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Excitatory Amino Acid Transporters (excitatory + amino_acid_transporter)
Selected AbstractsInhibition of the Activity of Excitatory Amino Acid Transporter 4 Expressed in Xenopus Oocytes After Chronic Exposure to EthanolALCOHOLISM, Issue 7 2008Seung-Yeon Yoo Background:, The extracellular glutamate concentration is tightly controlled by excitatory amino acid transporters (EAATs). EAAT4 is the predominant EAAT in the cerebellar Purkinje cells. Purkinje cells play a critical role in motor coordination and may be an important target for ethanol to cause motor impairments. We designed this study to determine the effects of chronic ethanol exposure on the activity of EAAT4 and evaluate the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in these effects. Methods:, EAAT4 was expressed in Xenopus oocytes following injection of EAAT4 mRNA. Oocytes were incubated with ethanol-containing solution for 24 to 96 hours. Membrane currents induced by l -aspartate were recorded using 2-electrode voltage clamps. Responses were quantified by integration of the current trace and reported in microCoulombs (,C). Results:, Ethanol dose- and time-dependently reduced EAAT4 activity. EAAT4 activity after a 96-hour exposure was significantly decreased compared to the control values at all concentrations tested (10 to 100 mM). Ethanol (50 mM) significantly decreased the Vmax (2.2 ± 0.2 ,C for control vs. 1.6 ± 0.2 ,C for ethanol, n = 18, p < 0.05) of EAAT4 for l -aspartate. Preincubation of ethanol-treated (50 mM for 96 hours) oocytes with phorbol-12-myrisate-13-acetate (100 nM for 10 minutes) abolished the ethanol-induced decrease in EAAT4 activity. While staurosporine (2 ,M for 1 hour) or chelerythrine (100 ,M for 1 hour) significantly decreased EAAT4 activity, no difference was observed in EAAT4 activity among the staurosporine, ethanol, or ethanol plus staurosporine groups. Similarly, EAAT4 activity did not differ among the chelerythrine, ethanol, or ethanol plus chelerythrine groups. Pretreatment of the oocytes with wortmannin (1 ,M for 1 hour) also significantly decreased EAAT4 activity. However, no difference was observed in the wortmannin, ethanol, or ethanol plus wortmannin groups. Conclusions:, The results of this study suggest that chronic ethanol exposure decreases EAAT4 activity and that PKC and PI3K may be involved in these effects. These effects of ethanol on EAAT4 may cause an increase in peri-Purkinje cellular glutamate concentration, and may be involved in cerebellar dysfunction and motor impairment after chronic ethanol ingestion. [source] Transcriptional regulation of human excitatory amino acid transporter 1 (EAAT1): cloning of the EAAT1 promoter and characterization of its basal and inducible activity in human astrocytesJOURNAL OF NEUROCHEMISTRY, Issue 6 2003Seon-Young Kim Abstract Excitatory amino acid transporter 1 (EAAT1) is one of the two glial glutamate transporters that clear the extracellular glutamate generated during neuronal signal transmission. Here, we cloned and characterized a 2.1-kb promoter region of human EAAT1 and investigated its function in the transcriptional regulation of the EAAT1 gene in human primary astrocytes. The full-length promoter region lacked TATA and CCAAT boxes and an initiator element, it contained several potential transcription factor-binding sites and it exhibited promoter activity in primary astrocytes and in several types of transformed cells. Consecutive 5,-deletion analysis of the EAAT1 promoter indicated the presence of negative and positive regulatory regions and a putative core promoter between ,57 bp and +20 bp relative to the transcription start site (TSS). The core promoter contained a single GC-box in position ,52/,39 and one E-box near the TSS and the GC-box site that was responsible for 90% of the basal promoter activity as determined by mutational analysis. Electrophoretic mobility shift, supershift and competition assays demonstrated binding of stimulating proteins (Sp) 1 and 3 to the GC-box and upstream stimulating factor (USF) 1 to the E-box. Treatment of primary human astrocytes with cellular modulators 8-bromo cyclic AMP and epidermal growth factor increased EAAT1 promoter activity in transient transfection assays and increased cellular EAAT1 mRNA expression and glutamate uptake by astrocytes. Conversely, tumor necrosis factor-, reduced both EAAT promoter activity and cellular EAAT1 mRNA expression. These results enable studies of transcriptional regulation of EAAT1 gene at the promoter level. [source] Glutamate spillover augments GABA synthesis and release from axodendritic synapses in rat hippocampusHIPPOCAMPUS, Issue 1 2010Misty M. Stafford Abstract Tight coupling between gamma-aminobutyric acid (GABA) synthesis and vesicle filling suggests that the presynaptic supply of precursor glutamate could dynamically regulate inhibitory synapses. Although the neuronal glutamate transporter excitatory amino acid transporter 3 (EAAT3) has been proposed to mediate such a metabolic role, highly efficient astrocytic uptake of synaptically released glutamate normally maintains low-extracellular glutamate levels. We examined whether axodendritic inhibitory synapses in stratum radiatum of hippocampal area CA1, which are closely positioned among excitatory glutamatergic synapses, are regulated by synaptic glutamate release via presynaptic uptake. Under conditions of spatially and temporally coordinated release of glutamate and GABA within pyramidal cell dendrites, blocking glial glutamate uptake enhanced quantal release of GABA in a transporter-dependent manner. These physiological findings correlated with immunohistochemical studies revealing expression of EAAT3 by interneurons and uptake of D-asparate into putative axodendritic inhibitory terminals only when glial uptake was blocked. These results indicate that spillover of glutamate between adjacent excitatory and inhibitory synapses can occur under conditions when glial uptake incompletely clears synaptically released glutamate. Our anatomical studies also suggest that perisomatic inhibitory synapses, unlike synapses within dendritic layers of hippocampus, are not capable of glutamate uptake and therefore transporter-mediated dynamic regulation of inhibition is a unique feature of axodendritic synapses that may play a role in maintaining a homeostatic balance of inhibition and excitation. © 2009 Wiley-Liss, Inc. [source] EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinaseJOURNAL OF NEUROCHEMISTRY, Issue 3 2007Jeyaganesh Rajamanickam Abstract EAAT4 (SLC1A6) is a Purkinje-Cell-specific post-synaptic excitatory amino acid transporter that plays a major role in clearing synaptic glutamate. EAAT4 abundance and function is known to be modulated by the serum and glucocorticoid inducible kinase (SGK) 1 but the precise mechanism of kinase action has not been defined yet. The present work aims to identify the molecular mechanism of EAAT4 modulation by the kinase. The EAAT4 sequence bears two putative SGK1 consensus sites (at Thr40 and Thr504) at the amino and carboxy terminus that are conserved among species. Expression studies in Xenopus oocytes demonstrated that EAAT4-mediated [3H] glutamate uptake and cell surface abundance are enhanced by co-expression of SGK1. Disruption of the SGK1 phosphorylation site at threonine 40 (T40AEAAT4) or of both phosphorylation sites (T40AT504AEAAT4) abrogated the effect of SGK1 on transporter function and expression. SGK1 modulates several transport proteins via inhibition of the ubiquitin ligase Nedd4-2. Co-expression of Nedd4-2 inhibited wild-type EAAT4 but not the T40AT504AEAAT4 mutant. Besides, RNA interference-mediated reduction of endogenous Nedd4-2 (xNedd4-2) expression increased the activity of the transporter. In conclusion, maximal glutamate transport modulation by SGK1 is accomplished by direct EAAT4 stimulation and to a lesser extent by inhibition of intrinsic Nedd4-2. [source] Transcriptional regulation of human excitatory amino acid transporter 1 (EAAT1): cloning of the EAAT1 promoter and characterization of its basal and inducible activity in human astrocytesJOURNAL OF NEUROCHEMISTRY, Issue 6 2003Seon-Young Kim Abstract Excitatory amino acid transporter 1 (EAAT1) is one of the two glial glutamate transporters that clear the extracellular glutamate generated during neuronal signal transmission. Here, we cloned and characterized a 2.1-kb promoter region of human EAAT1 and investigated its function in the transcriptional regulation of the EAAT1 gene in human primary astrocytes. The full-length promoter region lacked TATA and CCAAT boxes and an initiator element, it contained several potential transcription factor-binding sites and it exhibited promoter activity in primary astrocytes and in several types of transformed cells. Consecutive 5,-deletion analysis of the EAAT1 promoter indicated the presence of negative and positive regulatory regions and a putative core promoter between ,57 bp and +20 bp relative to the transcription start site (TSS). The core promoter contained a single GC-box in position ,52/,39 and one E-box near the TSS and the GC-box site that was responsible for 90% of the basal promoter activity as determined by mutational analysis. Electrophoretic mobility shift, supershift and competition assays demonstrated binding of stimulating proteins (Sp) 1 and 3 to the GC-box and upstream stimulating factor (USF) 1 to the E-box. Treatment of primary human astrocytes with cellular modulators 8-bromo cyclic AMP and epidermal growth factor increased EAAT1 promoter activity in transient transfection assays and increased cellular EAAT1 mRNA expression and glutamate uptake by astrocytes. Conversely, tumor necrosis factor-, reduced both EAAT promoter activity and cellular EAAT1 mRNA expression. These results enable studies of transcriptional regulation of EAAT1 gene at the promoter level. [source] Mercury compounds disrupt neuronal glutamate transport in cultured mouse cerebellar granule cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2005Elena Fonfrķa Abstract Cerebellar granule cells are targeted selectively by mercury compounds in vivo. Despite the affinity of mercury for thiol groups present in all cells, the molecular determinant(s) of selective cerebellar degeneration remain to be elucidated fully. We studied the effect of mercury compounds on neuronal glutamate transport in primary cultures of mouse cerebellar granule cells. Immunoblots probed with an antibody against the excitatory amino acid transporter (EAAT) neuronal glutamate transporter, EAAT3, revealed the presence of a specific band in control and mercury-treated cultures. Micromolar concentrations of both methylmercury and mercuric chloride increased the release of endogenous glutamate, inhibited glutamate uptake, reduced mitochondrial activity, and decreased ATP levels. All these effects were completely prevented by the nonpermeant reducing agent Tris-(2-carboxyethyl)phosphine (TCEP). Reduction of mitochondrial activity by mercuric chloride, but not by methylmercury, was inhibited significantly by 4,4,-diisothiocyanato-stilbene-2,2,-disulfonic acid (DIDS) and by reduced extracellular Cl, ion concentration. In addition, DIDS and low extracellular Cl, completely inhibited the release of glutamate induced by mercuric chloride, and produced a partial although significant reduction of that induced by methylmercury. We suggest that a direct inhibition of glutamate uptake triggers an imbalance in cell homeostasis, leading to neuronal failure and Cl, -regulated cellular glutamate efflux. Our results demonstrate that neuronal glutamate transport is a novel target to be taken into account when assessing mercury-induced neurotoxicity. © 2005 Wiley-Liss, Inc. [source] ENT1 Regulates Ethanol-Sensitive EAAT2 Expression and Function in AstrocytesALCOHOLISM, Issue 6 2010Jinhua Wu Background:, Equilibrative nucleoside transporter 1 (ENT1) and excitatory amino acid transporter 2 (EAAT2) are predominantly expressed in astrocytes where they are thought to regulate synaptic adenosine and glutamate levels. Because mice lacking ENT1 display increased glutamate levels in the ventral striatum, we investigated whether ENT1 regulates the expression and function of EAAT2 in astrocytes, which could contribute to altered glutamate levels in the striatum. Methods:, We examined the effect of ENT1 inhibition and overexpression on the expression of EAAT2 using quantitative real-time PCR and measured glutamate uptake activity in cultured astrocytes. We also examined the effect of 0 to 200 mM ethanol doses for 0 to 24 hours of ethanol exposure on EAAT2 expression and glutamate uptake activity. We further examined the effect of ENT1 knockdown by a specific siRNA on ethanol-induced EAAT2 expression. Results:, An ENT1-specific antagonist and siRNA treatments significantly reduced both EAAT2 expression and glutamate uptake activity while ENT1 overexpression up-regulated EAAT2 mRNA expression. Interestingly, 100 or 200 mM ethanol exposure increased EAAT2 mRNA expression as well as glutamate uptake activity. Moreover, we found that ENT1 knockdown inhibited the ethanol-induced EAAT2 up-regulation. Conclusions:, Our results suggest that ENT1 regulates glutamate uptake activity by altering EAAT2 expression and function, which might be implicated in ethanol intoxication and preference. [source] Physiological requirement for the glutamate transporter dEAAT1 at the adult Drosophila neuromuscular junctionDEVELOPMENTAL NEUROBIOLOGY, Issue 10 2006Thomas Rival Abstract L -Glutamate is the major excitatory neurotransmitter in the mammalian brain. Specific proteins, the Na+/K+ -dependent high affinity excitatory amino acid transporters (EAATs), are involved in the extracellular clearance and recycling of this amino acid. Type I synapses of the Drosophila neuromuscular junction (NMJ) similarly use L -glutamate as an excitatory transmitter. However, the localization and function of the only high-affinity glutamate reuptake transporter in Drosophila, dEAAT1, at the NMJ was unknown. Using a specific antibody and transgenic strains, we observed that dEAAT1 is present at the adult, but surprisingly not at embryonic and larval NMJ, suggesting a physiological maturation of the junction during metamorphosis. We found that dEAAT1 is not localized in motor neurons but in glial extensions that closely follow motor axons to the adult NMJ. Inactivation of the dEAAT1 gene by RNA interference generated viable adult flies that were able to walk but were flight-defective. Electrophysiological recordings of the thoracic dorso-lateral NMJ were performed in adult dEAAT1-deficient flies. The lack of dEAAT1 prolonged the duration of the individual responses to motor nerve stimulation and this effect was progressively increased during physiological trains of stimulations. Therefore, glutamate reuptake by glial cells is required to ensure normal activity of the Drosophila NMJ, but only in adult flies. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Oxidative and excitotoxic insults exert differential effects on spinal motoneurons and astrocytic glutamate transporters: Implications for the role of astrogliosis in amyotrophic lateral sclerosisGLIA, Issue 2 2009Chrissandra J. Zagami Abstract In amyotrophic lateral sclerosis (ALS) non-neuronal cells play key roles in disease etiology and loss of motoneurons via noncell-autonomous mechanisms. Reactive astrogliosis and dysfunctional transporters for L -glutamate [excitatory amino acid transporters, (EAATs)] are hallmarks of ALS pathology. Here, we describe mechanistic insights into ALS pathology involving EAAT-associated homeostasis in response to a destructive milieu, in which oxidative stress and excitotoxicity induce respectively astrogliosis and motoneuron injury. Using an in vitro neuronal-glial culture of embryonic mouse spinal cord, we demonstrate that EAAT activity was maintained initially, despite a loss of cellular viability induced by exposure to oxidative [3-morpholinosydnonimine chloride (SIN-1)] and excitotoxic [(S)-5-fluorowillardiine (FW)] conditions. This homeostatic response of EAAT function involved no change in the cell surface expression of EAAT1/2 at 0.5,4 h, but rather alterations in kinetic properties. Over this time-frame, EAAT1/2 both became more widespread across astrocytic arbors in concert with increased expression of glial fibrillary acidic protein (GFAP), although at 8,24 h there was gliotoxicity, especially with SIN-1 rather than FW. An opposite picture was found for motoneurons where FW, not SIN-1, produced early and extensive neuritic shrinkage and blebbing (,0.5 h) with somata loss from 2 h. We postulate that EAATs play an early homeostatic and protective role in the pathologic milieu. Moreover, the differential profiles of injury produced by oxidative and excitotoxic insults identify two distinct phases of injury which parallel important aspects of the pathology of ALS. © 2008 Wiley-Liss, Inc. [source] The position of an arginine residue influences substrate affinity and K+ coupling in the human glutamate transporter, EAAT1JOURNAL OF NEUROCHEMISTRY, Issue 2 2010Renae M. Ryan J. Neurochem. (2010) 114, 565,575. Abstract Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and extracellular glutamate levels are controlled by a family of transporters known as excitatory amino acid transporters (EAATs). The EAATs transport glutamate and aspartate with similar micromolar affinities and this transport is coupled to the movement of Na+, K+, and H+. The crystal structure of a prokaryotic homologue of the EAATs, aspartate transporter from Pyrococcus horokoshii (GltPh), has yielded important insights into the architecture of this transporter family. GltPh is a Na+ -dependent transporter that has significantly higher affinity for aspartate over glutamate and is not coupled to H+ or K+. The highly conserved carboxy-terminal domains of the EAATs and GltPh contain the substrate and ion binding sites, however, there are a couple of striking differences in this region that we have investigated to better understand the transport mechanism. An arginine residue is in close proximity to the substrate binding site of both GltPh and the EAATs, but is located in transmembrane domain (TM) 8 in the EAATs and hairpin loop 1 (HP1) of GltPh. Here we report that the position of this arginine residue can explain some of the functional differences observed between the EAATs and GltPh. Moving the arginine residue from TM8 to HP1 in EAAT1 results in a transporter that has significantly increased affinity for both glutamate and aspartate and is K+ independent. Conversely, moving the arginine residue from HP1 to TM8 in GltPh results in a transporter that has reduced affinity for aspartate. [source] Preferential vulnerability of mesencephalic dopamine neurons to glutamate transporter dysfunctionJOURNAL OF NEUROCHEMISTRY, Issue 2 2008Imane Nafia Abstract Nigral depletion of the main brain antioxidant GSH is the earliest biochemical event involved in Parkinson's disease pathogenesis. Its causes are completely unknown but increasing number of evidence suggests that glutamate transporters [excitatory amino acid transporters (EAATs)] are the main route by which GSH precursors may enter the cell. In this study, we report that dopamine (DA) neurons, which express the excitatory amino acid carrier 1, are preferentially affected by EAAT dysfunction when compared with non-DA neurons. In rat embryonic mesencephalic cultures, l -trans-pyrrolidine-2,4-dicarboxylate, a substrate inhibitor of EAATs, is directly and preferentially toxic for DA neurons by decreasing the availability of GSH precursors and lowering their resistance threshold to glutamate excitotoxicity through NMDA-receptors. In adult rat, acute intranigral injection of l -trans-pyrrolidine-2,4-dicarboxylate induces a large regionally selective and dose-dependent loss of DA neurons and ,-synuclein aggregate formation. These data highlight for the first time the importance of excitatory amino acid carrier 1 function for the maintenance of antioxidant defense in DA neurons and suggest its dysfunction as a candidate mechanism for the selective death of DA neurons such as occurring in Parkinson's disease. [source] Differing effects of substrate and non-substrate transport inhibitors on glutamate uptake reversalJOURNAL OF NEUROCHEMISTRY, Issue 6 2001Christopher M. Anderson Na+ -dependent excitatory amino acid transporters (EAATs) normally function to remove extracellular glutamate from brain extracellular space, but EAATs can also increase extracellular glutamate by reversal of uptake. Effects of inhibitors on EAATs can be complex, depending on cell type, whether conditions favor glutamate uptake or uptake reversal and whether the inhibitor itself is a substrate for the transporters. The present study assessed EAAT inhibitors for their ability to inhibit glutamate uptake, act as transporter substrates and block uptake reversal in astrocyte and neuron cultures. lthreo -,-hydroxyaspartate (l -TBHA), dlthreo -,-benzyloxyaspartate (dl -TBOA), ltrans -pyrrolidine-2,4-dicarboxylic acid (ltrans -2,4-PDC) (+/,)- cis -4-methy- trans -pyrrolidine-2,4-dicarboxylic acid (cis -4-methy- trans -2,4-PDC) and lantiendo -3,4-methanopyrrolidine-2,4-dicarboxylic acid (lantiendo -3,4-MPDC) inhibited l -[14C]glutamate uptake in astrocytes with equilibrium binding constants ranging from 17 µm (dl -TBOA and l -TBHA) , 43 µm (cis -4-methy- trans -2,4-PDC). Transportability of inhibitors was assessed in astrocytes and neurons. While l -TBHA, ltrans -2,4-PDC, cis -4-methy- trans -2,4-PDC and lantiendo -3,4-MPDC displayed significant transporter substrate activities in neurons and astrocytes, dl -TBOA was a substrate only in astrocytes. This effect of dl -TBOA was concentration-dependent, leading to complex effects on glutamate uptake reversal. At concentrations low enough to produce minimal dl -TBOA uptake velocity (, 10 µm), dl -TBOA blocked uptake reversal in ATP-depleted astrocytes; this blockade was negated at concentrations that drove substantial dl -TBOA uptake (> 10 µm). These findings indicate that the net effects of EAAT inhibitors can vary with cell type and exposure conditions. [source] Inhibition of the Activity of Excitatory Amino Acid Transporter 4 Expressed in Xenopus Oocytes After Chronic Exposure to EthanolALCOHOLISM, Issue 7 2008Seung-Yeon Yoo Background:, The extracellular glutamate concentration is tightly controlled by excitatory amino acid transporters (EAATs). EAAT4 is the predominant EAAT in the cerebellar Purkinje cells. Purkinje cells play a critical role in motor coordination and may be an important target for ethanol to cause motor impairments. We designed this study to determine the effects of chronic ethanol exposure on the activity of EAAT4 and evaluate the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in these effects. Methods:, EAAT4 was expressed in Xenopus oocytes following injection of EAAT4 mRNA. Oocytes were incubated with ethanol-containing solution for 24 to 96 hours. Membrane currents induced by l -aspartate were recorded using 2-electrode voltage clamps. Responses were quantified by integration of the current trace and reported in microCoulombs (,C). Results:, Ethanol dose- and time-dependently reduced EAAT4 activity. EAAT4 activity after a 96-hour exposure was significantly decreased compared to the control values at all concentrations tested (10 to 100 mM). Ethanol (50 mM) significantly decreased the Vmax (2.2 ± 0.2 ,C for control vs. 1.6 ± 0.2 ,C for ethanol, n = 18, p < 0.05) of EAAT4 for l -aspartate. Preincubation of ethanol-treated (50 mM for 96 hours) oocytes with phorbol-12-myrisate-13-acetate (100 nM for 10 minutes) abolished the ethanol-induced decrease in EAAT4 activity. While staurosporine (2 ,M for 1 hour) or chelerythrine (100 ,M for 1 hour) significantly decreased EAAT4 activity, no difference was observed in EAAT4 activity among the staurosporine, ethanol, or ethanol plus staurosporine groups. Similarly, EAAT4 activity did not differ among the chelerythrine, ethanol, or ethanol plus chelerythrine groups. Pretreatment of the oocytes with wortmannin (1 ,M for 1 hour) also significantly decreased EAAT4 activity. However, no difference was observed in the wortmannin, ethanol, or ethanol plus wortmannin groups. Conclusions:, The results of this study suggest that chronic ethanol exposure decreases EAAT4 activity and that PKC and PI3K may be involved in these effects. These effects of ethanol on EAAT4 may cause an increase in peri-Purkinje cellular glutamate concentration, and may be involved in cerebellar dysfunction and motor impairment after chronic ethanol ingestion. [source] Excitatory amino acid transporters EAAT-1 and EAAT-2 in temporal lobe and hippocampus in intractable temporal lobe epilepsyAPMIS, Issue 4 2009SINAN SARAC Intractable temporal lobe epilepsy (TLE) is an invalidating disease and many patients are resistant to medical treatment. Increased glutamate concentration has been found in epileptogenic foci and may induce local over-excitation and cytotoxicity; one of the proposed mechanisms involves reduced extra-cellular clearance of glutamate by excitatory amino acid transporters (EAAT-1 to EAAT-5). EAAT-1 and EAAT-2 are mainly expressed on astroglial cells for the reuptake of glutamate from the extra-cellular space. We have studied the expression of EAAT-1 and EAAT-2 in the hippocampus and temporal lobe in 12 patients with TLE by immunhistochemistry and densitometry. The expression of EAAT-1 and EAAT-2 was reduced to approximately 40% and 25%, respectively, in CA1 of the hippocampus. In the same area, an increased expression of glial fibrillary acid protein (GFAP) at 90% reflected molecular rearrangements and upregulation of GFAP in the existing astrocytes as Ki-67 staining failed to demonstrate any signs of astrocytic proliferation. The aetiology of the reduced expression of EAAT-1 and EAAT-2 remains unclear. The downregulation of EAAT-1 and EAAT-2 may be an adaptive response to neuronal death or it may be a causative event contributing to neuronal death. Further studies of the EAATs and their function are needed to clarify the mechanisms and significance of EAAT-1 and EAAT-2 disappearance in TLE. [source] | |||||||||||||