Rat Brain GABA Transporter Research Articles

On the presence of 3H-GABA uptake mechanism in bovine spermatozoa

In the present study, existence of 3H-GABA uptake mechanism in bovine spermatozoa and the modulation of 3H-GABA transport by GABA itself were evaluated. The hypothesis was tyrosine phosphorylation affects transporter (GAT) function. 3H-GABA uptake assays were performed on bovine spermatozoa and it resulted to be temperature- and time-dependent and K m was 1.48 μM. Uptake was inhibited by the metabolic inhibitor ouabain and different blockers of GAT-1 (β-alanine, l-DABA, nipecotic acid, tiagabine). Extracellular GABA up-regulated GABA transport, while the addition of SKF89976A, a high affinity inhibitor of the rat brain GABA transporter, reduced GABA uptake. Tyrosine phosphorylation affects transporter function since genistein, a broad-spectrum tyrosine kinase inhibitor, decreased 3H-GABA uptake. Reduction in uptake did not occur in the presence of daidzein, an inactive genistein analogue. Furthermore, the genistein-mediated reduction in transport could be prevented by the tyrosine phosphatase inhibitor pervanadate. The action of these drugs on GABA transport is likely mediated through the GABA transporter GAT-1 since SKF89976A blocked a majority of GABA uptake. Wash-out experiments indicated that the genistein effect was reversible. When the experiments were conducted using “ in vitro” capacitated spermatozoa there was no detectable uptake. Present results demonstrate that the carrier-mediated GABA uptake system in bovine spermatozoa modulates its function in response to extracellular GABA, that changes in lipid distribution and membrane composition which occur during capacitation eliminates GABA uptake and suggest the involvement of tyrosine phosphorylation in GABA transport.

Inhibition of the renal betaine transporter by calcium ions

Chronic upregulation of the renal betaine/GABA transporter (BGT1) by hypertonic stress has been well documented, but it is not known whether BGT1 can be regulated acutely after insertion in the basolateral plasma membrane. Related transporters, such as the rat brain GABA transporter, can be rapidly removed from the plasma membrane through activation of G protein-coupled receptors. The goal of the present study was to determine whether acute changes in extracellular and/or intracellular Ca(2+) will regulate BGT1 transport activity at the plasma membrane level in Madin-Darby canine kidney cells subjected to 24-h hypertonic stress. After brief pretreatment with a Ca(2+)-free solution, the addition of extracellular Ca(2+) in the transport assay produced dose-dependent inhibition of Na(+)-GABA cotransport. Maximum inhibition was 49% at 2 mM Ca(2+) (P < 0.05). Fura 2 imaging confirmed that addition of 2 mM Ca(2+) produced a transient increase in intracellular Ca(2+) that preceded transport inhibition. Acute inhibition of Na(+)-GABA cotransport was reproduced by addition of thapsigargin (5 microM) and ionomycin (10 microM). Amino acid transport system A, assayed as a control, was not inhibited. Brief treatment with phorbol esters reproduced the specific inhibition of Na(+)-GABA cotransport, and the inhibition was blocked by staurosporine. Surface biotinylation confirmed that the response to phorbol esters was accompanied by loss of BGT1 protein from the plasma membrane, and immunohistochemistry showed a shift to an intracellular distribution. We conclude that BGT1 can be inhibited acutely by extracellular Ca(2+) through a mechanism involving BGT1 protein internalization, and protein kinase C may play a role.

Intracellular Domains of a Rat Brain GABA Transporter That Govern Transport

Plasma membrane neurotransmitter transporters determine in part the concentration, time course, and diffusion of extracellular transmitter. Much has been learned about how substrate translocation through the transporter occurs; however, the precise way in which transporter structure maps onto transporter function has not yet been fully elucidated. Here, biochemical and electrophysiological approaches were used to test the hypothesis that intracellular domains of the rat brain GABA transporter (GAT1) contribute to the transport process. Injection of a peptide corresponding to the presumed fourth intracellular loop of the transporter (IL4) into oocytes expressing GAT1 greatly reduced both forward and reverse transport and reduced the transport rate in a dose-dependent manner. Coinjection of the IL4 peptide with a peptide corresponding to the N-terminal cytoplasmic tail of GAT1 reversed the IL4-mediated inhibition; this reversal, and direct binding between these two domains, was prevented by mutagenesis of charged residues in either the IL4 or N-terminal domains. Furthermore, syntaxin 1A, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein that inhibits GAT1 transport rates via interactions with the N-terminal tail of GAT1 was unable to regulate the GAT1 IL4 mutant. Together, these data suggest a model in which the GAT1 IL4 domain serves as a barrier for transport, and this barrier can be regulated through intra-molecular and inter-molecular interactions.

Syntaxin 1A inhibits GABA flux, efflux, and exchange mediated by the rat brain GABA transporter GAT1.

GABA transporters control extracellular GABA levels by coupling transmitter uptake to the sodium and chloride cotransport. The rat brain GABA transporter GAT1 and other members of this family are regulated by direct interactions with syntaxin 1A, a protein involved in vesicle docking and in the regulation of several ion channels and transporters. We have shown previously that syntaxin 1A exerts its effects on GAT1 by decreasing the net uptake of GABA and its associated ions through interactions with aspartic acid residues in the N-terminal tail of GAT1. This reduction in net uptake could be caused by many steps in the transport cycle, including substrate binding, substrate flux, substrate efflux, or reorientation of the unliganded transporter. To address this question, we performed GABA flux assays, measured flux- and efflux-associated ion currents, and assessed GABA exchange in multiple experimental systems expressing syntaxin 1A and wild-type GAT1 or GAT1 mutants. Syntaxin 1A caused similar reductions in forward and reverse transport that did not involve changes in apparent transport affinities for sodium, chloride, or GABA. The syntaxin 1A-mediated reduction in GABA flux and efflux was mimicked by mutations in GAT1 at the syntaxin 1A binding site. The binding site GAT1 mutant also caused a reduction in exchange. These data suggest that syntaxin 1A exerts its effects, directly or indirectly, on GAT1 function through interactions with GAT1's N-terminal tail and that the inhibition occurs at a step in the translocation process after substrate binding but which involves both unidirectional transport and transmitter exchange.

Upregulation of γ-aminobutyric acid transporter expression: role of alkylated γ-aminobutyric acid derivatives

Pregabalin [(S)-(+)-3-isobutylgaba] and gabapentin [1-(aminomethyl)cyclohexane acetic acid] are γ-aminobutyric acid (GABA) derivatives that are effective in the treatment of behavioural disorders, convulsions, epilepsy and hyperalgesia. The mechanisms underlying the diverse actions of these compounds in the brain have not been well elucidated. To test the hypothesis that these compounds exert some of their effects on GABA-ergic systems in the brain, we examined their role in regulating the rat brain GABA transporter GAT1, a plasma membrane protein involved in regulating synaptic transmitter levels. Prolonged incubation of hippocampal cultures, which endogenously express GAT1, with gabapentin and pregabalin caused a 2-fold increase in subsequent GABA uptake, which was concentration- and time-dependent. This increase in uptake was correlated with a redistribution of GAT1 protein from intracellular locations to the plasma membrane. Further experiments also suggested that the signal transduction cascade that modulates pregabalin-mediated GAT1 redistribution may involve pathways activated by specific GAT1 substrates and antagonists but does not involve protein kinase C and tyrosine kinases, two other pathways known to regulate GAT1 redistribution. These data suggest that pregabalin and gabapentin may exert some of their actions in the brain by altering GABAergic signalling.

Syntaxin 1A up-regulates GABA transporter expression by subcellular redistribution

Neurotransmitter transporters are regulated through a variety of signal transduction mechanisms which appear to operate in order to maintain appropriate levels of transmitter in the synaptic cleft. One such mechanism is the trafficking of the transporter in association with synaptic vesicle release machinery. This report examines the specifics of trafficking regulation of the rat brain GABA transporter GAT1 by syntaxin 1A, a plasma membrane component of the SNARE complex which is involved in vesicle membrane fusion. In hippocampal neurons, botulinum neurotoxin 1C, which specifically cleaves syntaxin 1A, down-regulates plasma membrane GAT1 levels as assessed by surface biotinylation, suggesting that syntaxin 1A is a positive regulator of GAT1 surface expression. The up-regulation correlates with a decrease in intracellular GAT1 levels and is complete within several minutes. These data suggest that syntaxin 1A mediates the redistribution of GAT1 on a time scale important for the rapid regulation of extracellular GABA levels. Expression of different syntaxin 1A constructs in Xenopus oocytes suggests that several portions of the syntaxin 1A molecule are required for the trafficking of GAT1. These data suggest that the trafficking of GAT1 will be subject to regulatory control by the many molecules known to interact with various domains of syntaxin 1A.

Multiple G Protein-Coupled Receptors Initiate Protein Kinase C Redistribution of GABA Transporters in Hippocampal Neurons

Neurotransmitter transporters function in synaptic signaling in part through the sequestration and removal of neurotransmitter from the synaptic cleft. A recurring theme of transporters is that many can be functionally regulated by protein kinase C (PKC); some of this regulation occurs via a redistribution of the transporter protein between the plasma membrane and the cytoplasm. The endogenous triggers that lead to PKC-mediated transporter redistribution have not been elucidated. G-protein-coupled receptors that activate PKC are likely candidates to initiate transporter redistribution. We tested this hypothesis by examining the rat brain GABA transporter GAT1 endogenously expressed in hippocampal neurons. Specific agonists of G-protein-coupled acetylcholine, glutamate, and serotonin receptors downregulate GAT1 function. This functional inhibition is dose-dependent, mimicked by PKC activators, and prevented by specific receptor antagonists and PKC inhibitors. Surface biotinylation experiments show that the receptor-mediated functional inhibition correlates with a redistribution of GAT1 from the plasma membrane to intracellular locations. These data demonstrate (1) that endogenous GAT1 function can be regulated by PKC via subcellular redistribution, and (2) that signaling via several different G-protein-coupled receptors can mediate this effect. These results raise the possibility that some effects of G-protein-mediated alterations in synaptic signaling might occur through changes in the number of transporters expressed on the plasma membrane and subsequent effects on synaptic neurotransmitter levels.

Protein Kinase C Regulates the Interaction between a GABA Transporter and Syntaxin 1A

Syntaxin 1A inhibits GABA uptake of an endogenous GABA transporter in neuronal cultures from rat hippocampus and in reconstitution systems expressing the cloned rat brain GABA transporter GAT1. Evidence of interactions between syntaxin 1A and GAT1 comes from three experimental approaches: botulinum toxin cleavage of syntaxin 1A, syntaxin 1A antisense treatments, and coimmunoprecipitation of a complex containing GAT1 and syntaxin 1A. Protein kinase C (PKC), shown previously to modulate GABA transporter function, exerts its modulatory effects by regulating the availability of syntaxin 1A to interact with the transporter, and a transporter mutant that fails to interact with syntaxin 1A is not regulated by PKC. These results suggest a new target for regulation by syntaxin 1A and a novel mechanism for controlling the machinery involved in both neurotransmitter release and reuptake.

Second Messengers, Trafficking-Related Proteins, and Amino Acid Residues that Contribute to the Functional Regulation of the Rat Brain GABA Transporter GAT1

Recent evidence indicates that several members of the Na+-coupled transporter family are regulated, and this regulation in part occurs by redistribution of transporters between intracellular locations and the plasma membrane. We elucidate components of this process for both wild-type and mutant GABA transporters (GAT1) expressed in Xenopus oocytes using a combination of uptake assays, immunoblots, and electrophysiological measurements of membrane capacitance, transport-associated currents, and GAT1-specific charge movements. At low GAT1 expression levels, activators of protein kinase C (PKC) induce redistribution of GAT1 from intracellular vesicles to the plasma membrane; at higher GAT1 expression levels, activators of PKC fail to induce this redistribution. However, coinjection of total rat brain mRNA with GAT1 permits PKC-mediated modulation at high transporter expression levels. This effect of brain mRNA on modulation is mimicked by coinjection of syntaxin 1a mRNA and is eliminated by injecting synaptophysin or syntaxin antisense oligonucleotides. Additionally, botulinum toxins, which inactivate proteins involved in vesicle release and recycling, reduce basal GAT1 expression and prevent PKC-induced translocation. Mutant GAT1 proteins, in which most or all of a leucine heptad repeat sequence was removed, display altered basal distribution and lack susceptibility to modulation by PKC, delineating one region of GAT1 necessary for its targeting. Thus, functional regulation of GAT1 in oocytes occurs via components common to transporters and to trafficking in both neural and non-neural cells, and suggests a relationship between factors that control neurotransmitter secretion and the components necessary for neurotransmitter uptake.

GABA uptake and release by a mammalian cell line stably expressing a cloned rat brain GABA transporter.

In order to facilitate study of the neuronal GABA transporter and provide a convenient system for potential drug screening, we have established a CHO cell line, designated 1F9, which stably expresses the cloned GABA transporter from rat brain (GAT-1). 1F9 cells transport GABA at levels approximately 300-fold higher than untransfected CHO cells, and GABA transport in these cells has the following properties: (1) a dependence on sodium and chloride ions; (2) higher sensitivity to neuronal subtype uptake inhibitors (DABA and ACHC) than to glial subtype inhibitors (beta-alanine and THPO); and (3) Km (2.5 microM) and IC50 values for various competitive ligands that are comparable with values determined in synaptosomes and brain slices. Given the fidelity with which the 1F9 cell line expresses these characteristics of the native neuronal GABA transporter, we have used it to further address GABA transporter activity. [3H]GABA uptake by 1F9 cells is inhibited approximately 50% by the chloride transport blockers DIDS and SITS. The GABA receptor agonists muscimol and baclofen also inhibit GABA transport; however, the receptor antagonists bicuculline and phaclofen have no effect. 1F9 cells also show release of [3H]GABA release is calcium independent, and is differentially affected by changes in the ion gradient, as well as by the presence of external substrates and uptake blockers. These experiments indicate that 1F9 cells provide a convenient system for the screening of GABA transport inhibitors.

Identification of domains of a cloned rat brain GABA transporter which are not required for its functional expression

The sodium and chloride coupled γ-aminobutyric acid (GABA) transporter purified from rat brain, belongs to a superfamily of neurotransmitter transporters. They are involved in the termination of synaptic transmission and are predicted to have 12 membrane spanning α-helices with both amino- and carboxyl-termini oriented toward the cytoplasm. In order to define the domains not required for functional expression, we have constructed and expressed a series of deletion mutants in GAT-1, the cDNA clone encoding for the transporter. Transporters truncated at either end until just a few amino-acids distance from the beginning of helix 1 and the end of helix 12, retain their ability to catalyze sodium and chloride-dependent GABA transport.

Partial purification of the human platelet 5-HT transporter and crossreactivity with antibodies to the rat brain GABA transporter

Partial purification of the human platelet 5-HT transporter and crossreactivity with antibodies to the rat brain GABA transporter

Partial purification of the human platelet 5-HT transporter and crossreactivity with antibodies to the rat brain GABA transporter

Partial purification of the human platelet 5-HT transporter and crossreactivity with antibodies to the rat brain GABA transporter