Biomedical subjects

N C Danbolt

Publications and source records attributed to N C Danbolt.

At least 19 recordsLinked to original sources

PubMed2006

Changes in glial glutamate transporters in human epileptogenic hippocampus: inadequate explanation for high extracellular glutamate during seizures.

Temporal lobe epilepsy (TLE) with hippocampal sclerosis is associated with high extracellular glutamate levels, which could trigger seizures. Down-regulation of glial glutamate transporters GLAST (EAAT1) and GLT-1 (EAAT2) in sclerotic hippocampi may account for such increases. Their distribution was compared immunohistochemically in non-sclerotic and sclerotic hippocampi and localized only in astrocytes, with weaker immunoreactivity for both transporters in areas associated with pronounced neuronal loss, especially in CA1, but no decrease or even an increase in areas with less neuronal loss, like CA2 and the subiculum in the sclerotic group. Such compensatory changes in immunoreactivity may account for the lack of differences between the groups in immunoblot studies as blots show the average concentrations in the samples. These data suggest that differences in glial glutamate transporter distribution between the two groups of hippocampi may be an insufficient explanation for the high levels of extracellular glutamate in sclerotic seizure foci observed through in vivo dialysis studies.

Adolescent↗

PubMed2006

Specificity controls for immunocytochemistry.

Antibodies have been in widespread use for more than three decades as invaluable tools for the specific detection of proteins or other molecules in biological samples. In spite of such a long experience, the field of immunocytochemistry is still troubled by spurious results due to insufficient specificity of antibodies or procedures used. The importance of keeping a high standard is increasing because massive sequencing of entire genomes leads to the identification of numerous new proteins. All the identified proteins and their variants will have to be localized precisely and quantitatively at high resolution throughout the development of all species. Consequently, antibody generation and immunocytochemical investigations will be done on a large scale. It will be economically important to secure an optimal balance between the risk of publishing erroneous data (which are expensive to correct) and the costs of specificity testing. Because proofs of specificity are never absolute, but rather represent failures to detect crossreactivity, there is no limit to the number of control experiments that can be performed. The aims of the present paper are to increase the awareness of the difficulties in proving the specificity of immunocytochemical labeling and to initiate a discussion on optimized standards. The main points are: (1) antibodies should be described properly, (2) the labeling obtained with an antibody to a single epitope needs additional verification and (3) the investigators should be required to outline in detail how they arrive at the conclusion that the immunocytochemical labeling is specific.

Antibodies, Monoclonal↗

PubMed2005

Specificity of antibodies: unexpected cross-reactivity of antibodies directed against the excitatory amino acid transporter 3 (EAAT3).

UNLABELLED: Specific antibodies are essential tools for identifying individual proteins in biological samples. While generation of antibodies is often straightforward, determination of the antibody specificity is not. Here we illustrate this by describing the production and characterization of antibodies to excitatory amino acid transporter 3 (EAAT3). We synthesized 13 peptides corresponding to parts of the EAAT3 sequence and immunized 6 sheep and 30 rabbits. All sera were affinity purified against the relevant immobilized peptide. Antibodies to the peptides were obtained in almost all cases. Immunoblotting with tissue extracts from wild type and EAAT3 knockout animals revealed that most of the antibodies did not recognize the native EAAT3 protein, and that some recognized other proteins. Several immunization protocols were tried, but strong reactions with EAAT3 were only seen with antibodies to the C-terminal peptides. In contrast, good antibodies were obtained to several parts of EAAT2. EAAT3 was only detected in neurons. However, rabbits immunized with an EAAT3-peptide corresponding to residues 479-498 produced antibodies that labeled axoplasm and microtubules therein particularly strongly. On blots, these antibodies recognized both EAAT3 and a slightly smaller, but far more abundant protein that turned out to be tubulin. The antibodies were fractionated on columns with immobilized tubulin. One fraction contained antibodies apparently specific for EAAT3 while another fraction contained antibodies recognizing both EAAT3 and tubulin despite the lack of primary sequence identity between the two proteins. Addition of free peptide to the incubation solution blocked immunostaining of both EAAT3 and tubulin. CONCLUSIONS: Not all antibodies to synthetic peptides recognize the native protein. The peptide sequence is more important than immunization protocol. The specificity of an antibody is hard to predict because cross-reactivity can be specific and to unrelated molecules. The antigen preabsorption test is of little value in testing the specificity of affinity purified antibodies.

Amino Acid Sequence↗

PubMed2004

Loss of glutamine synthetase in the human epileptogenic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy.

BACKGROUND: High extracellular glutamate concentrations have been identified as a likely trigger of epileptic seizures in mesial temporal lobe epilepsy (MTLE), but the underlying mechanism remains unclear. We investigated whether a deficiency in glutamine synthetase, a key enzyme in catabolism of extracellular glutamate in the brain, could explain the perturbed glutamate homoeostasis in MTLE. METHODS: The anteromedial temporal lobe is the focus of the seizures in MTLE, and surgical resection of this structure, including the hippocampus, leads to resolution of seizures in many cases. By means of immunohistochemistry, western blotting, and functional enzyme assays, we assessed the distribution, quantity, and activity of glutamine synthetase in the MTLE hippocampus. FINDINGS: In western blots, the expression of glutamine synthetase in the hippocampus was 40% lower in MTLE than in non-MTLE samples (median 44 [IQR 30-58] vs 69 [56-87]% of maximum concentration in standard curve; p=0.043; n=8 and n=6, respectively). The enzyme activity was lower by 38% in MTLE vs non-MTLE (mean 0.0060 [SD 0.0031] vs 0.0097 [0.0042] U/mg protein; p=0.045; n=6 and n=9, respectively). Loss of glutamine synthetase was particularly pronounced in areas of the MTLE hippocampus with astroglial proliferation, even though astrocytes normally have high content of the enzyme. Quantitative immunoblotting showed no significant change in the amount of EAAT2, the predominant glial glutamate transporter in the hippocampus. INTERPRETATION: A deficiency in glutamine synthetase in astrocytes is a possible molecular basis for extracellular glutamate accumulation and seizure generation in MTLE. Further studies are needed to define the cause, but the loss of glutamine synthetase may provide a new focus for therapeutic interventions in MTLE.

Adolescent↗

PubMed2001

Amyotrophic lateral sclerosis-linked glutamate transporter mutant has impaired glutamate clearance capacity.

We have investigated the functional impact of a naturally occurring mutation of the human glutamate transporter GLT1 (EAAT2), which had been detected in a patient with sporadic amyotrophic lateral sclerosis. The mutation involves a substitution of the putative N-linked glycosylation site asparagine 206 by a serine residue (N206S) and results in reduced glycosylation of the transporter and decreased uptake activity. Electrophysiological analysis of N206S revealed a pronounced reduction in transport rate compared with wild-type, but there was no alteration in the apparent affinities for glutamate and sodium. In addition, no change in the sensitivity for the specific transport inhibitor dihydrokainate was observed. However, the decreased rate of transport was associated with a reduction of the N206S transporter in the plasma membrane. Under ionic conditions, which favor the reverse operation mode of the transporter, N206S exhibited an increased reverse transport capacity. Furthermore, if coexpressed in the same cell, N206S manifested a dominant negative effect on the wild-type GLT1 activity, whereas it did not affect wild-type EAAC1. These findings provide evidence for a role of the N-linked glycosylation in both cellular trafficking and transport function. The resulting alteration in glutamate clearance capacity likely contributes to excitotoxicity that participates in motor neuron degeneration in amyotrophic lateral sclerosis.

ATP-Binding Cassette Transporters↗

PubMed2001

Glutamate uptake.

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.

ATP-Binding Cassette Transporters↗

PubMed2001

Selective down-regulation of the astrocyte glutamate transporters GLT-1 and GLAST within the medial thalamus in experimental Wernicke's encephalopathy.

Although earlier studies on thiamine deficiency have reported increases in extracellular glutamate concentration in the thalamus, a vulnerable region of the brain in this disorder, the mechanism by which this occurs has remained unresolved. Treatment with pyrithiamine, a central thiamine antagonist, resulted in a 71 and 55% decrease in protein levels of the astrocyte glutamate transporters GLT-1 and GLAST, respectively, by immunoblotting in the medial thalamus of day 14 symptomatic rats at loss of righting reflexes. These changes occurred prior to the onset of convulsions and pannecrosis. Loss of both GLT-1 and GLAST transporter sites was also confirmed in this region of the thalamus at the symptomatic stage using immunohistochemical methods. In contrast, no change in either transporter protein was detected in the non-vulnerable frontal parietal cortex. These effects are selective; protein levels of the astrocyte GABA transporter GAT-3 were unaffected in the medial thalamus. In addition, astrocyte-specific glial fibrillary acidic protein (GFAP) content was unchanged in this brain region, suggesting that astrocytes are spared in this disorder. Loss of GLT-1 or GLAST protein was not observed on day 12 of treatment, indicating that down-regulation of these transporters occurs within 48 h prior to loss of righting reflexes. Finally, GLT-1 content was positively correlated with levels of the neurofilament protein alpha-internexin, suggesting that early neuronal drop-out may contribute to the down-regulation of this glutamate transporter and subsequent pannecrosis. A selective, focal loss of GLT-1 and GLAST transporter proteins provides a rational explanation for the increase in interstitial glutamate levels, and may play a major role in the selective vulnerability of thalamic structures to thiamine deficiency-induced cell death.

ATP-Binding Cassette Transporters↗

PubMed2000

Transient expression of the glial glutamate transporters GLAST and GLT in hippocampal neurons in primary culture.

The extracellular glutamate concentration is kept low by glutamate transporters in the plasma membranes. Here we have studied the expression of the glutamate transporters GLAST, GLT and EAAC during the in vitro development of embryonic hippocampal neurons grown in a defined (serum free) medium. Immunochemistry studies showed that both the GLAST and GLT proteins are expressed in a subpopulation of neurons at the early, but not at the later stages of the cultures. Glial cells expressing the GLAST and GLT proteins were found at all stages. EAAC was only detected in neurons. This is one of the first evidence of a neuronal ability to express GLAST.

ATP-Binding Cassette Transporters↗

PubMed1999

Interindividual differences in the levels of the glutamate transporters GLAST and GLT, but no clear correlation with Alzheimer's disease.

Alzheimer's disease is a common progressive neurodegenerative disease of unknown etiology. Several different pathological processes have been identified in the brains of Alzheimer patients. To determine if reduced glutamate uptake is a contributing factor, we have measured the levels of the glutamate transporter proteins GLAST (EAAT1) and GLT (EAAT2) in human autopsy samples. The postmortem proteolysis of these proteins turned out to be fairly rapid. Brains from 10 Alzheimer and 10 control patients were therefore obtained with a relatively short postmortem delay (5 hr on average). GLT (N-terminal and central parts), GLAST (C-terminal), glial fibrillary acidic protein (GFAP) and inositol (1,4,5)-triphosphate (IP3)-receptor immunoreactivities were determined in the cingulate and inferior temporal gyri by immunoblotting. The Na+-dependent "binding" of D-[3H]aspartate and the glutamate uptake after solubilization and reconstitution in liposomes were determined for comparison. An individual variation in GLAST and GLT levels was found, but no significant correlation with Alzheimer's disease, except for a 14% lower ratio of N-terminal to central GLT immunoreactivity (P < 0.04). The levels of GLAST and GLT showed negative correlation in agreement with the idea that these proteins are differentially regulated. In conclusion, Alzheimer's disease brains can have both normal and reduced levels of GLAST and GLT.

ATP-Binding Cassette Transporters↗

PubMed1999

Expression of the glutamate transporters in human temporal lobe epilepsy.

Glutamate is the major excitatory neurotransmitter in the central nervous system and is implicated in the pathogenesis of neurodegenerative diseases. Five human glutamate transporters have been cloned and are responsible for the removal of potentially excitotoxic excess glutamate from the extracellular space. In this study we consider whether there are selective changes in the expression of the glutamate transporters in the medial temporal cortex and hippocampus from temporal lobe epilepsy patients, which might contribute to the development or maintenance of seizures. Since disruption of the glial transporter excitatory amino acid transporter 2 in mice results in lethal spontaneous seizures, we were interested primarily in studying changes in this transporter. Using in situ hybridization we show that there was no reduction in the level of excitatory amino acid transporter 2 encoding messenger RNA in the temporal lobe epilepsy cases compared to post mortem controls and indeed there was a relative increase in content of excitatory amino acid transporter 2 messenger RNA per cell in temporal lobe epilepsy cases. Western blotting showed that there was no change in the excitatory amino acid transporter 2 protein content in temporal lobe epilepsy cases as compared to post mortem controls. A small reduction in the level of the second astroglial transporter protein, excitatory amino acid transporter 1, was observed in temporal lobe epilepsy cases. Surprisingly, immunohistochemical experiments using a polyclonal antiexcitatory amino acid transporter 2 antibody, showed a different localization of this protein in epilepsy derived tissue as compared to post mortem controls although glial markers such as glial fibrillary acidic protein and glutamine synthase showed similar patterns of staining. However, repeating this experiment using control tissue from non-temporal lobe epilepsy biopsies demonstrated that this change in the excitatory amino acid transporter 2 transporter localization occurred post mortem. These data suggest that major changes in the level of expression of the glutamate transporters do not play an important role in the development of human temporal lobe epilepsy but may be implicated the aetiology of other types of epilepsy.

ATP-Binding Cassette Transporters↗

PubMed1999

SOD1 mutants linked to amyotrophic lateral sclerosis selectively inactivate a glial glutamate transporter.

The mechanism by which Cu2+/Zn2+ superoxide dismutase (SOD1) mutants lead to motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS) is unknown. We show that oxidative reactions triggered by hydrogen peroxide and catalyzed by A4V and I113T mutant but not wild-type SOD1 inactivated the glutamate transporter human GLT1. Chelation of the copper ion of the prosthetic group of A4V prevented GLT1 inhibition. GLT1 was a selective target of oxidation mediated by SOD1 mutants, and its reactivity was confined to the intracellular carboxyl-terminal domain. The antioxidant Mn(III)TBAP rescued GLT1 from inhibition. Because inactivation of GLT1 results in neuronal degeneration, we propose that toxic properties of SOD1 mutants lead to neuronal death via an excitotoxic mechanism in SOD1-linked FALS.

ATP-Binding Cassette Transporters↗

PubMed1999

Hippocampal GABA and glutamate transporter immunoreactivity in patients with temporal lobe epilepsy.

OBJECTIVE: Sodium-coupled transporters remove extracellular neurotransmitters and alterations in their function could enhance or suppress synaptic transmission and seizures. This study determined hippocampal gamma-aminobutyric acid (GABA) and glutamate transporter immunoreactivity (IR) in temporal lobe epilepsy (TLE) patients. METHODS: Hippocampal sclerosis (HS) patients (n = 25) and non-HS cases (mass lesion and cryptogenic; n = 20) were compared with nonseizure autopsies (n = 8). Hippocampal sections were studied for neuron densities along with IR for glutamate decarboxylase (GAD; presynaptic GABA terminals), GABA transporter-1 (GAT-1; presynaptic GABA transporter), GAT-3 (astrocytic GABA transporter), excitatory amino acid transporter 3 (EAAT3; postsynaptic glutamate transporter), and EAAT2-1 (glial glutamate transporters). RESULTS: Compared with autopsies, non-HS cases with similar neuron counts showed: 1) increased GAD IR gray values (GV) in the fascia dentata outer molecular layer (OML), hilus, and stratum radiatum; 2) increased GAT-1 OML GVs; 3) increased astrocytic GAT-3 GVs in the hilus and Ammon's horn; and 4) no IR differences for EAAT3-1. HS patients with decreased neuron densities demonstrated: 1) increased OML and inner molecular layer GAD puncta; 2) decreased GAT-1 puncta relative to GAD in the stratum granulosum and pyramidale; 3) increased GAT-1 OML GVs; 4) decreased GAT-3 GVs; 5) increased EAAT3 IR on remaining granule cells and pyramids; 6) decreased glial EAAT2 GVs in the hilus and CA1 stratum radiatum associated with neuron loss; and 7) increased glial EAAT1 GVs in CA2/3 stratum radiatum. CONCLUSIONS: Hippocampal GABA and glutamate transporter IR differ in TLE patients compared with autopsies. These data support the hypothesis that excitatory and inhibitory neurotransmission and seizure susceptibility could be altered by neuronal and glial transporters in TLE patients.

ATP-Binding Cassette Transporters↗

PubMed1998

The vesicular GABA transporter, VGAT, localizes to synaptic vesicles in sets of glycinergic as well as GABAergic neurons.

A transporter thought to mediate accumulation of GABA into synaptic vesicles has recently been cloned (McIntire et al., 1997). This vesicular GABA transporter (VGAT), the first vesicular amino acid transporter to be molecularly identified, differs in structure from previously cloned vesicular neurotransmitter transporters and defines a novel gene family. Here we use antibodies specific for N- and C-terminal epitopes of VGAT to localize the protein in the rat CNS. VGAT is highly concentrated in the nerve endings of GABAergic neurons in the brain and spinal cord but also in glycinergic nerve endings. In contrast, hippocampal mossy fiber boutons, which although glutamatergic are known to contain GABA, lack VGAT immunoreactivity. Post-embedding immunogold quantification shows that the protein specifically associates with synaptic vesicles. Triple labeling for VGAT, GABA, and glycine in the lateral oliva superior revealed a higher expression of VGAT in nerve endings rich in GABA, with or without glycine, than in others rich in glycine only. Although the great majority of nerve terminals containing GABA or glycine are immunopositive for VGAT, subpopulations of nerve endings rich in GABA or glycine appear to lack the protein. Additional vesicular transporters or alternative modes of release may therefore contribute to the inhibitory neurotransmission mediated by these two amino acids.

Animals↗

PubMed1998

The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain.

The role of transporters in shaping the glutamate concentration in the extracellular space after synaptic release is controversial because of their slow cycling and because diffusion alone gives a rapid removal. The transporter densities have been measured electrophysiologically, but these data are from immature brains and do not give precise information on the concentrations of the individual transporter subtypes. Here we show by quantitative immunoblotting that the numbers of the astroglial glutamate transporters GLAST (EAAT1) and GLT (EAAT2) are 3200 and 12,000 per micrometer3 tissue in the stratum radiatum of adult rat hippocampus (CA1) and 18,000 and 2800 in the cerebellar molecular layer, respectively. The total astroglial cell surface is 1.4 and 3.8 m2/cm3 in the two regions, respectively, implying average densities of GLAST and GLT molecules in the membranes around 2300 and 8500 micrometer-2 in the former and 4700 and 740 micrometer-2 in the latter region. The total concentration of glial glutamate transporters in both regions corresponds to three to five times the estimated number of glutamate molecules in one synaptic vesicle from each of all glutamatergic synapses. However, the role of glial glutamate transporters in limiting synaptic spillover is likely to vary between the two regions because of differences in the distribution of astroglia. Synapses are completely ensheathed and separated from each other by astroglia in the cerebellar molecular layer. In contrast, synapses in hippocampus (stratum radiatum) are only contacted by astroglia and are often found side by side without intervening glial processes.

ATP-Binding Cassette Transporters↗

PubMed1998

Neuronal expression of the glutamate transporter GLT-1 in hippocampal microcultures.

To address the question of the relative contributions of glial and neuronal glutamate transport in the vertebrate CNS, we studied the distribution of forebrain glutamate transporters in rat hippocampal microcultures, a preparation in which physiological functions of glutamate transporters have been well characterized. Two of the three transporters, GLAST (EAAT1) and EAAC1 (EAAT3), are localized to microculture glia and neurons, respectively, as expected. However, we find strong immunoreactivity for the third glutamate transporter GLT-1 (EAAT2), a putatively glial transporter, in microculture neurons and in a small subset of microculture glia. Indistinguishable immunohistochemical staining patterns for GLT-1 were obtained with antibodies directed against both the N terminal and C terminal of the GLT-1 protein. Double-labeling experiments suggest that neuronal GLT-1 protein is primarily localized to the dendrites of excitatory neurons. Neuronal electrogenic transport currents in response to D-aspartate applications were occluded by the selective GLT-1 inhibitor dihydrokainate. In contrast, glia exhibited a larger transporter current density than did neurons, and the glial transport current was less sensitive to dihydrokainate. Neuronal transport currents were potentiated less than were glial currents when the chaotropic anion thiocyanate was substituted for gluconate in the whole-cell recording pipette, consistent with the previously reported lower anion permeability of EAAC1 and GLT-1 compared with that of GLAST. After microculture glia were rendered nonviable, excitatory autaptic currents (EACs) were prolonged in the presence of dihydrokainate, suggesting that neuronal GLT-1 is capable of participating in the clearance of synaptically released glutamate. Our results suggest that the initially proposed characterization of GLT-1 as a purely glial transporter is too simplistic and that under certain conditions functional GLT-1 protein can be expressed in brain neurons. The study suggests that changes in GLT-1 levels that occur with pathology or experimental manipulations cannot be assumed to be glial.

ATP-Binding Cassette Transporters↗

PubMed1998

The glutamate transporter EAAT4 in rat cerebellar Purkinje cells: a glutamate-gated chloride channel concentrated near the synapse in parts of the dendritic membrane facing astroglia.

Antibodies to an excitatory amino acid transporter (EAAT4) label a glycoprotein of approximately 65 kDa strongly in the cerebellum and weakly in the forebrain. Cross-linking of cerebellar proteins with bis(sulfosuccinimidyl) suberate before solubilization causes dimer bands of EAAT4 and both dimer and trimer bands of the other glutamate transporters GLAST (EAAT1) and GLT (EAAT2) to appear on immunoblots. In contrast to GLAST, GLT, and EAAC (EAAT3), EAAT4 is unevenly distributed in the cerebellar molecular layer, being strongly expressed in parasagittal zones. It is located in cerebellar Purkinje cells, and the highest concentrations are seen in ones expressing high levels of zebrin II (aldolase C). The labeling of Purkinje cell spines and thin dendrites is stronger than that of large diameter dendrites and cell bodies. EAAT4 is present at low concentrations in the synaptic membrane, but is highly enriched in the parts of the dendritic and spine membranes facing astrocytes (which express GLAST and GLT) compared with parts facing neuronal membranes, suggesting a functional relationship with glial glutamate transporters. The presence of EAAT4 in intracellular cisterns and multivesicular organelles may reflect turnover of transporter in the plasma membrane. The total Purkinje cell spine surface and the EAAT4 concentration were found to be 1.1 m2/cm3 and 0.2 mg/cm3, respectively, in the molecular layer, corresponding to 1800 molecules/microm2. The juxtasynaptic location of EAAT4 may explain electrophysiological observations predicting the presence of a neuronal glutamate transporter near the release site at a Purkinje cell spine synapse. EAAT4 may function as a combined transporter and inhibitory glutamate receptor.

ATP-Binding Cassette Transporters↗

PubMed1998

Molecular cloning of the cDNA encoding pp36, a tyrosine-phosphorylated adaptor protein selectively expressed by T cells and natural killer cells.

Activation of T and natural killer (NK) cells leads to the tyrosine phosphorylation of pp36 and to its association with several signaling molecules, including phospholipase Cgamma-1 and Grb2. Microsequencing of peptides derived from purified rat pp36 protein led to the cloning, in rat and man, of cDNA encoding a T- and NK cell-specific protein with several putative Src homology 2 domain-binding motifs. A rabbit antiserum directed against a peptide sequence from the cloned rat molecule recognized tyrosine phosphorylated pp36 from pervanadate-treated rat thymocytes. When expressed in 293T human fibroblast cells and tyrosine-phosphorylated, pp36 associated with phospholipase Cgamma-1 and Grb2. Studies with GST-Grb2 fusion proteins demonstrated that the association was specific for the Src homology 2 domain of Grb-2. Molecular cloning of the gene encoding pp36 should facilitate studies examining the role of this adaptor protein in proximal signaling events during T and NK cell activation.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Inducible expression of the GLT-1 glutamate transporter in a CHO cell line selected for low endogenous glutamate uptake.

Inducible expression of the mammalian glial cell glutamate transporter GLT-1 has been established in a CHO cell line selected for low endogenous Na+-dependent glutamate uptake by [3H]aspartate suicide selection. Culturing the cells in doxycycline-containing medium, to activate GLT-1 expression via the Tet-On system, increased uptake of the GLT-1 substrate D-aspartate 280-fold, and increased cell size. Applying glutamate to whole-cell clamped, doxycycline-treated cells evoked a transporter-mediated current with characteristics appropriate for GLT-1. This cell line provides a useful tool for further examination of the electrical, biochemical and pharmacological properties of GLT-1, the most abundant glutamate transporter in the brain.

ATP-Binding Cassette Transporters↗