Γ-aminobutyric Acid Transporter Research Articles

Concentrative Export from the Endoplasmic Reticulum of the γ-Aminobutyric Acid Transporter 1 Requires Binding to SEC24D

Re-uptake of gamma-aminobutyric acid (GABA) into presynaptic specializations is mediated by the GABA transporter 1 (GAT1), a member of the SLC6 gene family. Here, we show that a motif in the COOH terminus of GAT1 ((566)RL(567)), which is conserved in SLC6 family members, is a binding site for the COPII coat component Sec24D. We also identified residues in Sec24D ((733)DD(734)) that are required to support the interaction with GAT1 and two additional family members, i.e. the transporters for serotonin and dopamine. We used three strategies to prevent recruitment of Sec24D to GAT1: knock-down of Sec24D by RNA interference, overexpression of Sec24D-VN (replacement of (733)DD(734) by (733)VN(734)), and mutation of (566)RL(567) to (566)AS(567) (GAT1-RL/AS). In each instance, endoplasmic reticulum (ER) export of GAT1 was impaired: in the absence of Sec24D or upon coexpression of dominant negative Sec24D-VN, GAT1 failed to undergo concentrative ER export; GAT1-RL/AS also accumulated in the ER and exerted a dominant negative effect on cell surface targeting of wild type GAT1. Our observations show that concentrative ER-export is contingent on a direct interaction of GAT1 with Sec24D; this also provides a mechanistic explanation for the finding that oligomeric assembly of transporters is required for their ER export: transporter oligomerization supports efficient recruitment of COPII components.

Reduced Anxiety and Depression-Like Behaviors in Mice Lacking GABA Transporter Subtype 1

Gamma-aminobutyric acid (GABA) transporter subtype 1 (GAT1), which transports extracellular GABA into presynaptic neurons, plays an important regulatory role in the function of GABAergic systems. However, the contributions of the GAT1 in regulating mental status are not fully understood. In this paper, we observed the behavioral alterations of GAT1 knockout (GAT1(-/-)) mice using several depression- and anxiety-related models (eg, the forced-swimming test and the tail-suspension test for testing depression-related behaviors; the open-field test, the dark-light exploration test, the emergence test, and the elevated plus maze (EPM) test for anxiety-related behaviors). Here we found that GAT1(-/-) mice showed a lower level of depression- and anxiety-like behaviors in comparison to wild-type mice. Furthermore, GAT1(-/-) mice exhibited measurable insensitivity to selected antidepressants and anxiolytics such as fluoxetine, amitriptyline, buspirone, diazepam, and tiagabine in the tail-suspension test and/or the EPM test. Moreover, the basal level of corticosterone was found to be significantly lower in GAT1(-/-) mice. These results showed that the absence of GAT1 affects mental status through enhancing the GABAergic system, as well as modifying the serotonergic system and the hypothalamic-pituitary-adrenal (HPA) activity in mice.

State-dependent Conformations of the Translocation Pathway in the Tyrosine Transporter Tyt1, a Novel Neurotransmitter:Sodium Symporter from Fusobacterium nucleatum

The gene of a novel prokaryotic member (Tyt1) of the neurotransmitter:sodium symporter (NSS) family has been cloned from Fusobacterium nucleatum. In contrast to eukaryotic and some prokaryotic NSSs, which contain 12 transmembrane domains (TMs), Tyt1 contains only 11 TMs, a characteristic shared by approximately 70% of prokaryotic NSS homologues. Nonetheless upon heterologous expression in an engineered Escherichia coli host, Tyt1 catalyzes robust Na+-dependent, highly selective l-tyrosine transport. Genetic engineering of Tyt1 variants devoid of cysteines or with individually retained endogenous cysteines at positions 18 or 238, at the cytoplasmic ends of TM1 and TM6, respectively, preserved normal transport activity. Whereas cysteine-less Tyt1 was resistant to the inhibitory effect of sulfhydryl-alkylating reagents, N-ethylmaleimide inhibited transport by Tyt1 variants containing either one or both of the endogenous cysteines, and this inhibition was altered by the substrates sodium and tyrosine, consistent with substrate-induced dynamics in the transport pathway. Our findings support a binding model of Tyt1 function in which an ordered sequence of substrate-induced structural changes reflects distinct conformational states of the transporter. This work identifies Tyt1 as the first functional bacterial NSS member putatively consisting of only 11 TMs and shows that Tyt1 is a suitable model for the study of NSS dynamics with relevance to structure/function relationships of human NSSs, including the dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid transporters.

Subcellular Redistribution of the Serotonin Transporter by Secretory Carrier Membrane Protein 2

The serotonin transporter (SERT) belongs to the SLC6 family of sodium- and chloride-dependent neurotransmitter transporters responsible for uptake of amino acids and biogenic amines from extracellular spaces. Their activities and subcellular distributions are regulated by various cellular mechanisms, including interactions with other proteins. Using the yeast two-hybrid approach we screened a human brain cDNA library and identified secretory carrier membrane protein 2 (SCAMP2) as a novel SERT-interacting protein. GST-pulldown assays confirmed the physical interaction between SCAMP2 and the N-terminal domain of SERT. In addition, SERT was found to form a complex with SCAMP2 as demonstrated by co-immunoprecipitation from a heterologous expression system and from rat brain homogenate. Co-expression of SERT and SCAMP2 in mammalian cells results in the subcellular redistribution of SERT with a decrease in cell surface SERT and a concomitant reduction in 5-HT uptake activity. Using confocal microscopy we show that in neuronal cells endogenous SERT co-localizes with SCAMP2 in discrete structures also containing the lipid raft marker flotillin-1 and the SNARE protein syntaxin 1A. In contrast, SERT immunoreactivity is clearly segregated from transferrin receptor-containing endosomes. A single amino acid mutation, cysteine-201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, abolished SCAMP2-mediated down-regulation of SERT, although this mutation had no effect on the physical interaction between SERT and SCAMP2. Taken together, our results suggest that SCAMP2 plays an important role in the regulation of the subcellular distribution of SERT.

Identification of a Lithium Interaction Site in the γ-Aminobutyric Acid (GABA) Transporter GAT-1

The sodium- and chloride-dependent electrogenic gamma-aminobutyric acid (GABA) transporter GAT-1, which transports two sodium ions together with GABA, is essential for synaptic transmission by this neurotransmitter. Although lithium by itself does not support GABA transport, it has been proposed that lithium can replace sodium at one of the binding sites but not at the other. To identify putative lithium selectivity determinants, we have mutated the five GAT-1 residues corresponding to those whose side chains participate in the sodium binding sites Na1 and Na2 of the bacterial leucine-transporting homologue LeuT(Aa). In GAT-1 and in most other neurotransmitter transporter family members, four of these residues are conserved, but aspartate 395 replaces the Na2 residue threonine 354. At varying extracellular sodium, lithium stimulated sodium-dependent transport currents as well as [3H]GABA uptake in wild type GAT-1. The extent of this stimulation was dependent on the GABA concentration. In mutants in which aspartate 395 was replaced by threonine or serine, the stimulation of transport by lithium was abolished. Moreover, these mutants were unable to mediate the lithium leak currents. This phenotype was not observed in mutants at the four other positions, although their transport properties were severely impacted. Thus at saturating GABA, the site corresponding to Na2 behaves as a low affinity sodium binding site where lithium can replace sodium. We propose that GABA participates in the other sodium binding site, just like leucine does in the Na1 site, and that at limiting GABA, this site determines the apparent sodium affinity of GABA transport.

A novel selective γ-aminobutyric acid transport inhibitor demonstrates a functional role for GABA transporter subtype GAT2/BGT-1 in the CNS

The system of GABA transporters in neural cells constitutes an efficient mechanism for terminating inhibitory GABAergic neurotransmission. This transport system is an important therapeutical target in epileptic disorders, but potentially also in other neurological disorders. Thus, selective intervention in GABA uptake has been the subject of extensive research for several decades. In a series of lipophilic diaromatic derivatives of ( RS)-3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole ( exo-THPO), N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[ d]isoxazol-3-ol (EF1502) turned out to be an equipotent inhibitor at the mouse transporters GAT1 and GAT2 (BGT-1) but inactive at GAT3 and GAT4. This novel pharmacological profile among GABA uptake inhibitors prompted a thorough investigation of the in vivo properties of this compound. These investigations have for the first time demonstrated a functional role for GABA transporter subtype GAT2/BGT-1, which points to the therapeutic relevance of inhibiting this transporter subtype. An overview of the development and characterisation of EF1502 is presented here.

AtGAT1, a High Affinity Transporter for γ-Aminobutyric Acid in Arabidopsis thaliana

Functional characterization of Arabidopsis thaliana GAT1 in heterologous expression systems, i.e. Saccharomyces cerevisiae and Xenopus laevis oocytes, revealed that AtGAT1 (At1g08230) codes for an H(+)-driven, high affinity gamma-aminobutyric acid (GABA) transporter. In addition to GABA, other omega-aminofatty acids and butylamine are recognized. In contrast to the most closely related proteins of the proline transporter family, proline and glycine betaine are not transported by AtGAT1. AtGAT1 does not share sequence similarity with any of the non-plant GABA transporters described so far, and analyses of substrate selectivity and kinetic properties showed that AtGAT1-mediated transport is similar but distinct from that of mammalian, bacterial, and S. cerevisiae GABA transporters. Consistent with a role in GABA uptake into cells, transient expression of AtGAT1/green fluorescent protein fusion proteins in tobacco protoplasts revealed localization at the plasma membrane. In planta, AtGAT1 expression was highest in flowers and under conditions of elevated GABA concentrations such as wounding or senescence.

Chronic morphine treatment increases the expression of vesicular glutamate transporter 1 in the mouse spinal cord

Long-term exposure to morphine results in tolerance to morphine-induced antinociception. Here, we found that mice tolerant to morphine exhibited the significant increase in the protein levels of the vesicular glutamate transporter 1 and the synaptic vesicle-specific small G protein Rab3A, but not vesicular glutamate transporter 2 and vesicular γ-aminobutyric acid transporter. These findings suggest that repeated treatment with morphine enhances excitatory synaptic transmission in the spinal cord, and in turn suppresses the morphine-induced antinociception.

Identification and characterization of a novel isoform of the vesicular γ-aminobutyric acid transporter with glucose-regulated expression in rat islets

Pancreatic islets are unique outside the nervous system in that they contain high levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), synthesized by the enzyme glutamic acid decarboxylase (GAD). Since the role that GABA plays in the islet and the mechanisms whereby the two major GAD isoforms (GAD65 and GAD67) function as diabetes-associated autoantigens are unknown, continued characterization of the islet GAD-GABA system is important. We previously demonstrated that the GABA and glycine transporter vesicular inhibitory amino acid transporter (VIAAT also known as VGAT) is present in rat islets. Here we identify a novel 52 kDa variant of VIAAT in rat islets: VIAAT-52 (V52). V52 is an amino-terminally truncated form of VIAAT (V57) that likely results from utilization of a downstream start site of translation. V57 and V52 display different patterns of post-translational modification and cellular expression. Our results have indicated that islet content of V52, but not V57, is responsive to changes in glucose concentration and other extracellular conditions. VIAAT is expressed in the islet alpha cells, but there have been conflicting findings regarding the presence of VIAAT in the beta cells. Here we have also provided additional evidence for the presence of VIAAT in islet beta cells and show that the beta cell line INS-1 expresses V57. V52 may be better adapted than V57 to the unique rat alpha cell GAD-GABA system, which lacks GAD65 and in which VIAAT traffics to secretory granules rather than just to synaptic microvesicles.

Novel parent structures for inhibitors of the murine GABA transporters mGAT3 and mGAT4

Searching for potent and subtype selective parent structures of the murine γ-aminobutyric acid (GABA) transporter subtypes mGAT3 and mGAT4 a series of amino acids was characterised in a uniform [3H]GABA uptake test system based on transiently expressed mGAT1-4. From several potent inhibitors showing IC50 values at mGAT3 and mGAT4 in the low μM range cis-4-aminocrotonic acid and (RS)-2,3-diaminopropionic acid turned out to be most subtype selective for these transporters. With (RS)-isoserine — a compound unknown as GAT inhibitor until now — one of the most potent amino acids selectively inhibiting mGAT3 and mGAT4 was found. Furthermore, (2-amino-1,3-thiazol-4-yl)acetic acid was identified as the first parent structure exhibiting a clear, though still moderate, selective inhibition of GABA uptake at mGAT3.

Proximity of Transmembrane Domains 1 and 3 of the γ-AminobutyricAcid Transporter GAT-1 Inferred from Paired CysteineMutagenesis

GAT-1 is a sodium- and chloride-dependent gamma-aminobutyric acid transporter and is the first identified member of a family of transporters that maintain low synaptic neurotransmitter levels and thereby enable efficient synaptic transmission. Because transmembrane domains 1 and 3 contain amino acid residues important for transport activity, we hypothesized that these domains may participate in the formation of the binding pocket of the transporter. Pairwise substitutions have been introduced in several predicted transmembrane domains and in the first extracellular loop of GAT-1. In the double mutant W68C/I143C, in which the cysteines were introduced at locations at the extracellular part of transmembrane domains 1 and 3, respectively, approximately 70% inhibition of transport was observed by cadmium with an IC50 of approximately 10 microm. This inhibition was not observed in the corresponding single mutants and also not in > 10 other double mutants, except for V67C/I143C, where the half-maximal effect was obtained at approximately 50 microm. The inhibition by cadmium was only observed when the cysteine pairs were introduced in the same polypeptide. Our results suggest that transmembrane domains 1 and 3 come in close proximity within the transporter monomer.

Transporter-associated Currents in the γ-Aminobutyric Acid Transporter GAT-1 Are Conditionally Impaired by Mutations of a Conserved Glycine Residue

To determine whether glycine residues play a role in the conformational changes during neurotransmitter transport, we have analyzed site-directed mutants of the gamma-aminobutyric acid (GABA) transporter GAT-1 in a domain containing three consecutive glycines conserved throughout the sodium- and chloride-dependent neurotransmitter transporter family. Only cysteine replacement of glycine 80 resulted in the complete loss of [(3)H]GABA uptake, but oocytes expressing this mutant exhibited the sodium-dependent transient currents thought to reflect a charge-moving conformational change. When sodium was removed and subsequently added back, the transients by G80C did not recover, as opposed to wild type, where recovery was almost complete. Remarkably, the transients by G80C could be restored after exposure of the oocytes to either GABA or a depolarizing pre-pulse. These treatments also resulted in a full recovery of the transients by the wild type. Whereas in wild type lithium leak currents are observed after prior sodium depletion, this was not the case for the glycine 80 mutants unless GABA was added or the oocytes were subjected to a depolarizing pre-pulse. Thus, glycine 80 appears essential for conformational transitions in GAT-1. When this residue is mutated, removal of sodium results in "freezing" the transporter in one conformation from which it can only exit by compensatory changes induced by GABA or depolarization. Our results can be explained by a model invoking two outward-facing states of the empty transporter and a defective transition between these states in the glycine 80 mutants.

Trafficking of the Plasma Membrane γ-Aminobutyric Acid Transporter GAT1: SIZE AND RATES OF AN ACUTELY RECYCLING POOL

Trafficking of the Plasma Membrane γ-Aminobutyric Acid Transporter GAT1: SIZE AND RATES OF AN ACUTELY RECYCLING POOL

Zinc inhibition of γ-aminobutyric acid transporter 4 (GAT4) reveals a link between excitatory and inhibitory neurotransmission

gamma-Aminobutyric acid (GABA) transporters (GATs) play an important role in inhibitory neurotransmission by clearing synaptically released GABA and by maintaining low resting levels of GABA in synaptic and extrasynaptic regions. In certain brain regions, vesicular zinc is colocalized and coreleased with glutamate and modulates the behavior of a number of channels, receptors, and transporters. We examined the effect of zinc on expressed GATs (GAT1, GAT2, GAT3, and GAT4) in Xenopus laevis oocytes by using tracer flux and electrophysiological methods. We show that zinc is a potent inhibitor of GAT4 (K(i) of 3 muM). Immunolocalization of GAT4 in the hippocampus revealed dense localization in the CA1 and CA3 regions of the hippocampus, regions which are known to be heavily populated by zinc-containing glutamatergic neurons. The results suggest a physiological role of synaptically released zinc in the hippocampus, because zinc released from hyperactive glutamatergic neurons may simultaneously bring about elevated GABAergic inhibition. Therefore, this mode of zinc function signifies a link between excitatory and inhibitory neurotransmission and may play a neuroprotective role against glutamate-induced excitotoxicity.

Excretion of Putrescine and Spermidine by the Protein Encoded by YKL174c (TPO5) in Saccharomyces cerevisiae

The properties of the protein encoded by YKL174c (TPO5) were studied. It was found that TPO5 excretes putrescine effectively and spermidine less effectively. Gamma-aminobutyric acid slightly inhibited the excretion of putrescine, but basic amino acids did not affect excretion, suggesting that TPO5 preferentially recognizes polyamines. Accordingly, yeast cells transformed with the plasmid encoding YKL174c (TPO5) were resistant to toxicity caused by 120 mm putrescine or by 3 mm spermidine, and a mutant with a disrupted YKL174c (TPO5) gene was sensitive to toxicity by 90 mm putrescine. The growth of this mutant was faster than that of the wild-type strain. In parallel, there was an increase in putrescine and spermidine content of the YKL174c (TPO5) mutant compared with wild-type. It is noted that TPO5 functions as a suppressor of cell growth by excreting polyamines. The level of YKL174c (TPO5) mRNA was increased by the addition of polyamines to the medium. The degree of induction of the mRNA was spermine > spermidine > putrescine. The subcellular localization of TPO5 was determined by immunostaining of hemagglutinin-tagged TPO5, and it was found on Golgi or post-Golgi secretory vesicles. Excretion of putrescine and spermidine by TPO5 was reduced in cells that have mutations in the secretory or endocytic pathways, indicating that both processes are involved in the excretion of polyamines.

P.4.026 The anticonflict and antidepressant-like effects of proproten are serotonin dependent

Two novel nipecotic acid derivatives, 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(4-methoxyphenyl)-4-piperidinol (NNC 05-2045) and 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methoxyphenyl)-4-piperidinol (NNC 05-2090) have been tested for inhibition of γ-amino butyric acid (GABA) transporters in synaptosomal preparations of rat cerebral cortex and inferior colliculus and found to differ markedly from gabitril® (tiagabine), a selective GAT-1 inhibitor. IC50 values for inhibition of [3H]GABA uptake into synaptosomes from cerebral cortex for NNC 05-2045 and NNC 05-2090 were 12±2 and 4.4±0.8 μM, respectively. In synaptosomes from inferior colliculus in the presence of 1 μM 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid (NNC 05-0711), a highly potent and selective GAT-1 inhibitor, IC50 values for inhibition of [3H]GABA uptake were 1.0±0.1 and 2.5±0.7 μM, respectively. A receptor profile showed that NNC 05-2045 has binding affinities to sigma-, α1- and D2-receptors of 113, 550 and 122 nM, respectively. NNC 05-2090 displayed α1- and D2-receptor affinity of 266 and 1632 nM, respectively. The anticonvulsant action of both compounds was tested in four rodent models after intra peritoneal (i.p.) injection. Both NNC 05-2045 and NNC 05-2090 dose-dependently inhibited sound-induced tonic and clonic convulsions in DBA/2 mice with ED50 values of 6 and 19 μmol/kg, respectively. NNC 05-2045 also antagonized sound-induced seizures in genetic epilepsy prone rats (GEP rats) with ED50 values against wild running, clonic and tonic convulsions of 33, 39 and 39 μmol/kg, respectively (NNC 05-2090 was not tested in GEP rats). Both NNC 05-2045 and NNC 05-2090 dose-dependently antagonized tonic hindlimb extension in the maximal electroshock (MES) test with ED50 values of 29 and 73 μmol/kg, respectively. In amygdala kindled rats NNC 05-2045 and NNC 05-2090 significantly (P<0.05) reduced generalized seizure severity (seizure grade 3–5) at highest doses (72–242 μmol/kg) and NNC 05-2090 also significantly reduced afterdischarge duration at these doses (P<0.05). These data show that inhibition of GABA uptake through non-GAT-1 transporters has different anticonvulsant effects than selective GAT-1 inhibitors (e.g. tiagabine) in that enhanced efficacy against MES and reduced efficacy against kindled seizures is observed. Although a contribution of adrenergic agonistic effects cannot be entirely ruled out, it is proposed that inhibition of GAT-3 (mouse GAT4) is primarily responsible for the anticonvulsant action of these two nipecotic acid derivatives in MES, amygdala kindled rats and in sound-induced seizures in GEP-rats and DBA/2 mice.

Novel Properties of a Mouse γ-Aminobutyric Acid Transporter (GAT4)

We expressed the mouse gamma-aminobutyric acid (GABA) transporter GAT4 (homologous to rat/ human GAT-3) in Xenopus laevis oocytes and examined its functional and pharmacological properties by using electrophysiological and tracer uptake methods. In the coupled mode of transport (Na+/ Cl-/GABA cotransport), there was tight coupling between charge flux and GABA flux across the plasma membrane (2 charges/GABA). Transport was highly temperature-dependent with a temperature coefficient (Q10) of 4.3. The GAT4 turnover rate (1.5 s(-l); -50 mV, 21 degrees C) and temperature dependence suggest physiological turnover rates of 15-20 s(-1). No uncoupled current was observed in the presence of Na+. In the absence of external Na+, GAT4 exhibited two distinct uncoupled currents. (i) A Cl- leak current (ICl(leak)) was observed when Na+ was replaced with choline or tetraethylammonium. The reversal potential of (ICl(leak)) followed the Cl- Nernst potential. (ii) A Li+ leak current (ILi(leak)) was observed when Na+ was replaced with Li+. Both leak currents were inhibited by Na+, and both were temperature-independent (Q10 approximately 1). The two leak modes appeared not to coexist, as Li+ inhibited (ICl(leak)). The results suggest the existence of cation- and anion-selective channel-like pathways in GAT4. Flufenamic acid inhibited GAT4 Na+/Cl-/GABA cotransport, ILi(leak), and ICl(leak), (Ki approximately 30 microM), and the voltage-induced presteady-state charge movements (Ki approximately 440 microM). Flufenamic acid exhibited little or no selectivity for GAT1, GAT2, or GAT3. Sodium and GABA concentration jicroumps revealed that slow Na+ binding to the transporter is followed by rapid GABA-induced translocation of the ligands across the plasma membrane. Thus, Na+ binding and associated conformational changes constitute the rate-limiting steps in the transport cycle.

Oligomerization of the γ-Aminobutyric Acid Transporter-1 Is Driven by an Interplay of Polar and Hydrophobic Interactions in Transmembrane Helix II

The available evidence indicates that members of the neurotransmitter:sodium symporter family form constitutive oligomers. Their second transmembrane helix (TM2) contains a leucine heptad repeat proposed to be involved in oligomerization. In artificial transmembrane segments, interhelical interactions are stabilized by polar residues. We searched for these hydrogen bond donors in TM2 by mutating the five polar residues in TM2 of the gamma-aminobutyric acid transporter-1 (GAT1). We tested the ability of the resulting mutants to oligomerize by fluorescence microscopy, Foerster resonance energy transfer, and beta-lactamase fragment complementation. Of all generated mutants, only Y86A- (but not Y86F-), E101A-, E101Q-, and E101D-GAT1 were judged by these criteria to be deficient in oligomerization and were retained intracellularly. The observations are consistent with a model where the leucine heptad repeat in TM2 drives a homophilic association that is stabilized by Tyr(86) and Glu(101); Tyr(86) participates in hydrophobic stacking. Glu(101) is in the a-position of the leucine heptad repeat (where positions 1-7 are denoted a-g, and each leucine is in the central d-position). Thus, Glu(101) is in the position predicted for the hydrogen bond donor (i.e. sandwiched between Leu(97) and Leu(104), which are one helical turn above and below Glu(101)). These key residues, namely Tyr(86) and Glu(101), are conserved in related transporters from archaeae to humans; they are therefore likely to support oligomeric assembly in transporter orthologs and possibly other proteins with multiple transmembrane segments.

Oxidative stress affects synaptosomal gamma-aminobutyric acid and glutamate transport in diabetic rats: the role of insulin.

Evidence suggests that oxidative stress is involved in the pathophysiology of diabetic complications and that insulin has a neuroprotective role in oxidative stress conditions. In this study, we evaluated the in vitro effect of insulin in the susceptibility to oxidative stress and in the transport of the amino acid neurotransmitters gamma-aminobutyric acid (GABA) and glutamate in a synaptosomal fraction isolated from male type 2 diabetic Goto-Kakizaki (GK) rat brain cortex. The ascorbate/Fe(2+)-induced increase in thiobarbituric acid reactive substances (TBARSs) was similar in Wistar and GK rats and was not reverted by insulin (1 micromol/l), suggesting that other mechanisms, rather than a direct effect in membrane lipid peroxidation, may mediate insulin neuroprotection. Diabetes did not affect GABA and glutamate transport, despite the significant decrease in membrane potential and ATP/ADP ratio, and insulin increased the uptake of both GABA and glutamate in GK rats. Upon oxidation, there was a decrease in the uptake of both neurotransmitters and an increase in extrasynaptosomal glutamate levels and in ATP/ADP ratio in GK rats. Insulin treatment reverted the ascorbate/Fe(2+)-induced decrease in GABA accumulation, with a decrease in extrasynaptosomal GABA. These results suggest that insulin modulates synaptosomal GABA and/or glutamate transport, thus having a neuroprotective role under oxidizing and/or diabetic conditions.

Two Discontinuous Segments in the Carboxyl Terminus Are Required for Membrane Targeting of the Rat γ-Aminobutyric Acid Transporter-1 (GAT1)

Like all members of the Na(+)/Cl(-)-dependent neurotransmitter transporter family, the rat gamma-aminobutyric acid transporter-1 (GAT1) is sorted and targeted to specialized domains of the cell surface. Here we identify two discontinuous signals in the carboxyl terminus of GAT1 that cooperate to drive surface expression. This conclusion is based on the following observations. Upon deletion of the last 37 amino acids, the resulting GAT1-Delta37 remained trapped in the endoplasmic reticulum. The presence of 10 additional residues (GAT1-Delta27) sufficed to support the interaction with the coat protein complex II component Sec24D; surface expression of GAT1-Delta27 reached 50% of the wild type level. Additional extensions up to the position -3 (GAT1-Delta3) did not further enhance surface expression. Thus the last three amino acids (AYI) comprise a second distal signal. The sequence AYI is reminiscent of a type II PDZ-binding motif; accordingly substituting Glu for Ile abrogated the effect of this motif. Neither the AYI motif nor the last 10 residues rescued the protein from intracellular retention when grafted onto GAT1-Delta37 and GAT1-Delta32; the AYI motif was dispensable for targeting of GAT1 to the growth cone of differentiating PC12 cells. We therefore conclude that the two segments act in a hierarchical manner such that the proximal motif ((569)VMI(571)) supports endoplasmic reticulum export of the protein and the distal AYI motif places GAT1 under the control of the exocyst.