Molecular cloning and expression of a high affinity L-proline transporter expressed in putative glutamatergic pathways of rat brain.

We have used the polymerase chain reaction (PCR) with degenerate oligonucleotides derived from two conserved regions of the norepinephrine and gamma-aminobutyric acid transporters to identify novel Na(+)-dependent transporters in rat brain. One PCR product hybridized to a 4.0 kb RNA concentrated in subpopulations of putative glutamatergic neurons including mitral cells of the olfactory bulb, pyramidal cells of layer V of the cerebral cortex, pyramidal cells of the piriform cortex, and pyramidal cells of field CA3 of the hippocampus. Transient expression of the cognate cDNA conferred Na(+)-dependent L-proline uptake in HeLa cells that was saturable (Km = 9.7 microM) and exhibited a pharmacological profile similar to that for high affinity L-proline transport in rat brain slices. The cloned transporter cDNA predicts a 637 aa protein with 12 putative transmembrane domains and exhibits 44%-45% amino acid sequence identity with other members of the emerging family of neurotransmitter transporters. These findings support a synaptic role for L-proline in specific excitatory pathways in the CNS.     

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.     

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.     

Changes in expression of neuronal and glial glutamate transporters in lead-exposed adult rat brain

Excitatory amino acid transporters (EAATs) are membrane-bound proteins localized in glial and neuronal cells which transport glutamate (Glu) in a process essential for terminating its action and protecting neurons from excitotoxic damage. Since Pb-induced neurotoxicity has a glutamatergic component and astrocytes serve as a cellular Pb deposition site, it was of interest to investigate the response of main glutamate transporters to short-term lead exposure in the adult rat brain (25mg/kg b.w. of lead acetate, i.p. for 3 days). We examined the expression of mRNA and protein of GLAST, GLT-1 and EAAC1 in homogenates obtained from cerebellum, hippocampus and forebrain. Molecular evidence is provided which indicates that, of the two glial transporters, GLT-1 is more susceptible than GLAST to the neurotoxic effect arising from Pb. RT-PCR analysis revealed highly decreased expression of GLT-1 mRNA in forebrain and hippocampus. In contrast, GLAST was overexpressed in forebrain and in cerebellum. In the case of EAAC1, the enhanced expression of mRNA and protein of transporter was observed only in forebrain. The results demonstrate regional differences in the expression of glutamate transporters after short-term exposure to Pb. In forebrain, downregulation of GLT-1 is compensated by enhanced expression of GLAST, while in hippocampus, the expression of both is lowered. This observation suggests that under conditions of Pb toxicity in adult rat brain, the hippocampus is most vulnerable to the excitotoxic cell damage arising from impaired clearance of the released glutamate.     

GABA Effects During Neuronal Differentiation of Stem Cells

Gamma-amino butyrate (GABA) is the most prevalent inhibitory neurotransmitter in the adult brain. In this review, we summarize the pharmacology and regulation of GABAergic transmission components (biosynthetic enzymes, receptors and transporters) in adult non-neurogenic brain regions. The effects of targeted mutations in genes relevant for GABAergic functions and how they influence specific neuronal circuits and pathological states are presented. We then review GABA actions on neuronal differentiation. During brain development, GABA has depolarizing activity in cerebrocortical neural precursors, controlling cell division and contributing to neuronal migration and maturation. In the adult forebrain there are two neurogenic regions exposed to synaptic and non-synaptic GABA release. Neural stem cells and neuronal progenitors express GABA receptors in subventricular and subgranular zones. GABA effects in these cells are very similar to those found in embryonic cortical precursor cells, and therefore it is possible that this amino acid has important roles during adult brain plasticity. Special issue article in honor of Dr. Ricardo Tapia.     

Expression of rat liver glutamine transporters in Xenopus laevis oocytes.

As a first step in attempting to isolate the Na(+)-dependent System N transporter from rat liver we have investigated the use of prophase-arrested oocytes from Xenopus laevis for the functional expression of rat liver glutamine transporters. Individual oocytes, defolliculated by collagenase treatment, were injected with 50 nl of a 1 mg.ml-1 solution of poly(A)+ RNA (mRNA) isolated from rat liver. 50 microM L-[3H]glutamine uptake was measured 1-5 days post-injection: after 48 h, poly(A)+ RNA-injected oocytes showed a 60 +/- 12% increase in Na(+)-dependent glutamine uptake compared to controls. This increased uptake showed characteristic features of hepatic System N: that is, it tolerated Li(+)-for-Na+ substitution and was inhibited by the System N substrate L-histidine (5 mM) in Li medium, unlike endogenous Na(+)-dependent glutamine transport. In subsequent experiments rat liver poly(A)+ RNA, size-fractionated by density gradient fractionation, was injected into oocytes. Injection of poly(A)+ RNA of 1.9-2.8 kilobases (kb) in size resulted in a significant stimulation of Na(+)-dependent glutamine transport to 0.362 +/- 0.080 pmol.min-1/oocyte from 0.178 +/- 0.060 pmol.min-1/oocyte in vehicle-injected oocytes (p less than 0.01). A lighter fraction, with poly(A)+ RNA of less than 1.9 kilobases size resulted in a similar increase in Na(+)-dependent glutamine uptake which was largely Li(+)-tolerant: Li(+)-stimulated glutamine uptake in oocytes injected with this fraction increased to 0.230 +/- 0.070 pmol.min-1/oocyte from 0.098 +/- 0.029 pmol.min-1/oocyte in controls (p less than 0.05). This enhanced rate of Li(+)-stimulated glutamine uptake was inhibited 28 and 70%, respectively, by 1 and 5 mM L-histidine. Na(+)-independent uptake of glutamine rose by 72 +/- 12% in oocytes injected with poly(A)+ RNA of 2.8-3.6 kb (p less than 0.001). These results demonstrate that glutamine transporters, with characteristics associated with hepatic Systems N, L, and A (or ASC), can be expressed in X. laevis oocytes injected with specific size fractions of rat liver mRNA.     

Effect of sodium lithium and proton concentrations on the electrophysiological properties of the four mouse GABA transporters expressed in Xenopus oocytes

Mouse GABA transporters belong to the family of Na(+) and Cl(-) dependent neurotransmitter transporter. GABA transport, by these family members, was shown to be electrogenic and driven by sodium ions. It was demonstrated that, as in several other transporters, sodium binding and release by GAT1, GAT3 and BGT-1, the canine homolog of GAT2, resulted in the appearance of presteady-state currents. In this work we show that each of the four GABA transporters exhibit unique presteady-state currents when expressed in Xenopus oocytes. The properties of the presteady-state currents correspond to the transporters affinities to Na(+). At 100 mM GAT1 exhibited symmetric presteady-state currents at all imposed potentials, whereas GAT2 exhibited asymmetric presteady-state currents exclusively at negative imposed potentials, GAT3 or GAT4 exhibited presteady-state currents predominantly at positive imposed potentials. GABA uptake by GAT2 and GAT4 was much more sensitive to external pH than GAT1 and GAT3. Reducing the external Na(+) concentration rendered the GABA uptake activity by GAT1 and GAT3 to be sensitive to pH. Lowering the external pH reduced the Na(+) affinity of GAT1. Substitution of the external Na(+) to Li(+) resulted in the appearance of leak currents exclusively at negative potentials in Xenopus oocyte expressing GAT1 and GAT3. Low Na(+) concentrations inhibited the leak currents of GAT1 but Na(+) had little effect on the leak currents of GAT3. Washing of occluded Na(+) in GAT1 enhanced the leak currents. Similarly addition of GABA in the presence of 80 mM Li(+), that presumably accelerated the release of the bound Na(+), also induced the leak currents. Conversely, addition of GABA to GAT3 expressing oocytes, in the presence of 80 mM Li(+), inhibited the leak currents.     

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.     

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.     

Calcium-Independent γ-Aminobutyric Acid Release from Growth Cones: Role of γ-Aminobutyric Acid Transport

Neuronal growth cones isolated in bulk from neonatal rat forebrain have uptake and K(+)-stimulated release mechanisms for gamma-aminobutyric acid (GABA). Up to and including postnatal day 5, the K(+)-stimulated release of [3H]GABA and endogenous GABA is Ca2+ independent. At these ages, isolated growth cones neither contain synaptic vesicles nor stain for synaptic vesicle antigens. Here we examined the possibility that the release mechanism underlying Ca2(+)-independent GABA release from isolated growth cones is by reversal of the plasma membrane GABA transporter. The effects of two GABA transporter inhibitors, nipecotic acid and an analogue of nipecotic acid, SKF 89976-A, on K(+)-stimulated release of [3H]GABA from superfused growth cones were examined. Nipecotic acid both stimulated basal [3H]GABA release and enhanced K(+)-stimulated release of [3H]GABA, which indicates that this agent can stimulate GABA release and is, therefore, not a useful inhibitor with which to test the role of the GABA transporter in K(+)-stimulated GABA release from growth cones. In contrast, SKF 89976-A profoundly depressed both basal and K(+)-stimulated [3H]GABA release. This occurred at similar concentrations at which uptake was blocked. These observations provide evidence for a major role of the GABA transporter in GABA release from neuronal growth cones.     

Amino acid neurotransmitter transporters: Structure,function, and molecular diversity

Many biologically active compounds including neurotransmitters, metabolic precursors, and certain drugs are accumulated intracellularly by transporters that are coupled to the transmembrane Na⁺ gradient. Amino acid neurotransmitter transporters play a key role in the regulation of extracellular amino acid concentrations and termination of neurotransmission in the CNS

1 Abbreviations: CNS, central nervous system; GABA, γ-aminobutyric acid; cDNA, complementary deoxyribonucleic acid; mRNA, messenger ribonucleic acid; NMDA, N-methyl-D-aspartate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; DAG, diacyl glycerol; R59022, DAG kinase inhibitor; AA, arachidonic acid; ACHC, cis-3-aminocyclohexanecarboxylic acid; GAT-A, ACHC-sensitive GABA transporter; GAT-B, β-alanine-sensitive GABA transporter; GLY-1 and GLYT-1, glycine transporters; PROT-1, proline transporter; BGT-1, betaine transporter.

. Transporters for the major amino acid neurotransmitters glutamate, GABA, and glycine are found in both neurons and glial cells. Recent work has resulted in the identification of cDNAs encoding several amino acid neurotransmitter transport proteins, all of which belong to the Na⁺-and Cl^?-dependent transporter gene family. The diversity of this family suggests a degree of transporter heterogeneity that is greater than that indicated by biochemical and pharmacological studies.     

Manganese disrupts astrocyte glutamine transporter expression and function

Glutamine (Gln) plays an important role in brain energy metabolism and as a precursor for the synthesis of neurotransmitter glutamate and GABA. Previous studies have shown that astrocytic Gln transport is impaired following manganese (Mn) exposure. The present studies were performed to identify the transport routes and the respective Gln transporters contributing to the impairment. Rat neonatal cortical primary astrocytes treated with Mn displayed a significant decrease in Gln uptake mediated by the principle Gln transporting systems, N and ASC. Moreover, systems N, ASC and L were less efficient in Gln export after Mn treatment. Mn treatment caused a significant reduction of both in mRNA expression and protein levels of SNAT3 (system N), SNAT2 (system A) and LAT2 (system L), and lowered the protein but not mRNA expression of ASCT2 (system ASC). Mn exposure did not affect the expression of the less abundant systems N transporter SNAT5 and the system L transporter LAT1, at either the mRNA or protein level. Hence, Mn-induced decrease of inward and outward Gln transport can be largely ascribed to the loss of the specific Gln transporters. Consequently, deregulation of glutamate homeostasis and its diminished availability to neurons may lead to impairment in glutamatergic neurotransmission, a phenomenon characteristic of Mn-induced neurotoxicity.     

A cognate dopamine transporter-like activity endogenously expressed in a COS-7 kidney-derived cell line.

The activity of the dopamine transporter is an important mechanism for the maintenance of normal dopaminergic homeostasis by rapidly removing dopamine from the synaptic cleft. In kidney-derived COS-7, COS-1 and HEK-293 but not in other mammalian cell lines (CHO, Y1, Ltk-), we have characterized a putative functional dopamine transporter displaying a high affinity (Km approximately 250 nM) and a low capacity (approximately 0.1 pmol/10(5) cells/min) for [3H]dopamine uptake. Uptake displayed a pharmacological profile clearly indicative of the neuronal dopamine transporter. Estimated Ki values of numerous substrates and inhibitors for the COS-dopamine transporter and the cloned human neuronal transporter (human dopamine transporter) correlate well with the exception of a few notable compounds, including the endogenous neurotransmitter dopamine, the dopamine transporter inhibitor GBR 12,909 and the dopaminergic agonist apomorphine. As with native neuronal and cloned dopamine transporters, the uptake velocity was sodium-sensitive and reduced by phorbol ester pre-treatment. Two mRNA species of 3.8 and 4.0 kb in COS-7 cells were revealed by Northern blot analysis similar in size to that seen in native neuronal tissue. A reverse-transcribed PCR analysis confirmed the existence of a processed dopamine transporter. However, no immunoreactive proteins of expected dopamine transporter molecular size or [3H]WIN 35,428 binding activity were detected. A partial cDNA of 1.3 kb, isolated from a COS-1 cDNA library and encoding transmembrane domains 1-6, displayed a deduced amino acid sequence homology of approximately 96% to the human dopamine transporter. Taken together, the data suggest the existence of a non-neuronal endogenous high affinity dopamine uptake system sharing strong functional and molecular homology to that of the cloned neuronal dopamine transporter.     

The Family of Na+/Cl− Neurotransmitter Transporters

Abstract: The termination of neurotransmission is achieved by rapid uptake of the released neurotransmitter by specific high-affinity neurotransmitter transporters. Most of these transporters are encoded by a family of genes (Na⁺/Cl⁻ transporters) having a similar membrane topography of 12 transmembrane helices. An evolutionary tree revealed five distinct subfamilies: γ-aminobutyric acid transporters, monoamine transporters, amino acid transporters, "orphan" transporters, and the recently discovered bacterial transporters. The bacterial transporters that belong to this family may help to develop heterologous expression systems with the aim of solving the three-dimensional structure of these membrane proteins. Some of the neurotransmitter transporters have been implicated as important sites for drug action. Monoamine transporters, for example, are targeted by major classes of antidepressants, psychostimulants, and antihypertensive drugs. Localization of individual transporters in specific cells and brain areas is pertinent to understanding their contribution to neurotransmission and their potential as targets for drugs. The most important questions in the field include resolving the mechanism of neurotransmitter transport, the structure of the transporters, and the interaction of each transporter in complex neurological activities.     

Chinese hamster ovary mRNA-dependent, Na(+)-independent L-leucine transport in Xenopus laevis oocytes.

In freshly prepared uninjected folliculated oocytes, Na(+)-independent leucine uptake is mediated predominantly by a system L-like transport system. Removal of follicular cells, however, results in an irreversible loss of this transport activity. When total poly(A)+ mRNA derived from Chinese hamster ovary (CHO) cells was injected into prophase-arrested stage V or VI Xenopus laevis oocytes, enhanced expression of Na(+)-independent leucine transport was observed. The injected mRNAs associated with increased levels of leucine uptake were between 2 and 3 kb in length. The newly expressed leucine transport activity exhibited important differences from the known characteristics of system L, which is the dominant Na(+)-independent leucine transporter in CHO cells as well as in freshly isolated folliculated oocytes. The CHO mRNA-dependent leucine uptake in oocytes was highly sensitive to the cationic amino acids lysine, arginine, and and ornithine (> 95% inhibition). As with the leucine uptake, an enhanced lysine uptake was also observed in size-fractionated CHO mRNA-injected oocytes. The uptakes of leucine and lysine were mutually inhibitable, suggesting that the newly expressed transporter was responsible for uptakes of both leucine and lysine. The inhibition of uptake of lysine by leucine was Na+ independent, thus clearly distinguishing it from the previously reported endogenous system y+ activity. Furthermore, the high sensitivity to tryptophan of the CHO mRNA-dependent leucine transport was in sharp contrast to the properties of the recently cloned leucine transport-associated gene from rat kidney tissue, although leucine transport from both sources was sensitive to cationic amino acids. Our results suggest that there may be a family of leucine transporters operative in different tissues and possibly under different conditions.     

Cloning and Expression of a Betaine/GABA Transporter from Human Brain

Abstract: A cDNA clone encoding a human γ-aminobutyric acid (GABA) transporter has been isolated from a brain cDNA library, and its functional properties have been examined in mammalian cells. The nucleotide sequence predicts a transporter with 614 amino acids and 12 putative transmembrane domains. The highest degree of amino acid identity is with a betaine/GABA transporter originally cloned from the dog termed BGT-1 (91%) and a related transporter from mouse brain (87%). These identities are similar to those for species homologues of other neurotransmitter transporters and suggest that the new clone represents the human homologue of BGT-1. The transporter displays high affinity for GABA (IC₅₀ of 30 µM) and is also sensitive to phloretin, l -2,4-diaminobutyric acid, and hypotaurine (IC₅₀ values of ～150–400 µM). The osmolyte betaine is ～25-fold weaker than GABA, displaying an IC₅₀ of ～1 mM. The relative potencies of these inhibitors at human BGT-1 differ from those of mouse and dog BGT-1. Northern blot analysis reveals that BGT-1 mRNA is widely distributed throughout the human brain. The cloning of the human homologue of BGT-1 will further our understanding of the roles of GABA and betaine in neural function.