Cloning and expression of a bovine glutamate transporter

doi:10.1016/0169-328X(94)00244-9

Molecular Brain Research

Volume 28, Issue 2, February 1995, Pages 343-348

https://doi.org/10.1016/0169-328X(94)00244-9 Get rights and content

Abstract

We have isolated a 3845 base-pair cDNA (BNGLUAS) encoding a bovine glutamate transporter (bovine GLAST) by screening a bovine retina cDNA library with an oligonucleotide probe corresponding to conserved regions of known glutamate transporters. The cDNA sequence predicted a protein of 542 amino acids and displayed 96% and 97% amino acid identity with the rat GLAST/GluT-1 and human GLAST, respectively. Expression of the bovine GLAST in Xenopus oocytes revealed Na⁺-dependent [¹⁴C]l-glutamate uptake and electrogenic glutamate uptake.

References (21)

J.L. Arriza et al.
Cloning and expression of a human neutral amino acid transporter with structural similarity to the glutamate transporter gene family
J. Biol. Chem.
(1993)
D.R. Blakely et al.
Expression of neurotransmitter transport from rat brain mRNA in Xenopus laevis oocytes
H. Brew et al.
Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells
Nature
(1987)
M. Casado et al.
Phosphorylation and modulation of brain glutamate transporters by protein kinase C
J. Biol. Chem.
(1993)
D. Eisenberg et al.
The hydrophobic moment detects periodicity in protein hydrophobicity
S. Eliasof et al.
Characterization of the glutamate transporter in retinal cones of the tiger salamander
J. Neurosci.
(1993)
Y. Kanai et al.
Primary structure and functional characterization of a high-affinity glutamate transporter
Nature
(1992)
M. Kozak
An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs
Nucleic Acids Res.
(1987)
J. Kyte et al.
A simple method for displaying the hydropathic character of a protein
J. Mol. Biol.
(1982)
G.J. McBean et al.
Neurotoxicity of l-glutamate and dl-threo-3-hydroxyaspartate
J. Neurochem.
(1985)

There are more references available in the full text version of this article.

Cited by (24)

Topical and systemic drug delivery to the posterior segments
2005, Advanced Drug Delivery Reviews
The posterior segments of the eye are exquisitely protected from the external environment. This poses unique and fairly challenging hurdles for drug delivery. It is somewhat dogmatic that topical ocular delivery is insufficient to achieve therapeutic drug levels in the posterior segments. However, some drugs are currently challenging this dogma. In this review we investigate the constraints and challenges of drug delivery to the posterior segment. Additionally, we outline several potential absorption pathways that may potentially be exploited to deliver drug to the back of the eye. Data on several compounds that achieve therapeutic posterior segment concentrations after topical dosing is presented. Finally, the issues surrounding systemic delivery to the posterior segment are reviewed.
Glutamate uptake
2001, Progress in Neurobiology
Citation Excerpt :
The family of Na+- and Cl−-coupled neurotransmitter transporters (for GABA, glycine, dopamine, serotonin, noradrenaline, proline, taurine) represents a completely different protein family (for review, see Borowsky and Hoffman, 1995; Malandro and Kilberg, 1996; Povlock and Amara, 1997; Nelson, 1998; Masson et al., 1999; Saier, 1999). Analogues of the five glutamate transporters have been sequenced from a number of species including man (Shashidharan and Plaitakis, 1993; Arriza et al., 1994; Kanai et al., 1994; Kawakami et al., 1994; Manfras et al., 1994; Shashidharan et al., 1994a,b), mouse (Tanaka, 1993a; Kirschner et al., 1994a,b; Mukainaka et al., 1995; Sutherland et al., 1995; Maeno-Hikichi et al., 1997), rat (Kanai et al., 1995; Kiryu et al., 1995; Bjørås et al., 1996; Velaz-Faircloth et al., 1996; Lin et al., 1998b; Grewer et al., 2000) cow (Inoue et al., 1995), dog (Sato et al., 2000) and salamander (Eliasof et al., 1998a) as well as nematodes (Kawano et al., 1996, 1997; Radice and Lustigman, 1996) and insects (Donly et al., 1997; Seal et al., 1998; Besson et al., 1999; Kawano et al., 1999; Donly et al., 2000; Kucharski et al., 2000). A partial sequence has been obtained from Aplysia californica (Levenson et al., 2000).
Brain tissue has a remarkable ability to accumulate glutamate. This ability is due to glutamate transporter proteins present in the plasma membranes of both glial cells and neurons. The transporter proteins represent the only (significant) mechanism for removal of glutamate from the extracellular fluid and their importance for the long-term maintenance of low and non-toxic concentrations of glutamate is now well documented. In addition to this simple, but essential glutamate removal role, the glutamate transporters appear to have more sophisticated functions in the modulation of neurotransmission. They may modify the time course of synaptic events, the extent and pattern of activation and desensitization of receptors outside the synaptic cleft and at neighboring synapses (intersynaptic cross-talk). Further, the glutamate transporters provide glutamate for synthesis of e.g. GABA, glutathione and protein, and for energy production. They also play roles in peripheral organs and tissues (e.g. bone, heart, intestine, kidneys, pancreas and placenta). Glutamate uptake appears to be modulated on virtually all possible levels, i.e. DNA transcription, mRNA splicing and degradation, protein synthesis and targeting, and actual amino acid transport activity and associated ion channel activities. A variety of soluble compounds (e.g. glutamate, cytokines and growth factors) influence glutamate transporter expression and activities. Neither the normal functioning of glutamatergic synapses nor the pathogenesis of major neurological diseases (e.g. cerebral ischemia, hypoglycemia, amyotrophic lateral sclerosis, Alzheimer's disease, traumatic brain injury, epilepsy and schizophrenia) as well as non-neurological diseases (e.g. osteoporosis) can be properly understood unless more is learned about these transporter proteins. Like glutamate itself, glutamate transporters are somehow involved in almost all aspects of normal and abnormal brain activity.
Inherited defects of sodium-dependent glutamate transport mediated by glutamate/aspartate transporter in canine red cells due to a decreased level of transporter protein expression
2000, Journal of Biological Chemistry
Citation Excerpt :
TheK m value for l-glutamate obtained from a Lineweaver-Burk plot (Fig. 3 B) was 36.3 μm. This value was slightly higher than that estimated for the uptake in canine red cells at 37 °C (7–14 μm; Refs. 12 and 13), whereas lower affinity was reported for other mammalian GLAST homologues in oocytes (70–80 μm) (1, 28-30). Several structural analogues of l-glutamate were tested for their inhibitory effects on glutamate transport to compare the pharmacological properties of canine GLAST and canine red cells (TableI).
Canine red cells have a high affinity Na⁺/K⁺-dependent glutamate transporter. We herein demonstrate that this transport is mediated by the canine homologue of glutamate/aspartate transporter (GLAST), one of the glutamate transporter subtypes abundant in the central nervous system. We also demonstrate that GLAST is the most ubiquitous glutamate transporter among the transporter subtypes that have been cloned to date. The GLAST protein content was extremely reduced in variant red cells, low glutamate transport (LGlut) red cells characterized by an inherited remarkable decrease in glutamate transport activity. All LGluT dogs carried a missense mutation of Gly⁴⁹² to Ser (G492S) in either the heterozygous or homozygous state. The GLAST protein with G492S mutation was fully functional in glutamate transport in Xenopus oocytes. However, G492S GLAST exhibited a marked decrease in activity after the addition of cycloheximide, while the wild type showed no significant change, indicating that G492S GLAST was unstable compared with the wild-type transporter. Moreover, LGluT dogs, but not normal dogs, heterozygous for the G492S mutation showed a selective decrease in the accumulation of GLAST mRNA from the normal allele. Based on these findings, we conclude that a complicated heterologous combination of G492S mutation and some transcriptional defect contributes to the pathogenesis of the LGluT red cell phenotype.
Molecular cloning and distinct developmental expression pattern of spliced forms of a novel zinc finger gene wiz in the mouse cerebellum
1998, Molecular Brain Research
In the course of a study conducted to identify the mouse homologue of Drosophila eyes absent (eya), we isolated a novel mouse cDNA fragment which show little homology to eya but encodes a protein with Krüppel (C₂H₂)-type zinc finger motifs. By further screening using this cDNA fragment as a probe, we obtained the short and long forms of full-length cDNAs, which were apparently alternatively spliced products from one gene. Since both mRNAs encode proteins with widely-interspaced zinc finger motifs, we termed this gene wiz and refer to the short and long wiz transcripts as wizS and wizL, respectively. In situ hybridization studies using the probe against the region common to wizS and wizL showed that these mRNAs were expressed abundantly in the granule cell layers of the mouse cerebellum, the olfactory bulb, and the dentate gyrus, whereas the same technique using the probe against only wizL could not detect positive signals in the developing cerebellum, indicating that there is no expression of wizL mRNA there. Northern blot and in situ hybridization analyses demonstrated that the extracerebellar regions expressed both wizS and wizL mRNAs from the midgestational period to adulthood. The finding that two types of wiz transcripts (wizS and wizL) are expressed with different developmental patterns might indicate separate transcription functions in the cerebellar granule cells and the extracerebellar regions.
Cellular distribution and kinetic properties of high-affinity glutamate transporters
1998, Brain Research Bulletin
L-glutamic acid is a key chemical transmitter of excitatory signals in the nervous system. The termination of glutamatergic transmission occurs via uptake of glutamate by a family of high-affinity glutamate transporters that utilize the Na⁺/K⁺ electrochemical gradient as a driving force. The stoichiometry of a single translocation cycle is still debatable, although all proposed models stipulate an inward movement of a net positive charge. This electrogenic mechanism is capable of translocating the neurotransmitter against its several thousandfold concentration gradient, therefore, keeping the resting glutamate concentration below the treshold levels. The five cloned transporters (GLAST/EAAT1, GLT1/EAAT2, EAAC1/EAAT3, EAAT4, and EAAT5) exhibit distinct distribution patterns and kinetic properties in different brain regions, cell types, and reconstitution systems. Moreover, distinct pharmacological profiles were revealed among the species homologues. GLAST and GLT1, the predominant glutamate transporters in the brain, are coexpressed in astroglial processess, whereas neuronal carriers are mainly located in the dendrosomatic compartment. Some of these carrier proteins may possess signal transducing properties, distinct from their transporter activity. Some experimental conditions and several naturally occurring and synthetic compounds are capable of regulating the expression of glutamate transporters. However, selective pharmacological tools interfering with the individual glutamate carriers have yet to be developed.
Properties and localization of glutamate transporters
1998, Progress in Brain Research
The glutamate transporters in the plasma membranes of astrocytes and neurons are essential for the normal functioning of the nervous system. They represent the only mechanism capable of quickly removing glutamate from the extracellular fluid. It is important to maintain a low concentration of glutamate extracellularly for two reasons. First, glutamate is the major excitatory neurotransmitter and a high signal-to-noise ratio requires the removal of extracellular glutamate so that the concentration fluctuates with synaptic release. Second, glutamate is highly toxic to neurons expressing glutamate receptors and glutamate receptors are found on most neurons and even on many glial cells. There is experimental evidence for the idea that the transporters may be actively involved in the regulation of synaptic transmission because they can modify the time course of synaptic events. The sodium-dependent glutamate transporters use the transmembrane gradients of sodium, potassium, and pH as driving forces.

View all citing articles on Scopus

View full text

Article preview

Molecular Brain Research

Abstract

References (21)

Cloning and expression of a human neutral amino acid transporter with structural similarity to the glutamate transporter gene family

J. Biol. Chem.

Expression of neurotransmitter transport from rat brain mRNA in Xenopus laevis oocytes

Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells

Nature

Phosphorylation and modulation of brain glutamate transporters by protein kinase C

J. Biol. Chem.

The hydrophobic moment detects periodicity in protein hydrophobicity

Characterization of the glutamate transporter in retinal cones of the tiger salamander

J. Neurosci.

Primary structure and functional characterization of a high-affinity glutamate transporter

Nature

An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs

Nucleic Acids Res.

A simple method for displaying the hydropathic character of a protein

J. Mol. Biol.

Neurotoxicity of l-glutamate and dl-threo-3-hydroxyaspartate

J. Neurochem.

Cited by (24)

Topical and systemic drug delivery to the posterior segments

Glutamate uptake

Inherited defects of sodium-dependent glutamate transport mediated by glutamate/aspartate transporter in canine red cells due to a decreased level of transporter protein expression

Molecular cloning and distinct developmental expression pattern of spliced forms of a novel zinc finger gene wiz in the mouse cerebellum

Cellular distribution and kinetic properties of high-affinity glutamate transporters

Properties and localization of glutamate transporters

Short communicationCloning and expression of a bovine glutamate transporter

Abstract

Cloning and expression of a human neutral amino acid transporter with structural similarity to the glutamate transporter gene family

J. Biol. Chem.

Expression of neurotransmitter transport from rat brain mRNA in Xenopus laevis oocytes

Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells

Nature

Phosphorylation and modulation of brain glutamate transporters by protein kinase C

J. Biol. Chem.

The hydrophobic moment detects periodicity in protein hydrophobicity

Characterization of the glutamate transporter in retinal cones of the tiger salamander

J. Neurosci.

Primary structure and functional characterization of a high-affinity glutamate transporter

Nature

An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs

Nucleic Acids Res.

A simple method for displaying the hydropathic character of a protein

J. Mol. Biol.

Neurotoxicity of l-glutamate and dl-threo-3-hydroxyaspartate

J. Neurochem.

Short communication
Cloning and expression of a bovine glutamate transporter