Role of Glutamate and GABA Transporters in Development of Pentylenetetrazol-Kindling

Kindling is a form of epileptogenesis that can be induced with pentylenetetrazol (PTZ). We undertook this study to evaluate the contribution of glutamate and GABA transporters to the process of PTZ kindling. Rats were injected i.p. three times per week with PTZ (40 mg/kg) until they were fully kindled. In rats who achieved full kindling, measurement of hippocampal glutamate and GABA transporters within 24 h by western blot showed that GLAST, GLT-1, and EAAC1 were elevated significantly. However, fully kindled rats at 30 days after their last seizure had no change in either glutamate or GABA transporters proteins. These sequential observations suggest that glutamate transporters may contribute to the occurrence of seizures, but were not associated with maintenance of epileptogenesis. During this experiment, we collected data from animals that had kindled easily and animals who were resistant to kindling. Easily-kindled rats reached full kindling with less than five injections of PTZ. Kindling resistant animals failed to achieve full kindling even after administration of 12 consecutive injections of PTZ. Levels of EAAC1 and GAT-1 in easily-kindled rats were decreased by 30% when compared to kindling resistant animals at 30 days after the last PTZ injection. Since decreased EAAC1 and GAT-1 would diminish GABA function, less quantity of these proteins would appear to be associated with the convulsive threshold at the beginning of kindling development. We wonder if glutamate and GABA transporters might be operant in a convulsion threshold set factor or as a pace factor for kindling.     

Overexpression of γ－aminobutyric acid transporter subtype I leads to susceptibility to kainic acid－induced seizure in transgenic mice

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABA(A) receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters.     

Reduction of glial glutamate transporters in the parietal cortex and hippocampus of the EL mouse

There is extensive experimental evidence indicating a crucial role for glutamate in epileptogenesis and epileptic activity. The glial glutamate transporters GLT1 and GLAST are proposed to account for the majority of extracellular glutamate re-uptake. In the present study, polyclonal antibodies specific to GLT1 and GLAST were generated and characterized, revealing distribution patterns for the two transporters confirming those previously reported. In situ hybridization and immunoblotting were then used to compare levels of these two transporters in the parietal cortex and hippocampus of unstimulated and stimulated EL mice with DDY control mice. Additionally, HPLC determined tissue glutamate concentrations in the same regions of these animals. These experiments revealed reductions in GLT1 mRNA and protein in the parietal cortex of unstimulated and stimulated EL mice compared with DDY controls, accompanied by an increase in tissue glutamate concentration in the stimulated EL mice group. GLT1 mRNA was also reduced in the CA3 hippocampal subfield of both unstimulated and stimulated EL mice. GLAST protein was reduced in the hippocampus of the stimulated EL mice group, while no changes in GLAST mRNA or protein were detected in the parietal cortex of EL mice when compared with DDY controls. The glial glutamate transporter down-regulation reported here may play a role in seizure initiation, spread and maintenance in the EL mouse.     

Selective down-regulation of the astrocyte glutamate transporters GLT-1 and GLAST within the medial thalamus in experimental Wernicke's encephalopathy

Although earlier studies on thiamine deficiency have reported increases in extracellular glutamate concentration in the thalamus, a vulnerable region of the brain in this disorder, the mechanism by which this occurs has remained unresolved. Treatment with pyrithiamine, a central thiamine antagonist, resulted in a 71 and 55% decrease in protein levels of the astrocyte glutamate transporters GLT-1 and GLAST, respectively, by immunoblotting in the medial thalamus of day 14 symptomatic rats at loss of righting reflexes. These changes occurred prior to the onset of convulsions and pannecrosis. Loss of both GLT-1 and GLAST transporter sites was also confirmed in this region of the thalamus at the symptomatic stage using immunohistochemical methods. In contrast, no change in either transporter protein was detected in the non-vulnerable frontal parietal cortex. These effects are selective; protein levels of the astrocyte GABA transporter GAT-3 were unaffected in the medial thalamus. In addition, astrocyte-specific glial fibrillary acidic protein (GFAP) content was unchanged in this brain region, suggesting that astrocytes are spared in this disorder. Loss of GLT-1 or GLAST protein was not observed on day 12 of treatment, indicating that down-regulation of these transporters occurs within 48 h prior to loss of righting reflexes. Finally, GLT-1 content was positively correlated with levels of the neurofilament protein alpha-internexin, suggesting that early neuronal drop-out may contribute to the down-regulation of this glutamate transporter and subsequent pannecrosis. A selective, focal loss of GLT-1 and GLAST transporter proteins provides a rational explanation for the increase in interstitial glutamate levels, and may play a major role in the selective vulnerability of thalamic structures to thiamine deficiency-induced cell death.     

Na(+)-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood-brain barrier. A mechanism for glutamate removal.

Na(+)-dependent transporters for glutamate exist on astrocytes (EAAT1 and EAAT2) and neurons (EAAT3). These transporters presumably assist in keeping the glutamate concentration low in the extracellular fluid of brain. Recently, Na(+)-dependent glutamate transport was described on the abluminal membrane of the blood-brain barrier. To determine whether the above-mentioned transporters participate in glutamate transport of the blood-brain barrier, total RNA was extracted from bovine cerebral capillaries. cDNA for EAAT1, EAAT2, and EAAT3 was observed, indicating that mRNA was present. Western blot analysis demonstrated all three transporters were expressed on abluminal membranes, but none was detectable on luminal membranes of the blood-brain barrier. Measurement of transport kinetics demonstrated voltage dependence, K(+)-dependence, and an apparent K(m) of 14 microM (aggregate of the three transporters) at a transmembrane potential of -61 mV. Inhibition of glutamate transport was observed using inhibitors specific for EAAT2 (kainic acid and dihydrokainic acid) and EAAT3 (cysteine). The relative activity of the three transporters was found to be approximately 1:3:6 for EAAT1, EAAT2, and EAAT3, respectively. These transporters may assist in maintaining low glutamate concentrations in the extracellular fluid.     

Long-term alterations in glutamate receptor and transporter expression following early-life seizures are associated with increased seizure susceptibility

Prolonged seizures in early childhood are associated with an increased risk of development of epilepsy in later life. The mechanism(s) behind this susceptibility to later development of epilepsy is unclear. Increased synaptic activity during development has been shown to permanently alter excitatory neurotransmission and could be one of the mechanisms involved in this increased susceptibility to the development of epilepsy. In the present study we determine the effect of status-epilepticus induced by lithium/pilocarpine at postnatal day 10 (P10 SE) on the expression of glutamate receptor and transporter mRNAs in hippocampal dentate granule cells and protein levels in dentate gyrus of these animals in adulthood. The results revealed a decrease in glutamate receptor 2 (GluR2) mRNA expression and protein levels as well as an increase in protein levels for the excitatory amino acid carrier 1 (EAAC1) in P10 SE rats compared to controls. Expression of glutamate receptor 1 (GluR1) mRNA was decreased in both P10 SE rats and identically handled, lithium-injected littermate controls compared to naive animals, and GluR1 protein levels were significantly lower in lithium-controls than in naive rats, suggesting an effect of either the handling or the lithium on GluR1 expression. These changes in EAA receptors and transporters were accompanied by an increased susceptibility to kainic acid induced seizures in P10 SE rats compared to controls. The current data suggest that early-life status-epilepticus can result in permanent alterations in glutamate receptor and transporter gene expression, which may contribute to a lower seizure threshold.     

Functional Defects in the External and Internal Thin Gates of the γ-Aminobutyric Acid (GABA) Transporter GAT-1 Can Compensate Each Other

The GABA transporter GAT-1 belongs to the neurotransmitter:sodium:symporters which are crucial for synaptic transmission. GAT-1 mediates electrogenic transport of GABA together with sodium and chloride. Structure-function studies indicate that the bacterial homologue LeuT, which possess extra- and intracellular thin gates, is an excellent model for this class of neurotransmitter transporters. We recently showed that a conserved aspartate residue of GAT-1, Asp-451, whose LeuT equivalent participates in its thin extracellular gate, is functionally irreplaceable in GAT-1. Only the D451E mutant exhibited residual transport activity but with an elevated apparent sodium affinity as a consequence of an increased proportion of outward-facing transporters. Because during transport the opening and closing of external and internal gates should be tightly coupled, we have addressed the question of whether mutations of the intracellular thin gate residues Arg-44 and Asp-410 can compensate for the effects of their extracellular counterparts. Mutation of Asp-410 to glutamate resulted in impaired transport activity and a reduced apparent affinity for sodium. However, the transport activity of the double mutant D410E/D451E was increased by approximately 10-fold of that of each of the single mutants. Similar compensatory effects were also seen when other combinations of intra- and extracellular thin gate mutants were analyzed. Moreover, the introduction of D410E into the D451E background resulted in lower apparent sodium affinity than that of D451E alone. Our results indicate that a functional interaction of the external and internal gates of GAT-1 is essential for transport.     

GABA release and uptake measured in crude synaptosomes from Genetic Absence Epilepsy Rats from Strasbourg (GAERS).

GABA release and uptake were examined in Genetic Absence Epilepsy Rats from Strasbourg and in non-epileptic control animals, using crude synaptosomes prepared from the cerebral cortex and thalamus. Uptake of [3H]GABA over time was reduced in thalamic synaptosomes from epileptic rats, compared to controls. The affinity of the uptake process in thalamic synaptosomes was lower in epileptic animals. NNC-711, a ligand for the GAT-1 uptake protein, reduced synaptosomal uptake by more than 95%; beta-alanine, an inhibitor selective for the uptake proteins GAT-2 and -3, did not significantly reduce synaptosomal uptake. Autoradiography studies using [3H]tiagabine, a ligand selective for GAT-1, revealed no differences between the strains in either affinity or levels of binding. Ethanolamine O-sulphate (100 microM), a selective inhibitor of GABA-transaminase, did not affect uptake levels. Aminooxyacetic acid (10-100 microM), an inhibitor of GABA-transaminase and, to a lesser extent, glutamate decarboxylase, caused an increase in measured uptake in both thalamic and cortical synaptosomes, in both strains. We found no difference in in vitro basal or KCl-stimulated endogenous GABA release between epileptic and control rats. These results indicate that GABA uptake in the thalamus of Genetic Absence Epilepsy Rats from Strasbourg was reduced, compared to control animals. The lower uptake affinity in the epileptic animals probably contributed to the reduction in uptake over time. Uptake appeared to be mediated primarily by the 'neuronal' transporter GAT-1. Autoradiography studies revealed no differences in the number or affinity of this uptake protein. It is therefore possible that altered functional modulation of GAT-1 caused the decrease in uptake shown in the epileptic animals. Inhibition of GABA-transaminase activity had no effect on measured GABA uptake, whereas a reduction in glutamate decarboxylase activity may have affected measured uptake levels.     

Restoration of the luteinizing hormone surge in middle-aged female rats by altering the balance of GABA and glutamate transmission in the medial preoptic area

Hypothalamic glutamate and gamma-aminobutyric acid (GABA) neurotransmission are involved in the ovarian hormone-induced GnRH-LH surge in rodents. We previously reported that middle-aged rats have significantly less glutamate release in the medial preoptic area than young rats on the day of the LH surge. The present study tested the hypothesis that the delayed and attenuated LH surge in ovariohysterectomized middle-aged rats primed with ovarian steroids results from reduced hypothalamic glutamate and increased GABA(A) neurotransmission. Microdialysis results show that middle-aged rats with attenuated LH surges had reduced extracellular glutamate and increased extracellular GABA levels in the medial preoptic area compared with young rats. Blocking GABA(A) receptors with bicuculline or inhibiting synaptic glutamate reuptake with L-trans-pyrrolidine-2,4-dicarboxylic acid increased extracellular Glu in the medial preoptic area and partially restored LH surge amplitude in middle-aged rats without altering LH surge onset. Complete recovery of LH surge amplitude was observed in middle-aged rats treated with the combination of bicuculline and L-trans-pyrrolidine-2,4-dicarboxylic acid. This treatment also restored the extracellular glutamate:GABA ratio in the medial preoptic area of middle-aged rats to the level of young rats. Immunoblot analysis revealed that estradiol and progesterone treatment reduced SLC32A1(formerly known as vesicular GABA transporter) levels and increased SLC17A6 (formerly known as vesicular glutamate transporter 2) levels in the anterior hypothalamus of ovariohysterectomized young but not middle-aged rats. These data suggest that both reduced availability of glutamate and increased activation of GABA(A) receptors under estrogen-positive feedback conditions contribute to the age-related delay in onset and attenuated amplitude of the LH surge.     

Molecular heterogeneity of the gamma-aminobutyric acid (GABA) transport system. Cloning of two novel high affinity GABA transporters from rat brain.

cDNA clones encoding two novel gamma-aminobutyric acid (GABA) transporters (designated GAT-2 and GAT-3) have been isolated from rat brain, and their functional properties have been examined in mammalian cells. The transporters display high affinity for GABA (Km approximately 10 microM) and exhibit pharmacological properties distinct from the previously cloned neuronal GABA transporter (GAT-1). Both transporters require sodium and chloride for transport activity. The nucleotide sequences of GAT-2 and GAT-3 predict proteins of 602 and 627 amino acids, respectively, which can be modeled with 12 transmembrane domains, similar to the topology proposed for other cloned neurotransmitter transporters. Localization studies indicate that both transporters are present in brain and retina, while GAT-2 is also present in peripheral tissues. The cloning of these transporter genes from rat brain reveals previously undescribed heterogeneity in GABA transporters.     

Differential expression of the GABA transporters GAT-1 and GAT-3 in brains of rats,cats, monkeys and humans

The homeostasis of GABA is critical to normal brain function. Extracellular levels of GABA are regulated mainly by plasmalemmal gamma-aminobutyric acid (GABA) transporters. Whereas the expression of GABA transporters has been extensively studied in rodents, validation of this data in other species, including humans, has been limited. As this information is crucial for our understanding of therapeutic options in human diseases such as epilepsy, we have compared, by immunocytochemistry, the distributions of the GABA transporters GAT-1 and GAT-3 in rats, cats, monkeys and humans. We demonstrate subtle differences between the results reported in the literature and our results, such as the predominance of GAT-1 labelling in neurons rather than astrocytes in the rat cortex. We note that the optimal localisation of GAT-1 in cats, monkeys and humans requires the use of an antibody against the human sequence carboxyl terminal region of GAT-1 rather than against the slightly different rat sequence. We demonstrate that GAT-3 is localised mainly to astrocytes in hindbrain and midbrain regions of rat brains. However, in species such as cats, monkeys and humans, additional strong immunolabelling of oligodendrocytes has also been observed. We suggest that differences in GAT distribution, especially the expression of GAT-3 by oligodendrocytes in humans, must be accommodated in extrapolating rodent models of GABA homeostasis to humans.Grant support was provided by the National Health and Medical Research Council (Australia) grant nos. 210127 and 102448, and a Senior Research Fellowship to David Pow.     

GAT-1 regulates both tonic and phasic GABA(A) receptor-mediated inhibition in the cerebral cortex

γ-Aminobutyric acid 1 (GAT-1) is the most copiously expressed GABA transporter; we studied its role in phasic and tonic inhibition in the neocortex using GAT-1 knockout (KO) mice. Immunoblotting and immunocytochemical studies showed that GAT-2 and GAT-3 levels in KOs were unchanged and that GAT-3 was not redistributed in KOs. Moreover, the expression of GAD65/67 was increased, whereas that of GABA or VGAT was unchanged. Microdialysis studies showed that in KOs spontaneous extracellular release of GABA and glutamate was comparable in WT and KO mice, whereas KCl-evoked output of GABA, but not of glutamate, was significantly increased in KOs. Recordings from layer II/III pyramids revealed a significant increase in GABA_AR-mediated tonic conductance in KO mice. The frequency, amplitude and kinetics of spontaneous inhibitory post-synaptic currents (IPSCs) were unchanged, whereas the decay time of evoked IPSCs was significantly prolonged in KO mice. In KO mice, high frequency stimulation of GABAergic terminals induced large GABA_AR-mediated inward currents associated with a reduction in amplitude and decay time of IPSCs evoked immediately after the train. The recovery process was slower in KO than in WT mice. These studies show that in the cerebral cortex of GAT-1 KO mice GAT-3 is not redistributed and GADs are adaptively changed and indicate that GAT-1 has a prominent role in both tonic and phasic GABA_AR-mediated inhibition, in particular during sustained neuronal activity.     

Expression of GABA transporters,GAT-1 and GAT-3, in the cerebral cortex and thalamus of the rat during postnatal development

The cellular and subcellular localization of two GABA transporters, GAT-1 and GAT-3, was investigated using immunocytochemical methods in the rat cerebral cortex and thalamus during postnatal development. The distribution of the transporters is compared with that of the neuronal marker GABA, and with that of vimentin and of glial fibrillary acidic protein, which identify immature and mature astrocytes, respectively. Our observations show that the two transporters are already expressed at birth in both brain areas with the same cellular localization as in adult rats, as GAT-1 is present in growth cones and terminals only in the cortex, whereas both transporters are expressed in astrocytes in the cortex and thalamus. The distribution of GAT-1 and GAT-3 undergoes postnatal changes reflecting in general the neurogenetic events of the neocortex and thalamus and, more specifically, the maturation of GABAergic innervation. The adult-like pattern of expression is achieved in the third postnatal week in the cortex and in the second postnatal week in the thalamus. The early expression of GAT-1 in GABAergic terminals confirms previous studies showing the existence of neuronal mechanisms of GABA uptake from the embryonic stages. As for the glial localization, the precocious existence of two astrocytic GABA transporters suggests that they operate through different functional mechanisms from birth, whereas their exclusively glial expression in the thalamus indicates that the astroglia plays a major role in the transport, recycling and metabolism of thalamic GABA.     

GABA is toxic for mouse striatal neurones through a transporter-mediated process

In the present study, GABA was shown to induce a necrotic neuronal death in cultured striatal neurones from mouse embryos. This effect did not depend on the activation of GABA(A), GABA(B) or GABA(C) receptors as it was neither antagonized by bicuculline, saclofen or picrotoxin, respectively, nor reproduced by the GABA receptor agonists, muscimol and baclofen. Excluding the participation of glutamate, GABA neurotoxicity persisted in the presence of either the antagonists of ionotropic and metabotropic glutamate receptors or glutamate pyruvate transaminase, which induces an immediate catabolism of glutamate. A GABA transport-associated process is involved in GABA neurotoxicity as nipecotic acid and NO 711, two inhibitors of the high-affinity neuronal GABA transporters (GAT-1, in particular), completely prevented the neurotoxic effect of GABA. The activation of a subset of G proteins is also implicated in the GABA transport-mediated neuronal death as GABA neurotoxicity was completely suppressed when striatal neurones were pre-treated with pertussis toxin. Further demonstrating the specificity of this neurotoxic process, GABA-induced neurotoxicity was not observed in cortical neurones which, in contrast to striatal neurones, are largely represented by glutamatergic neurones. In conclusion, our study suggests that glutamate is not the sole neurotransmitter that can be responsible for brain damage and that GABA neurotoxicity involves both GABA transport and G protein transduction pathways.     

Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation

Background

Glutamate and ??-aminobutyric acid (GABA) transporters play important roles in balancing excitatory and inhibitory signals in the brain. Increasing evidence suggest that they may act concertedly to regulate extracellular levels of the neurotransmitters.

Results

Here we present evidence that glutamate uptake-induced release of GABA from astrocytes has a direct impact on the excitability of pyramidal neurons in the hippocampus. We demonstrate that GABA, synthesized from the polyamine putrescine, is released from astrocytes by the reverse action of glial GABA transporter (GAT) subtypes GAT-2 or GAT-3. GABA release can be prevented by blocking glutamate uptake with the non-transportable inhibitor DHK, confirming that it is the glutamate transporter activity that triggers the reversal of GABA transporters, conceivably by elevating the intracellular Na⁺ concentration in astrocytes. The released GABA significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. Blockade of the Glu/GABA exchange mechanism increases the duration of seizure-like events in the low-[Mg²⁺] in vitro model of epilepsy. Under in vivo conditions the increased GABA release modulates the power of gamma range oscillation in the CA1 region, suggesting that the Glu/GABA exchange mechanism is also functioning in the intact hippocampus under physiological conditions.

Conclusions

The results suggest the existence of a novel molecular mechanism by which astrocytes transform glutamatergic excitation into GABAergic inhibition providing an adjustable, in situ negative feedback on the excitability of neurons.     

Relationships between glutamine, glutamate, and GABA in nerve endings under Pb-toxicity conditions

Glutamine (Gln), glutamate (Glu) and gamma-amino butyric acid (GABA) are essential amino acids for brain metabolism and function. Astrocytic-derived glutamine is the precursor of the two most important neurotransmitters: glutamate, an excitatory neurotransmitter, and GABA, an inhibitory neurotransmitter. In addition to their roles in neurotransmission these neurotransmitters act as alternative metabolic substrates that enable metabolic coupling between astrocytes and neurons. The relationships between Gln, Glu and GABA were studied under lead (Pb) toxicity conditions using synaptosomal fractions obtained from adult rat brains to investigate the cause of Pb neurotoxicity-induced seizures. We have found that diminished transport of [(14)C]GABA occurs after Pb treatment. Both uptake and depolarization-evoked release decrease by 40% and 30%, respectively, relative to controls. Lower expression of glutamate decarboxylase (GAD), the GABA synthesizing enzyme, is also observed. In contrast to impaired synaptosomal GABA function, the GABA transporter GAT-1 protein is overexpressed (possibly as a compensative mechanism). Furthermore, similar decreases in synaptosomal uptake of radioactive glutamine and glutamate are observed. However, the K(+)-evoked release of Glu increases by 20% over control values and the quantity of neuronal EAAC1 transporter for glutamate reaches remarkably higher levels after Pb treatment. In addition, Pb induces decreased activity of phosphate-activated glutaminase (PAG), which plays a role in glutamate metabolism. Most noteworthy is that the overexpression and reversed action of the EAAC1 transporter may be the cause of the elevated extracellular glutamate levels. In addition to the impairment of synaptosomal processes of glutamatergic and GABAergic transport, the results indicate perturbed relationships between Gln, Glu and GABA that may be the cause of altered neuronal-astrocytic interactions under conditions of Pb neurotoxicity.     

Cryptic expression of functional glutamate transporters in the developing rodent brain

The co-ordinate functioning of neurons and glia is required for glutamate-mediated neurotransmission. In this study, we show by immunocytochemical detection of D-aspartate uptake, that functional glutamate transporters are present in the developing CNS of fetal and neonatal rats, including forebrain, midbrain and hindbrain, at least as early as embryonic day 12 (E12). Use of the transport inhibitor dihydrokainic acid revealed a significant role for GLT-1 in the uptake process.Immunolabelling for the glutamate transporters GLAST, GLT-1alpha and GLT-1v showed that each of these proteins are expressed early in development and appear to be restricted to glial-like cells throughout the development period examined (except in the retina, where neuronal elements were also labelled). Our capacity to detect very early expression of the variant forms of GLT-1 contrasts with other studies, a feature that we attribute to the use of antigen-recovery techniques that unmask protein epitopes that are otherwise undetectable. These studies illustrate the widespread presence of functional glutamate transporters in the developing CNS, in many cases before the onset of periods of synaptogenesis and indicate that regulation of extracellular glutamate by multiple excitatory amino acid transporters might be crucial in early CNS development.     

Brain-derived neurotrophic factor (BDNF) enhances GABA transport by modulating the trafficking of GABA transporter-1 (GAT-1) from the plasma membrane of rat cortical astrocytes

The γ-aminobutyric acid (GABA) transporters (GATs) are located in the plasma membrane of neurons and astrocytes and are responsible for termination of GABAergic transmission. It has previously been shown that brain derived neurotrophic factor (BDNF) modulates GAT-1-mediated GABA transport in nerve terminals and neuronal cultures. We now report that BDNF enhances GAT-1-mediated GABA transport in cultured astrocytes, an effect mostly due to an increase in the V(max) kinetic constant. This action involves the truncated form of the TrkB receptor (TrkB-t) coupled to a non-classic PLC-γ/PKC-δ and ERK/MAPK pathway and requires active adenosine A(2A) receptors. Transport through GAT-3 is not affected by BDNF. To elucidate if BDNF affects trafficking of GAT-1 in astrocytes, we generated and infected astrocytes with a functional mutant of the rat GAT-1 (rGAT-1) in which the hemagglutinin (HA) epitope was incorporated into the second extracellular loop. An increase in plasma membrane of HA-rGAT-1 as well as of rGAT-1 was observed when both HA-GAT-1-transduced astrocytes and rGAT-1-overexpressing astrocytes were treated with BDNF. The effect of BDNF results from inhibition of dynamin/clathrin-dependent constitutive internalization of GAT-1 rather than from facilitation of the monensin-sensitive recycling of GAT-1 molecules back to the plasma membrane. We therefore conclude that BDNF enhances the time span of GAT-1 molecules at the plasma membrane of astrocytes. BDNF may thus play an active role in the clearance of GABA from synaptic and extrasynaptic sites and in this way influence neuronal excitability.     

Molecular cloning and functional characterization of a GABA transporter from the CNS of the cabbage looper, Trichoplusia ni.

A cDNA encoding a GABA transporter in the caterpillar Trichoplusia ni has been cloned and expressed in baculovirus-infected insect cells. The cDNA contains an ORF encoding a 608-residue protein, designated TrnGAT. Hydropathy analysis of the deduced amino acid sequence suggests 12 transmembrane domains, a structure similar to that of all other cloned Na+/Cl(-)-dependent GABA transporters. The deduced amino acid sequence shows high identity with a GABA transporter (MasGAT) expressed in the embryo of Manduca sexta. Expression of TrnGAT mRNA was detected only in the brain. Sf21 cells infected with recombinant baculovirus exhibited a 20- to 30-fold increase in [3H]GABA uptake compared to control-infected cells. Several blockers of GABA uptake were used to determine the pharmacological profile of TrnGAT. Although most similar to mammalian neuronal GABA transporter GAT-1 in its kinetic properties, stoichiometry of ionic dependence and pharmacological properties, TrnGAT may be distinguished from mammalian GAT-1 by the inability of cyclic GABA analogues, such as nipecotic acid and its derivatives, to inhibit GABA uptake by the insect protein. The unique pharmacology of TrnGAT suggests that the GABA transport system in the lepidopteran CNS could be a useful target in the future development of rapidly-acting neuroactive agents used to control agriculturally-important insects.