RESUMO
The recently discovered interaction between Presenilin 1 (PS1), a catalytic subunit of γ-secretase responsible for generating amyloid-ß peptides, and GLT-1, a major glutamate transporter in the brain (EAAT2), provides a mechanistic link between these two key factors involved in Alzheimer's disease (AD) pathology. Modulating this interaction can be crucial to understand the consequence of such crosstalk in AD context and beyond. However, the interaction sites between these two proteins are unknown. Herein, we utilized an alanine scanning approach coupled with FRET-based fluorescence lifetime imaging microscopy to identify the interaction sites between PS1 and GLT-1 in their native environment within intact cells. We found that GLT-1 residues at position 276 to 279 (TM5) and PS1 residues at position 249 to 252 (TM6) are crucial for GLT-1-PS1 interaction. These results have been cross validated using AlphaFold Multimer prediction. To further investigate whether this interaction of endogenously expressed GLT-1 and PS1 can be prevented in primary neurons, we designed PS1/GLT-1 cell-permeable peptides (CPPs) targeting the PS1 or GLT-1 binding site. We used HIV TAT domain to allow for cell penetration which was assayed in neurons. First, we assessed the toxicity and penetration of CPPs by confocal microscopy. Next, to ensure the efficiency of CPPs, we monitored the modulation of GLT-1-PS1 interaction in intact neurons by fluorescence lifetime imaging microscopy. We saw significantly less interaction between PS1 and GLT-1 with both CPPs. Our study establishes a new tool to study the functional aspect of GLT-1-PS1 interaction and its relevance in normal physiology and AD models.
Assuntos
Transportador 2 de Aminoácido Excitatório , Presenilina-1 , Animais , Humanos , Camundongos , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Sítios de Ligação , Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Neurônios/metabolismo , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Ligação Proteica , Peptídeos/metabolismoRESUMO
Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2.
Assuntos
Transportador 2 de Aminoácido Excitatório/química , Ácido Glutâmico , Animais , Sítios de Ligação , Encéfalo/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Transportador 3 de Aminoácido Excitatório/genética , Transportador 3 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Mamíferos/metabolismo , Neuroglia/metabolismoRESUMO
OBJECTIVE: Mutations in the gene solute carrier family member 1A2 (SLC1A2) encoding the excitatory amino acid transporter 2 (EAAT2) are associated with severe forms of epileptic encephalopathy. EAAT2 is expressed in glial cells and presynaptic nerve terminals and represents the main l-glutamate uptake carrier in the mammalian brain. It does not only function as a secondary active glutamate transporter, but also as an anion channel. How naturally occurring mutations affect these two transport functions of EAAT2 and how such alterations cause epilepsy is insufficiently understood. METHODS: Here we studied the functional consequences of three disease-associated mutations, which predict amino acid exchanges p.Gly82Arg (G82R), p.Leu85Pro (L85P), and p.Pro289Arg (P289R), by heterologous expression in mammalian cells, biochemistry, confocal imaging, and whole-cell patch-clamp recordings of EAAT2 l-glutamate transport and anion current. RESULTS: G82R and L85P exchange amino acid residues that contribute to the formation of the EAAT anion pore. They enlarge the pore diameter sufficiently to permit the passage of l-glutamate and thus function as l-glutamate efflux pathways. The mutation P289R decreases l-glutamate uptake, but increases anion currents despite a lower membrane expression. SIGNIFICANCE: l-glutamate permeability of the EAAT anion pore is an unexpected functional consequence of naturally occurring single amino acid substitutions. l-glutamate efflux through mutant EAAT2 anion channels will cause glutamate excitotoxicity and neuronal hyperexcitability in affected patients. Antagonists that selectively suppress the EAAT anion channel function could serve as therapeutic agents in the future.
Assuntos
Encefalopatias , Transportador 2 de Aminoácido Excitatório , Aminoácidos/metabolismo , Animais , Ânions/metabolismo , Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Humanos , Mamíferos/metabolismo , Mutação/genéticaRESUMO
Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system. Once released, it binds to specific membrane receptors and transporters activating a wide variety of signal transduction cascades, as well as its removal from the synaptic cleft in order to avoid its extracellular accumulation and the overstimulation of extra-synaptic receptors that might result in neuronal death through a process known as excitotoxicity. Although neurodegenerative diseases are heterogenous in clinical phenotypes and genetic etiologies, a fundamental mechanism involved in neuronal degeneration is excitotoxicity. Glutamate homeostasis is critical for brain physiology and Glutamate transporters are key players in maintaining low extracellular Glutamate levels. Therefore, the characterization of Glutamate transporters has been an active area of glutamatergic research for the last 40 years. Transporter activity its regulated at different levels: transcriptional and translational control, transporter protein trafficking and membrane mobility, and through extensive post-translational modifications. The elucidation of these mechanisms has emerged as an important piece to shape our current understanding of glutamate actions in the nervous system.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/química , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Ácido Glutâmico/metabolismo , Transmissão Sináptica/fisiologia , Sistema X-AG de Transporte de Aminoácidos/genética , Animais , Transportador 1 de Aminoácido Excitatório/química , Transportador 1 de Aminoácido Excitatório/genética , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Humanos , Processamento de Proteína Pós-Traducional/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Excitatory amino acid transporter 2 (EAAT2), the gene of which is known as solute carrier family 1 member 2 (SLC1A2), is an important membrane-bound transporter that mediates approximately 90% of the transport and clearance of l-glutamate at synapses in the central nervous system (CNS). Transmembrane domain 2 (TM2) of EAAT2 is close to hairpin loop 2 (HP2) and far away from HP1 in the inward-facing conformation. In the present study, 14 crucial amino acid residues of TM2 were identified via alanine-scanning mutations. Further analysis in EAAT2-transfected HeLa cells in vitro showed that alanine substitutions of these residues resulted in a decrease in the efficiency of trafficking/targeting to the plasma membrane and/or reduced functionality of membrane-bound, which resulted in impaired transporter activity. After additional mutations, the transporter activities of some alanine-substitution mutants recovered. Specifically, the P95A mutant decreased EAAT2-associated anion currents. The Michaelis constant (Km ) values of the mutant proteins L85A, L92A and L101A were increased significantly, whereas R87 and P95A were decreased significantly, indicating that the mutations L85A, L92A and L101A reduced the affinity of the transporter and the substrate, whereas R87A and P95A enhanced this affinity. The maximum velocity (Vmax) values of all 14 alanine mutant proteins were decreased significantly, indicating that all these mutations reduced the substrate transport rate. These results suggest that critical residues in TM2 affect not only the protein expression and membrane-bound localization of EAAT2, but also its interactions with substrates. Additionally, our findings elucidate that the P95A mutant decreased EAAT2-related anion currents. Our results indicate that the TM2 of EAAT2 plays a vital role in the transport process. The key residues in TM2 affect protein expression in the membrane, substrate transport and the anion currents of EAAT2.
Assuntos
Aminoácidos , Ânions/metabolismo , Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/metabolismo , Domínios e Motivos de Interação entre Proteínas , Aminoácidos/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , Células HeLa , Humanos , Cinese , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Excitatory amino acid transporters (EAATs) are prototypical dual function proteins that function as coupled glutamate/Na+/H+/K+ transporters and as anion-selective channels. Both transport functions are intimately intertwined at the structural level: Secondary active glutamate transport is based on elevator-like movements of the mobile transport domain across the membrane, and the lateral movement of this domain results in anion channel opening. This particular anion channel gating mechanism predicts the existence of mutant transporters with changed anion channel properties, but without alteration in glutamate transport. We here report that the L46P mutation in the human EAAT2 transporter fulfills this prediction. L46 is a pore-forming residue of the EAAT2 anion channels at the cytoplasmic entrance into the ion conduction pathway. In whole-cell patch clamp recordings, we observed larger macroscopic anion current amplitudes for L46P than for WT EAAT2. Rapid l-glutamate application under forward transport conditions demonstrated that L46P does not reduce the transport rate of individual transporters. In contrast, changes in selectivity made gluconate permeant in L46P EAAT2, and nonstationary noise analysis revealed slightly increased unitary current amplitudes in mutant EAAT2 anion channels. We used unitary current amplitudes and individual transport rates to quantify absolute open probabilities of EAAT2 anion channels from ratios of anion currents by glutamate uptake currents. This analysis revealed up to 7-fold increased absolute open probability of L46P EAAT2 anion channels. Our results reveal an important determinant of the diameter of EAAT2 anion pore and demonstrate the existence of anion channel gating processes outside the EAAT uptake cycle.
Assuntos
Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Mutação Puntual , Transporte Biológico , Transportador 2 de Aminoácido Excitatório/química , Humanos , Técnicas de Patch-Clamp , ProbabilidadeRESUMO
Excitatory Amino Acid Transporters (EAATs) are plasma membrane proteins responsible for maintenance of low extracellular concentrations of glutamate in the CNS. Dysfunction in their activity is implicated in various neurological disorders. Glutamate transport by EAATs occurs through the movement of the central transport domain relative to the scaffold domain in the EAAT membrane protein. Previous studies suggested that residues located within the interface of these two domains in EAAT2, the main subtype of glutamate transporter in the brain, are involved in regulating transport rates. We used mutagenesis, structure-function relationship, surface protein expression and electrophysiology studies, in transfected COS-7 cells and oocytes, to examine residue glycine at position 298, which is located within this interface. Mutation G298A results in increased transport rate without changes in surface expression, suggesting a more hydrophobic and larger alanine results in facilitated transport movement. The increased transport rate does not involve changes in sodium affinity. Electrophysiological currents show that G298A increase both transport and anion currents, suggesting faster transitions through the transport cycle. This work identifies a region critically involved in setting the glutamate transport rate.
Assuntos
Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Transportador 2 de Aminoácido Excitatório/química , Feminino , Proteínas Associadas à Matriz Nuclear/química , Estrutura Secundária de Proteína , Transporte Proteico/fisiologia , Especificidade por Substrato/fisiologia , XenopusRESUMO
Excitatory amino acid transporters (EAATs) represent a protein family that is an emerging drug target with great therapeutic potential for managing central nervous system disorders characterized by dysregulation of glutamatergic neurotransmission. As such, it is of significant interest to discover selective modulators of EAAT2 function. Here, we applied computational methods to identify specific EAAT2 inhibitors. Utilizing a homology model of human EAAT2, we identified a binding pocket at the interface of the transport and trimerization domain. We next conducted a high-throughput virtual screen against this site and identified a selective class of EAAT2 inhibitors that were tested in glutamate uptake and whole-cell electrophysiology assays. These compounds represent potentially useful pharmacological tools suitable for further exploration of the therapeutic potential of EAAT2 and may provide molecular insights into mechanisms of allosteric modulation for glutamate transporters.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Doenças do Sistema Nervoso Central/tratamento farmacológico , Transportador 2 de Aminoácido Excitatório/antagonistas & inibidores , Sistema X-AG de Transporte de Aminoácidos/química , Sistema X-AG de Transporte de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Transporte Biológico/efeitos dos fármacos , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/patologia , Biologia Computacional , Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/genética , Humanos , Ligação Proteica/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Interface Usuário-ComputadorRESUMO
SLC1A2 is a trimeric transporter essential for clearing glutamate from neuronal synapses. Recurrent de novo SLC1A2 missense variants cause a severe, early onset developmental and epileptic encephalopathy via an unclear mechanism. We demonstrate that all 3 variants implicated in this condition localize to the trimerization domain of SLC1A2, and that the Leu85Pro variant acts via a dominant negative mechanism to reduce, but not eliminate, wild-type SLC1A2 protein localization and function. Finally, we demonstrate that treatment of a 20-month-old SLC1A2-related epilepsy patient with the SLC1A2-modulating agent ceftriaxone did not result in a significant change in daily spasm count. ANN NEUROL 2019;85:921-926.
Assuntos
Epilepsia Generalizada/diagnóstico , Epilepsia Generalizada/genética , Transportador 2 de Aminoácido Excitatório/genética , Variação Genética/genética , Sequência de Aminoácidos , Ceftriaxona/uso terapêutico , Pré-Escolar , Epilepsia Generalizada/tratamento farmacológico , Transportador 2 de Aminoácido Excitatório/química , Feminino , Células HEK293 , Humanos , Lactente , Recém-Nascido , Masculino , Estrutura Secundária de ProteínaRESUMO
In the brain, glutamate transporters terminate excitatory neurotransmission by removing this neurotransmitter from the synapse via cotransport with three sodium ions into the surrounding cells. Structural studies have identified the binding sites of the three sodium ions in glutamate transporters. The residue side-chains directly interact with the sodium ions at the Na1 and Na3 sites and are fully conserved from archaeal to eukaryotic glutamate transporters. The Na2 site is formed by three main-chain oxygens on the extracellular reentrant hairpin loop HP2 and one on transmembrane helix 7. A glycine residue on HP2 is located closely to the three main-chain oxygens in all glutamate transporters, except for the astroglial transporter GLT-1, which has a serine residue at that position. Unlike for WT GLT-1, substitution of the serine residue to glycine enables sustained glutamate transport also when sodium is replaced by lithium. Here, using functional and simulation studies, we studied the role of this serine/glycine switch on cation selectivity of substrate transport. Our results indicate that the side-chain oxygen of the serine residues can form a hydrogen bond with a main-chain oxygen on transmembrane helix 7. This leads to an expansion of the Na2 site such that water can participate in sodium coordination at Na2. Furthermore, we found other molecular determinants of cation selectivity on the nearby HP1 loop. We conclude that subtle changes in the composition of the two reentrant hairpin loops determine the cation specificity of acidic amino acid transport by glutamate transporters.
Assuntos
Transportador 2 de Aminoácido Excitatório/metabolismo , Sódio/metabolismo , Sítios de Ligação , Cátions/metabolismo , Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/genética , Transportador 3 de Aminoácido Excitatório/química , Transportador 3 de Aminoácido Excitatório/genética , Transportador 3 de Aminoácido Excitatório/metabolismo , Glicina/metabolismo , Células HeLa , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Serina/metabolismoRESUMO
Excitatory amino acid transporters (EAATs) regulate glutamatergic signal transmission by clearing extracellular glutamate. Dysfunction of these transporters has been implicated in the pathogenesis of various neurological disorders. Previous studies have shown that venom from the spider Parawixia bistriata and a purified compound (Parawixin1) stimulate EAAT2 activity and protect retinal tissue from ischemic damage. In the present study, the EAAT2 subtype specificity of this compound was explored, employing chimeric proteins between EAAT2 and EAAT3 transporter subtypes and mutants to characterize the structural region targeted by the compound. This identified a critical residue (Histidine-71 in EAAT2 and Serine-45 in EAAT3) in transmembrane domain 2 (TM2) to be important for the selectivity between EAAT2 and EAAT3 and for the activity of the venom. Using the identified residue in TM2 as a structural anchor, several neighboring amino acids within TM5 and TM8 were identified to also be important for the activity of the venom. This structural domain of the transporter lies at the interface of the rigid trimerization domain and the central substrate-binding transport domain. Our studies suggest that the mechanism of glutamate transport enhancement involves an interaction with the transporter that facilitates the movement of the transport domain. We identified a domain (purple star) in the glutamate transporter EAAT2 that is important for transport stimulation through a spider venom, and suggest a mechanism for enhanced transporter function through facilitated substrate translocation (arrow). Because the dysfunction of glutamate transporters is implicated in the pathogenesis of neurological disorders, understanding the mechanisms of enhanced transport could have therapeutic implications.
Assuntos
Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/metabolismo , Modelos Moleculares , Multimerização Proteica , Animais , Transporte Biológico/efeitos dos fármacos , Células COS , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Transportador 2 de Aminoácido Excitatório/efeitos dos fármacos , Transportador 2 de Aminoácido Excitatório/genética , Ácido Glutâmico/metabolismo , Humanos , Mutação/genética , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Venenos de Aranha/química , Toxinas Biológicas/análise , Toxinas Biológicas/farmacologia , TransfecçãoRESUMO
Excitatory amino acid transporter 2, also known as glial glutamate transporter type 1 (GLT-1), plays an important role in maintaining suitable synaptic glutamate concentrations. Reentrant helical hairpin loop (HP) 2, as the extracellular gate, has been shown to participate in the binding of substrate and ions. Several residues in transmembrane domain (TM) 5 have been shown to be involved in the construction of the transport pathway. However, the spatial relationship between HP2 and TM5 during the recycling of glutamate has not yet been clarified. We introduced cysteine residue pairs in HP2 and TM5 of cysteine-less-GLT-1 by using site-directed mutagenesis in order to assess the proximity of HP2 and TM5. A significant decrease in substrate uptake was seen in the I283C/S443C and S287C/S443C mutants when the oxidative cross-linking agent copper(II) (1,10-phenanthroline)3 (CuPh) was used. The inhibitory effect of CuPh on the transport activity of the S287/S443C mutant was increased after the application of glutamate or potassium. In contrast, an apparent protection of the transport activity of the I283C/S443C mutant was observed after glutamate or potassium addition. The membrane-impermeable sulfhydryl reagent (2-trimethylammonium) methanethiosulfonate (MTSET) was used to detect the aqueous permeability of each single mutant. The aqueous permeability of the I283C mutant was identical to that of the S443C mutant. The sensitivity of I283C and S443C to MTSET was attenuated by glutamate and potassium. All these data indicate that there is a complex relative motion between TM5 and HP2 during the transport cycle.
Assuntos
Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Aspártico/farmacologia , Ditiotreitol/metabolismo , Ácido Glutâmico/farmacologia , Células HeLa , Humanos , Cinética , Mesilatos/química , Mesilatos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Cloreto de Sódio/farmacologia , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismoRESUMO
Dysregulation of glutamate handling ensuing downregulation of expression and activity levels of the astroglial glutamate transporter EAAT2 is implicated in excitotoxic degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). We previously reported that EAAT2 (a.k.a. GLT-1) is cleaved by caspase-3 at its cytosolic carboxy-terminus domain. This cleavage results in impaired glutamate transport activity and generates a proteolytic fragment (CTE) that we found to be post-translationally conjugated by SUMO1. We show here that this sumoylated CTE fragment accumulates in the nucleus of spinal cord astrocytes of the SOD1-G93A mouse model of ALS at symptomatic stages of disease. Astrocytic expression of CTE, artificially tagged with SUMO1 (CTE-SUMO1) to mimic the native sumoylated fragment, recapitulates the nuclear accumulation pattern of the endogenous EAAT2-derived proteolytic fragment. Moreover, in a co-culture binary system, expression of CTE-SUMO1 in spinal cord astrocytes initiates extrinsic toxicity by inducing caspase-3 activation in motor neuron-derived NSC-34 cells or axonal growth impairment in primary motor neurons. Interestingly, prolonged nuclear accumulation of CTE-SUMO1 is intrinsically toxic to spinal cord astrocytes, although this gliotoxic effect of CTE-SUMO1 occurs later than the indirect, noncell autonomous toxic effect on motor neurons. As more evidence on the implication of SUMO substrates in neurodegenerative diseases emerges, our observations strongly suggest that the nuclear accumulation in spinal cord astrocytes of a sumoylated proteolytic fragment of the astroglial glutamate transporter EAAT2 could participate to the pathogenesis of ALS and suggest a novel, unconventional role for EAAT2 in motor neuron degeneration.
Assuntos
Transportador 2 de Aminoácido Excitatório/toxicidade , Neurônios Motores/efeitos dos fármacos , Proteína SUMO-1/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Astrócitos/metabolismo , Axônios/fisiologia , Axônios/ultraestrutura , Caspase 3/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Células Cultivadas , Técnicas de Cocultura , Transportador 2 de Aminoácido Excitatório/química , Imunofluorescência , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Análise em Microsséries , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Reação em Cadeia da Polimerase em Tempo Real , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
BACKGROUND: GLT-1 is a glial glutamate transporter which maintains low synaptic concentrations of the excitatory neurotransmitter enabling efficient synaptic transmission. Based on the crystal structure of the bacterial homologue Glt(Ph), it has been proposed that the reentrant loop HP2, which connects transmembrane domains (TM) 7 and 8, moves to open and close access to the binding pocket from the extracellular medium. However the conformation change between TM5 and TM8 during the transport cycle is not clear yet. We used paired cysteine mutagenesis in conjunction with treatments with Copper(II)(1,10-Phenanthroline)(3) (CuPh), to verify the predicted proximity of residues located at these structural elements of GLT-1. METHODOLOGY/PRINCIPAL FINDINGS: To assess the proximity of transmembrane domain (TM) 5 relative to TM8 during transport by the glial glutamate transporter GLT-1/EAAT2, cysteine pairs were introduced at the extracellular ends of these structural elements. A complete inhibition of transport by Copper(II)(1,10-Phenanthroline)(3) is observed in the double mutants I295C/I463C and G297C/I463C, but not in the corresponding single mutants. Glutamate and potassium, both expected to increase the proportion of inward-facing transporters, significantly protected against the inhibition of transport activity of I295C/I463C and G297C/I463C by CuPh. Transport by the double mutants I295C/I463C and G297C/I463C also was inhibited by Cd(2+). CONCLUSIONS/SIGNIFICANCE: Our results suggest that TM5 (Ile-295, Gly-297) is in close proximity to TM8 (Ile-463) in the mammalian transporter, and that the spatial relationship between these domains is altered during the transport cycle.
Assuntos
Cisteína/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Mutagênese , Cristalografia por Raios X , Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/genética , Células HeLa , Humanos , Compostos de Sulfidrila/metabolismoRESUMO
The glial excitatory amino acid transporter 2 (EAAT2) mediates a majority of glutamate re-uptake in human CNS and, consequently, is associated with a variety of signaling and pathological processes. While our understanding of the function, mechanism and structure of this integral membrane protein is increasing, little if any mass spectrometric (MS) data is available for any of the EAATs specifically, and for only a few mammalian plasma membrane transporters in general. A protocol to express and purify functional EAAT2 in sufficient quantities to carry out MS-based peptide mapping as needed to study ligand-transporter interactions is described. A 6xHIS epitope was incorporated into the N-terminus of human EAAT2. The recombinant protein was expressed in high levels in mammalian HEK 293T cells, where it exhibited the pharmacological properties of the native transporter. EAAT2 was purified from isolated cell membranes in a single step using nickel affinity chromatography. In-gel and in-solution trypsin digestions were conducted on the isolated protein and then analyzed by MALDI-TOF and LC-MS/MS mass spectrometry. Overall, 89% sequence coverage of the protein was achieved with these methods. In particular, an 88 amino acid tryptic peptide covering the presumed substrate binding domains HP1, TMD7, HP2, and TMD8 domains of EAAT2 was also identified after N-deglycosylation. Beyond the specific applicability to EAAT2, this study provides an efficient, simple and scalable approach to express, purify, digest and characterize integral membrane transporter proteins by mass spectrometry.
Assuntos
Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/genética , Expressão Gênica , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Transportador 2 de Aminoácido Excitatório/isolamento & purificação , Transportador 2 de Aminoácido Excitatório/metabolismo , Humanos , Dados de Sequência Molecular , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismoRESUMO
Glial glutamate transporter-1 (GLT-1) plays an essential role in the maintenance of glutamate homeostasis and is involved in the development and maintenance of pathological pain. The present study was undertaken (1) to observe the anti-nociceptive effects of ceftriaxone (Cef) in a chronic neuropathic pain model induced by chronic constrictive nerve injury (CCI) of the sciatic nerve and (2) to identify the role of spinal GLT-1 in the process. CCI induced significant thermal hyperalgesia and mechanical allodynia, which began from postoperative day 3 and lasted to day 21. This long-term hyperalgesia was accompanied by significant down-regulation of GLT-1 expression in the L4-L6 segments of the spinal dorsal horn, as revealed by immunohistochemistry and Western blot. Intraperitoneal preventive and therapeutic administration of Cef effectively prevented or reversed, respectively, the development of thermal hyperalgesia, mechanical allodynia, and GLT-1 down-regulation in the spinal dorsal horn. To further determine whether the above anti-nociceptive effects of Cef are a result of the up-regulation of spinal GLT-1 expression and its function, we further observed the effects of intrathecal administration of Cef in the same model. It was found that intrathecal administration of Cef led to the specific up-regulation of GLT-1 expression and glutamate uptake ((3)H-glutamate) in the spinal dorsal horn, and similar anti-nociceptive effects to those of intraperitoneal administration of Cef. The above effects of intrathecal Cef administration were all significantly inhibited by intrathecal administration of GLT-1 antisense oligodeoxynucleotides (As-ODNs). These results indicate that Cef plays an anti-nociceptive role by up-regulating spinal GLT-1 expression and its function.
Assuntos
Analgésicos/administração & dosagem , Ceftriaxona/administração & dosagem , Limiar da Dor/efeitos dos fármacos , Ciática/tratamento farmacológico , Animais , Doença Crônica , Modelos Animais de Doenças , Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Hiperalgesia/classificação , Hiperalgesia/prevenção & controle , Masculino , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Limiar da Dor/fisiologia , Estimulação Física/efeitos adversos , Ratos , Ratos Sprague-Dawley , Tempo de Reação/efeitos dos fármacos , Ciática/patologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Fatores de Tempo , Trítio/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Glutamate transporter 1 (GLT1) in glial cells removes glutamate that diffuses from the synaptic cleft into the extracellular space. Previously, we have shown the distribution of glutamatergic neurons in the central nervous system (CNS) of the pigeon. In the present study, we identified cDNA sequence of the pigeon GLT1, and mapped the distribution of the mRNA-expressing cells in CNS to examine whether GLT1 is associated with glutamatergic terminal areas. The cDNA sequence of the pigeon GLT1 consisted of 1889bp nucleotides and the amino acids showed 97% and 87% identity to the chicken and human GLT1, respectively. In situ hybridization autoradiograms revealed GLT1 mRNA expression in glial cells and produced regional differences of GLT1 mRNA distribution in CNS. GLT1 mRNA was expressed preferentially in the pallium than the subpallium. Moderate expression was seen in the hyperpallium, Field L, mesopallium, and hippocampal formation. In the thalamus, moderate expression was found in the ovoidal nucleus, rotundal nucleus, triangular nucleus, and lateral spiriform nucleus, while the dorsal thalamic nuclei were weak. In the brainstem, the isthmic nuclei, optic tectum, vestibular nuclei, and cochlear nuclei expressed moderately, but the cerebellar cortex showed strong expression. Bergmann glial cells expressed GLT1 mRNA very strongly. The results indicate that cDNA sequence of the pigeon GLT1 is comparable with that of the mammalian GLT1, and a large number of GLT1 mRNA-expressing areas correspond with areas where AMPA-type glutamate receptors are located. Avian GLT1 in glial cells probably maintain microenvironment of glutamate concentration around synapses as in mammalian GLT1.
Assuntos
Sistema Nervoso Central/metabolismo , Columbidae/metabolismo , Transportador 2 de Aminoácido Excitatório/genética , RNA Mensageiro/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Mapeamento Encefálico , Sistema Nervoso Central/citologia , Columbidae/anatomia & histologia , DNA Complementar/biossíntese , DNA Complementar/genética , Evolução Molecular , Transportador 2 de Aminoácido Excitatório/química , Feminino , Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Hibridização In Situ , Masculino , Mamíferos/genética , Dados de Sequência Molecular , Neuroglia/citologia , Neuroglia/metabolismo , Filogenia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Medula Espinal/citologia , Medula Espinal/metabolismoRESUMO
The glutamate transporter GLT1 is expressed in at least two isoforms, GLT1a and GLT1b, which differ in their C termini. As GLT1 is an oligomeric protein, we have investigated whether GLT1a and GLT1b might associate as hetero-oligomers. Differential tagging (HA-GLT1a and YFP-GLT1b) revealed that these isoforms form complexes that could be immunoprecipitated when co-expressed in heterologous systems. The association of GLT1a and GLT1b was also observed in mixed primary cultures of rat brain and in the adult rat brain, where specific antibodies for GLT1a immunoprecipitated GLT1b and vice versa. Dual immunofluorescence in mixed cultures demonstrated the partial co-localization of both isoforms in neurons and in glial cells. Because GLT1b interacts with an organizer of post-synaptic densities, PSD-95, we examined the capacity of GLT1a to associate with this protein. GLT1a was immunoprecipitated from the rat brain in protein complexes that contained not only GLT1b but also PSD-95 and NMDAR. The interaction between GLT1a with PSD-95 and NMDAR was reproduced in transfected COS7 cells and it appears to be indirect as it requires the presence of GLT1b. These results indicate that the major isoform of the glutamate transporter, GLT1a, can acquire the capacity to interact with PDZ proteins through its inclusion in hetero-oligomers containing GLT1b.
Assuntos
Encéfalo/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Processamento Alternativo/genética , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Técnicas de Cocultura , Proteína 4 Homóloga a Disks-Large , Cães , Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/genética , Ácido Glutâmico/metabolismo , Imunoprecipitação , Neuroglia/metabolismo , Polímeros/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Membranas Sinápticas/genética , Membranas Sinápticas/metabolismo , Transmissão Sináptica/genéticaRESUMO
The L-glutamate transporter GLT-1 is an abundant central nervous system (CNS) membrane protein of the excitatory amino acid transporter (EAAT) family that controls extracellular L-glutamate levels and is important in limiting excitotoxic neuronal death. Using reverse transcription-polymerase chain reaction, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N and C termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI, and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected human embryonic kidney (HEK) 293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent L-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (tagged with V5, hemagglutinin, or FLAG) into the second extracellular domain of each isoform allowed coimmunoprecipitation and time-resolved Förster resonance energy transfer (tr-FRET) studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms is able to combine to form homomeric and heteromeric assemblies, each of which is expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.
Assuntos
Transportador 2 de Aminoácido Excitatório/química , Animais , Células Cultivadas , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/fisiologia , Humanos , Camundongos , Isoformas de Proteínas , Proteína Quinase C/fisiologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Sodium-dependent glutamate uptake is essential for limiting excitotoxicity, and dysregulation of this process has been implicated in a wide array of neurological disorders. The majority of forebrain glutamate uptake is mediated by the astroglial glutamate transporter, GLT-1. We and others have shown that this transporter undergoes endocytosis and degradation in response to activation of protein kinase C (PKC), however, the mechanisms involved remain unclear. In the current study, transfected C6 glioma cells or primary cortical cultures were used to show that PKC activation results in incorporation of ubiquitin into GLT-1 immunoprecipitates. Mutation of all 11 lysine residues in the amino and carboxyl-terminal domains to arginine (11R) abolished this signal. Selective mutation of the seven lysine residues in the carboxyl terminus (C7K-R) did not eliminate ubiquitination, but it completely blocked PKC-dependent internalization and degradation. Two families of variants of GLT-1 were prepared with various lysine residues mutated to arginine. Analyses of these constructs indicated that redundant lysine residues in the carboxyl terminus were sufficient for the appearance of ubiquitinated product and degradation of GLT-1. Together these data define a novel mechanism by which the predominant forebrain glutamate transporter can be rapidly targeted for degradation.