Production of specific antibodies against GABA transporter subtypes (GAT1, GAT2, GAT3) and their application to immunocytochemistry

doi:10.1016/0169-328X(94)90072-8

Molecular Brain Research

Volume 26, Issues 1–2, October 1994, Pages 47-54

https://doi.org/10.1016/0169-328X(94)90072-8 Get rights and content

Abstract

Polyclonal subtype-specific antibodies were developed against three subtypes of GABA transporters (GAT1, GAT2 and GAT3). By immunoblot analysis, each antibody detected a single band that could be blocked by absorption of the antibody with the respective antigen. GAT2 was found in various tissues, while GAT1 and GAT3 were detected only in the brain. GAT1 was distributed throughout the brain with the highest amount in the olfactory bulb, CA3 region of the hippocampus, layer I of the cerebral cortex, piriform cortex, superior colliculus, interpeduncular nucleus and nucleus spinal tract of the trigeminal nerve, while the GAT3 was densely found in the olfactory bulb, thalamus, hypothalamus, pons and medulla, globus pallidus, central gray, substantia nigra, deep cerebellar nuclei and nucleus spinal tract of the trigeminal nerve but not in the hippocampus, cerebral cortex, caudate-putamen and cerebellar cortex. GAT2 immunoreactivity was faint throughout the brain but was concentrated in the arachnoid and ependymal cells. Both GAT1 and GAT3 were found in the neuropil but not in the cell bodies nor in the white matter. These results suggest that GAT1, GAT2 and GAT3 are expressed in different cells and that GAT1 and GAT3 are involved in distinct GABAergic transmission while GAT2 may be related to non-neuronal function.

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The GABA transporter GAT1 regulates brain inhibitory neurotransmission and it is considered a potential therapeutic target for the treatment of wide spectrum of neurological diseases including epilepsy, stroke and autism. Syntenin-1 binds to syntaxin 1A, which is known to regulate the plasma membrane insertion of several neurotransmitter transporters. Previously, a direct interaction of syntenin-1 with the glycine transporter GlyT2 was reported. Here, we show that the GABA transporter GAT1 also directly interacts with syntenin-1, involving both unidentified protein interaction interface and the GAT1 C-terminal PDZ binding motif interacting mainly with syntenin-1 PDZ domain 1. The PDZ interaction was eliminated by the mutation of GAT1 isoleucine 599 and tyrosine 598 located in PDZ positions 0 and -1, respectively. This indicates an unconventional PDZ interaction and possible regulation of the transporter PDZ motif via tyrosine phosphorylation. Whole syntenin-1 protein fused to GST protein and immobilised on glutathione resin coprecipitated intact GAT1 transporter from an extract of GAT1 transfected neuroblastoma N2a cells. This coprecipitation was inhibited by tyrosine phosphatases inhibitor pervanadate. The fluorescence tagged GAT1 and syntenin-1 colocalized upon coexpression in N2a cells. The above results show that syntenin-1 might be, in addition to GlyT2, directly involved in the trafficking of GAT1 transporter.
γ-Aminobutyric acid transporters as relevant biological target: Their function, structure, inhibitors and role in the therapy of different diseases
2020, International Journal of Biological Macromolecules
γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the nervous system. It plays a crucial role in many physiological processes. Upon release from the presynaptic element, it is removed from the synaptic cleft by reuptake due to the action of GABA transporters (GATs). GATs belong to a large SLC6 protein family whose characteristic feature is sodium-dependent relocation of neurotransmitters through the cell membrane. GABA transporters are characterized in many contexts, but their spatial structure is not fully known. They are divided into four types, which differ in occurrence and role. Herein, the special attention was paid to these transporting proteins. This comprehensive review presents the current knowledge about GABA transporters. Their distribution in the body, physiological functions and possible utilization in the therapy of different diseases were fully discussed. The important structural features were described based on published data, including sequence analysis, mutagenesis studies, and comparison with known SLC6 transporters for leucine (LeuT), dopamine (DAT) and serotonin (SERT). Moreover, the most important inhibitors of GABA transporters of various basic scaffolds, diverse selectivity and potency were presented.
GAT-3 Dysfunction Generates Tonic Inhibition in External Globus Pallidus Neurons in Parkinsonian Rodents
2018, Cell Reports
The external globus pallidus (GP) is a key GABAergic hub in the basal ganglia (BG) circuitry, a neuronal network involved in motor control. In Parkinson’s disease (PD), the rate and pattern of activity of GP neurons are profoundly altered and contribute to the motor symptoms of the disease. In rodent models of PD, the striato-pallidal pathway is hyperactive, and extracellular GABA concentrations are abnormally elevated in the GP, supporting the hypothesis of an alteration of neuronal and/or glial clearance of GABA. Here, we discovered the existence of persistent GABAergic tonic inhibition in GP neurons of dopamine-depleted (DD) rodent models. We showed that glial GAT-3 transporters are downregulated while neuronal GAT-1 function remains normal in DD rodents. Finally, we showed that blocking GAT-3 activity in vivo alters the motor coordination of control rodents, suggesting that GABAergic tonic inhibition in the GP contributes to the pathophysiology of PD.
Localization and function of GABA transporters in the globus pallidus of parkinsonian monkeys
2010, Experimental Neurology
Citation Excerpt :
These high-affinity transporters are thought to constrain the extent of diffusion of GABA from the release sites and, thus, the length of time which the transmitter spends in the synaptic cleft and in the extrasynaptic space. Of the four identified GAT genes (GAT-1, GAT-2, GAT-3 and B-GAT, Borden, 1996; Dalby, 2003), only GAT-1 and GAT-3 mRNA or protein expressions have been described in the pallidum (Durkin et al., 1995; Ikegaki et al., 1994; Ng et al., 2000; Wang and Ong, 1999; Yasumi et al., 1997). We have previously shown that GAT-1 and GAT-3 are expressed in glia, and to a lesser extent in pre-terminal axons, and that both transporters modulate GABA levels and neuronal activity in the monkey GPe and GPi (Galvan et al., 2005).
The GABA transporters GAT-1 and GAT-3 are abundant in the external and internal segments of the globus pallidus (GPe and GPi, respectively). We have shown that pharmacological blockade of either of these transporters results in decreased neuronal firing, and in elevated levels of extracellular GABA in normal monkeys. We now studied whether the electrophysiologic and biochemical effects of local intra-pallidal injections of GAT-1 and GAT-3 blockers, or the subcellular localization of these transporters, are altered in monkeys rendered parkinsonian by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The subcellular localization of the transporters in GPe and GPi, studied with electron microscopy immunoperoxidase, was similar to that found in normal animals: i.e., GAT-3 immunoreactivity was mostly confined to glial processes, while GAT-1 labeling was expressed in unmyelinated axons and glial processes. A combined injection/recording device was used to record the extracellular activity of single neurons in GPe and GPi, before, during and after administration of small volumes (1 µl) of either the GAT-1 inhibitor, SKF-89976A hydrochloride (720 ng), or the GAT-3 inhibitor, (S)-SNAP-5114 (500 ng). In GPe, the effects of GAT-1 or GAT-3 blockade were similar to those seen in normal monkeys. However, unlike the findings in the normal state, the firing of most neurons was not affected by blockade of either transporter in GPi. These results suggest that, after dopaminergic depletion, the functions of GABA transporters are altered in GPi; without major changes in their subcellular localization.
Cloning and characterization of a functional human γ-aminobutyric acid (GABA) transporter, human GAT-2
2007, Journal of Biological Chemistry
Citation Excerpt :
Furthermore expression in liver (46) and in eye and the gastrointestinal system has been reported (57). The rGAT-2 has been detected in brain and retina, and in peripheral tissues it has been found in kidney, liver, heart, spleen, pancreas, and adrenal gland (11, 36, 39). The expression of mGAT3 has been found to be more restricted with low abundance in adult mouse brain and higher levels of expression in kidney and liver.
Plasma membrane γ-aminobutyric acid (GABA) transporters act to terminate GABA neurotransmission in the mammalian brain. Intriguingly four distinct GABA transporters have been cloned from rat and mouse, whereas only three functional homologs of these transporters have been cloned from human. The aim of this study therefore was to search for this fourth missing human transporter. Using a bioinformatics approach, we successfully identified and cloned the full-length cDNA of a so far uncharacterized human GABA transporter (GAT). The predicted protein displays high sequence similarity to rat GAT-2 and mouse GAT3, and in accordance with the nomenclature for rat GABA transporters, we therefore refer to the transporter as human GAT-2. We used electrophysiological and cell-based methods to demonstrate that this protein is a functional transporter of GABA. The transport was saturable and dependent on both Na⁺ and Cl^–. Pharmacologically the transporter is distinct from the other human GABA transporters and similar to rat GAT-2 and mouse GAT3 with high sensitivity toward GABA and β-alanine. Furthermore the GABA transport inhibitor (S)-SNAP-5114 displayed some inhibitory activity at the transporter. Expression analysis by reverse transcription-PCR showed that GAT-2 mRNA is present in human brain, kidney, lung, and testis. The finding of the human GAT-2 demonstrates for the first time that the four plasma membrane GABA transporters identified in several mammalian species are all conserved in human. Furthermore the availability of human GAT-2 enables the use of all human clones of the GABA transporters in drug development programs and functional characterization of novel inhibitors of GABA transport.
Anticonvulsant and anxiolytic activity of FrPbAII, a novel GABA uptake inhibitor isolated from the venom of the social spider Parawixia bistriata (Araneidae: Araneae)
2006, Brain Research
Citation Excerpt :
In an elegant review, Dalby (2003) points particularities of all subtypes of GABA transporters, and describes their regional distribution. Therefore, in situ, labeling for GAT-1 mRNA was detected in the limbic system, basal forebrain, the rostrocaudal extent of the cortex and in the cerebellar cortex (Liu et al., 1993; Swan et al., 1994; Ikegaki et al., 1994; Minelli et al., 1995; Durkin et al., 1995). In contrast, GAT-3 mRNA is expressed predominantly in the thalamus, hypothalamus, a minor part of the pyriform cortex (amygdala region), inferior colliculus, pons, brainstem and deep cerebellar nuclei (Ikegaki et al., 1994).
This study was aimed at determining the effects of FrPbAII (174 Da), a novel isolated component from Parawixia bistriata spider venom, in the CNS of Wistar rats. Considering that FrPbAII inhibits the high affinity GABAergic uptake in a dose-dependent manner, its anxiolytic and anticonvulsant effects were analyzed in well-established animal models. Injection of FrPbAII in the rat hippocampus induced a marked anxiolytic effect, increasing the occupancy in the open arms of the elevated plus maze (EC₅₀ = 0.09 μg/μl) and increasing the time spent in the lit area of the light–dark apparatus (EC₅₀ = 0.03 μg/μl). Anxiolytic effects were also observed considering the number of entries in the open arms of the EPM and in the lit compartment of the light–dark box. Interestingly, when microinjected bilaterally in the SNPr of freely moving rats, FrPbAII (0.6 μg/μl) effectively prevented seizures induced by the unilateral GABAergic blockade of Area tempestas (bicuculline, 0.75 μg/μl). This anticonvulsant effect was similar to that evoked by muscimol (0.1 μg/μl) and baclofen (0.6 μg/μl), but differed from that of the specific GAT1 inhibitor, nipecotic acid (0.7 μg/μl). This difference could be accounted either for the parallel action of FrPbAII over glycinergic transporters or to an inspecific activity on GABAergic transporters. Data from the present investigation might be pointing to a novel compound with interesting and yet unexplored pharmacological potential.

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Research reportProduction of specific antibodies against GABA transporter subtypes (GAT1, GAT2, GAT3) and their application to immunocytochemistry

Abstract

FEBS Lett.

J. Biol. Chem.

Neuron

Biochem. Pharmacol.

Brain Res.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Research report
Production of specific antibodies against GABA transporter subtypes (GAT1, GAT2, GAT3) and their application to immunocytochemistry