied by electrophysiological and ion flux measurement. Addition of LOOH to the incubation mixture of the oocytes raised the membrane potential and decreased the membrane resistance of the oocytes. These effects of LOOH on the oocytes were reversed within a few hours by incubation with frog Ringer solution. Addition of LOOH also caused an increase of Li+ and 45Ca2+ uptake into the oocytes. However, production of alkoxy radicals by the addition of FeCl2 to the incubation mixture containing LOOH did not accelerate the damage to the oocytes by LOOH. So essential toxicity is caused possibly by an increase in the membrane permeability resulting from disturbance of the lipid bilayer arrangement, not from production of active alkoxy radicals during decomposition of LOOH. Nicotinic acetylcholine and gamma-aminobutyric acid receptors were synthesized in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and rat brain. LOOH noncompetitively inhibited the function of these receptors and also increased the rate of desensitization of the receptors.     

Expression of GABA and glycine receptors by messenger RNAs from the developing rat cerebral cortex

The ontogenesis of mRNAs coding for GABA and glycine receptors in the cerebral cortex of the rat was examined by extracting poly(A)+ mRNA from the brains of embryonic, postnatal or adult rats and injecting it into Xenopus oocytes. The ability of a messenger to express functional receptors was then assayed by measuring the membrane currents elicited by the agonists. The size of the GABA-induced current increased progressively with age, being undetectable in oocytes injected with mRNA from embryonic day 15 and reaching a maximum in oocytes injected with mRNA from postnatal day 30. In contrast, the glycine-induced response was negligible in oocytes injected with mRNA from the cerebral hemispheres of embryos 15 days old; it increased sharply to a maximum with newborn animals and then decreased with age to become very small with mRNA from adult cortex. GABA and glycine receptors induced by mRNA from the cerebral cortex of all ages are associated with chloride channels.     

Serotonin receptors induced by exogenous messenger RNA in Xenopus oocytes

When poly(A)+-mRNA, extracted from rat brain, was injected into Xenopus laevis oocytes, it induced the appearance of serotonin receptors in the oocyte membrane. Application of serotonin to injected oocytes elicited, after a long delay, oscillations in membrane current. The equilibrium potential of this current corresponded with the chloride equilibrium potential. It appears that rat brain mRNA encodes the translation of serotonin receptors into the oocyte membrane. The combination of serotonin with these receptors leads to the opening of membrane channels.     

Zn2+对爪蟾卵母细胞表达鲫鱼脑GABA受体的调制作用

爪蟾卵母细胞注射鲫鱼脑ｍＲＮＡ后表达的ＧＡＢＡ受体中约８５％为ＧＡＢＡＡ受体。约１５％的成分为ＧＡＮＡＣ受体。本文利用双电极电压箝方法结合药物灌流研究了Ｚｎ６２＋对这两型受体的作用。我们观察到了Ｚｎ＾２＋对它们的调制都是可抑制性的,可逆的。     

Cross-talk and co-trafficking between rho1/GABA receptors and ATP-gated channels

Gamma-aminobutyric-acid (GABA) and ATP ionotropic receptors represent two structurally and functionally different classes of neurotransmitter-gated channels involved in fast synaptic transmission. We demonstrate here that, when the inhibitory rho1/GABA and the excitatory P2X2 receptor channels are co-expressed in Xenopus oocytes, activation of one channel reduces the currents mediated by the other one. This reciprocal inhibitory cross-talk is a receptor-mediated phenomenon independent of agonist cross-modulation, membrane potential, direction of ionic flux, or channel densities. Functional interaction is disrupted when the cytoplasmic C-terminal domain of P2X2 is deleted or in competition experiments with minigenes coding for the C-terminal domain of P2X2 or the main intracellular loop of rho1 subunits. We also show a physical interaction between P2X2 and rho1 receptors expressed in oocytes and the co-clustering of these receptors in transfected hippocampal neurons. Co-expression with P2X2 induces retargeting and recruitment of mainly intracellular rho1/GABA receptors to surface clusters. Therefore, molecular and functional cross-talk between inhibitory and excitatory ligand-gated channels may regulate synaptic strength both by activity-dependent current occlusion and synaptic receptors co-trafficking.     

Potentiation and Inhibition of Ionotropic Neurotransmitter Receptors Expressed in Xenopus Oocyte by Linoleic Acid and Its Hydroperoxide

Abstract: To study the effects of lipid hydroperoxide on ionotropic neurotransmitter receptors, γ-aminobutyric acid (GABA), N -methyl- d -aspartate (NMDA), and non-NMDA receptors (GABARs, NMDARs, and non-NMDARs, respectively) were expressed in Xenopus oocytes that received an injection of mRNA prepared from rat whole brain. Linoleic acid (LA) and its hydroperoxide 13- l -hydroperoxylinoleic acid (LOOH) prepared with soybean lipoxygenase inhibited the response of GABARs in the presence of GABA at high concentrations. The inhibition was stronger when the inhibitors were perfused 1 min before a mixture of GABA and the inhibitors than when they were perfused simultaneously with GABA. On the other hand, only LOOH potentiated the response of GABARs in the presence of GABA at low concentrations, possibly increasing the affinity of GABA to the receptors. Both LA and LOOH accelerated the rate of desensitization of GABARs, but LOOH did not affect their equilibrium between the active and desensitized form of the receptors. They also inhibited the response of NMDARs in a noncompetitive manner but barely inhibited the response of non-NMDARs in the presence of kainate at various concentrations. These results suggest the possibility that production of lipid hydroperoxide modulates the neural transmission in the brain, especially through GABARs.     

Effects of bisphenol A and its derivatives on the response of GABA(A) receptors expressed in Xenopus oocytes.

To study the effects of bisphenol-A (BPA) known to have estrogenic actions, and its derivatives, 3,5-dimethylphenol (DMP) and p-t-butylphenol (TBP), on ionotropic gamma-aminobutyric acid (GABA) receptors, GABA(A) receptors were expressed in Xenopus oocytes by injecting both poly(A)+ RNA prepared from rat whole brain and cRNAs synthesized from cloned cDNAs of alpha1 and beta1 subunit of the bovine receptors, and their electrical responses were measured by the voltage clamping method. BPA caused the potentiation and inhibition of the former receptor-responses, while it caused only inhibition of the latter ones. In the presence of low concentrations of GABA, DMP and TBP potentiated the responses of both receptors. DMP and TBP also increased the rate of decay of the response, possibly by desensitization of the receptors when GABA solution was continuously bath-applied. Diethyl terephthalate (DTP), which is also known to have estrogenic actions, had little effect on both the responses and the decay of both receptors.     

Potentiation of GABAA receptors expressed in Xenopus oocytes by perfume and phytoncid.

To study the effects of perfume and phytoncid on GABAA receptors, ionotropic GABAA receptors were expressed in Xenopus oocytes by injecting mRNAs that had been prepared from rat whole brain. Essential oil, perfume and such phytoncid as leaf alcohol, hinokitiol, pinene, eugenol, citronellol and citronellal potentiated the response in the presence of GABA at low concentrations (10 and 30 microM), possibly because they bound to the potentiation-site in GABAA receptors and increased the affinity of GABA to the receptors. Since it is known that the potentiation of GABAA receptors by benzodiazepine, barbiturate, steroids and anesthetics induces the anxiolytic, anticonvulsant and sedative activity or anesthetic effect, these results suggest the possibility that the intake of perfume or phytoncid through the lungs, the skin or the intestines modulates the neural transmission in the brain through ionotropic GABAA receptors and changes the frame of the human mind, as alcohol or tobacco does.     

A GABA(A) receptor of defined subunit composition and positioning: concatenation of five subunits

We show that the five subunits of a gamma-aminobutyric acid type A receptor (GABA(A) receptor) can be concatenated to yield a functional receptor. This concatenated receptor alpha(1)-beta(2)-alpha(1)-gamma(2)-beta(2) has the advantage of a known subunit arrangement. Most of its functional properties are not significantly different from a receptor formed by individual subunits. Extent of expression amounted to about 40% of that of non-concatenated receptors in Xenopus oocytes, after injection of oocytes with comparable amounts of cRNA coding for concatenated and non-concatenated receptors. The ability to express receptors consisting of five subunits enables detailed studies of GABA(A) receptor subtype selective compounds.     

Subunit-specific coupling between gamma-aminobutyric acid type A and P2X2 receptor channels

ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central synapses, where they co-activate excitatory P2X and inhibitory GABAA (GABA type A) receptors. We report here that co-activation of P2X2 and various GABAA receptors, co-expressed in Xenopus oocytes, leads to a functional cross-inhibition dependent on GABAA subunit composition. Sequential applications of GABA and ATP revealed that alphabeta- or alphabetagamma-containing GABAA receptors inhibited P2X2 channels, whereas P2X2 channels failed to inhibit gamma-containing GABAA receptors. This functional cross-talk is independent of membrane potential, changes in current direction, and calcium. Non-additive responses observed between cation-selective GABAA and P2X2 receptors further indicate the chloride independence of this process. Overexpression of minigenes encoding either the C-terminal fragment of P2X2 or the intracellular loop of the beta3 subunit disrupted the functional cross-inhibition. We previously demonstrated functional and physical cross-talk between rho1 and P2X2 receptors, which induced a retargeting of rho1 channels to surface clusters when co-expressed in hippocampal neurons (Boue-Grabot, E., Emerit, M. B., Toulme, E., Seguela, P., and Garret, M. (2004) J. Biol. Chem. 279, 6967-6975). Co-expression of P2X2 and chimeric rho1 receptors with the C-terminal sequences of alpha2, beta3, or gamma2 subunits indicated that only rho1-beta3 and P2X2 channels exhibit both functional cross-inhibition in Xenopus oocytes and co-clustering/retargeting in hippocampal neurons. Therefore, the C-terminal domain of P2X2 and the intracellular loop of beta GABAA subunits are required for the functional interaction between ATP- and GABA-gated channels. This gamma subunit-dependent cross-talk may contribute to the regulation of synaptic activity.     

Study of the nematode putative GABA type-A receptor subunits: evidence for modulation by ivermectin

Two alleles of the HG1 gene, which encodes a putative GABA receptor alpha/gamma subunit, were isolated from Haemonchus contortus. These two alleles were shown previously to be associated with ivermectin susceptibility (HG1A) and resistance (HG1E), respectively. Sequence analysis indicates that they differ in four amino acids. To explore the functional properties of the two alleles, a full-length cDNA encoding the beta subunit, a key functional component of the GABA receptor, was isolated from Caenorhabditis elegans (gab-1, corresponding to the GenBank locus ZC482.1) and coexpressed in Xenopus oocytes with the HG1 alleles. When gab-1 was coexpressed with either the HG1A allele or the HG1E allele in Xenopus oocytes, gamma-aminobutyric acid (GABA)-responsive channels with different sensitivity to the agonist were formed. The effects of ivermectin on the hetero-oligomeric receptors were determined. Application of ivermectin alone had no effect on the receptors. However, when coapplied with 10 micro m GABA, ivermectin potentiated the GABA-evoked current of the GAB-1/HG1A receptor, but attenuated the GABA response of the GAB-1/HG1E receptor. We demonstrated that the coexpressed HG1 and GAB-1 receptors are GABA-responsive, and provide evidence for the possible involvement of GABA receptors in the mechanism of ivermectin resistance.     

Aminomethyl-2,6-difluorophenols as a novel class of increased lipophilicity GABA(C) receptor antagonists

3- and 4-(Aminomethyl)-2,6-difuorophenols were tested for activity against the three major classes of GABA receptors. 4-(Amninomethyl)-2,6difluorophenol was shown to be a competitive and somewhat selective antagonist at p1 GABA(C) receptors expressed in Xenopus oocytes (K(B) = 75.5 microM with a 95% Confidence Interval range of 75.2 microM to 75.8 microM). This is the first in a novel class of increased lipophilicity GABA(C) receptor antagonists with little activity at alpha1beta2gamma2 GABA(A) and GABA(B) receptors.