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1.
Neuron ; 21(6): 1487-98, 1998 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-9883740

RESUMEN

To investigate the structural determinants underlying transport by the glutamate transporter EAAT1, we mutated each of 24 highly conserved residues (P392 to Q415) to cysteine. A majority of these substituted cysteines react with the sulfhydryl-modifying reagent MTSEA, suggesting that they reside in an aqueous environment. The impermeant reagents MTSES and MTSET react with residues at each end of the domain (A395C and A414C), supporting a model that places these residues near the extracellular surface. Substrates and inhibitors block the reaction between MTS derivatives and A395C, and the cosubstrate, sodium, slows reaction of MTSEA with Y405C and E406C. From these results, we propose that this domain forms a reentrant membrane loop at the cell surface and may comprise part of the translocation pore for substrates and cotransported ions.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sistema de Transporte de Aminoácidos X-AG , Animales , Transporte Biológico , Células COS , Secuencia Conservada , Cisteína , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sodio/metabolismo , Termodinámica , Transfección
2.
Neuron ; 25(3): 695-706, 2000 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-10774736

RESUMEN

Excitatory amino acid transporters (EAATs) function as both substrate transporters and ligand-gated anion channels. Characterization of the transporter's general topology is the first requisite step in defining the structural bases for these distinct activities. While the first six hydrophobic domains can be readily modeled as conventional transmembrane segments, the organization of the C-terminal hydrophobic domains, which have been implicated in both substrate and ion interactions, has been controversial. Here, we report the results of a comprehensive evaluation of the C-terminal topology of EAAT1 determined by the chemical modification of introduced cysteine residues. Our data support a model in which two membrane-spanning domains flank a central region that is highly accessible to the extracellular milieu and contains at least one reentrant loop domain.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Aminoácidos/metabolismo , Cisteína/química , Secuencia de Aminoácidos , Sistema de Transporte de Aminoácidos X-AG , Animales , Biotina , Células COS , Metanosulfonato de Etilo/análogos & derivados , Espacio Extracelular/química , Espacio Extracelular/metabolismo , Humanos , Indicadores y Reactivos , Activación del Canal Iónico/fisiología , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/fisiología , Estructura Terciaria de Proteína
3.
Handb Exp Pharmacol ; (175): 137-50, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16722234

RESUMEN

The expression of vesicular glutamate transporters (VGLUTs) 1 and 2 accounts for the ability of most traditionally accepted excitatory neurons to release glutamate by exocytosis. However, several cell populations (serotonin and dopamine neurons) have been demonstrated to release glutamate in vitro and do not obviously express these transporters. Rather, these neurons express a novel, third isoform that in fact appears confined to neurons generally associated with a transmitter other than glutamate. They include serotonin and possibly dopamine neurons, cholinergic interneurons in the striatum, and GABAergic interneurons of the hippocampus and cortex. Although the physiological role of VGLUT3 remains largely conjectural, several observations in vivo suggest that the glutamate release mediated by VGLUT3 has an important role in synaptic transmission, plasticity, and development.


Asunto(s)
Encéfalo/metabolismo , Ácido Glutámico/metabolismo , Serotonina/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismo , Acetilcolina/metabolismo , Animales , Dopamina/metabolismo , Humanos , Neuroglía/metabolismo , Neuronas/metabolismo , Transmisión Sináptica/fisiología , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo
4.
Neuroscience ; 248: 95-111, 2013 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-23727452

RESUMEN

Using specific riboprobes, we characterized the expression of vesicular glutamate transporter (VGLUT)1-VGLUT3 transcripts in lumbar 4-5 (L4-5) dorsal root ganglions (DRGs) and the thoracolumbar to lumbosacral spinal cord in male BALB/c mice after a 1- or 3-day hindpaw inflammation, or a 7-day sciatic nerve axotomy. Sham animals were also included. In sham and contralateral L4-5 DRGs of injured mice, VGLUT1-, VGLUT2- and VGLUT3 mRNAs were expressed in ∼45%, ∼69% or ∼17% of neuron profiles (NPs), respectively. VGLUT1 was expressed in large and medium-sized NPs, VGLUT2 in NPs of all sizes, and VGLUT3 in small and medium-sized NPs. In the spinal cord, VGLUT1 was restricted to a number of NPs at thoracolumbar and lumbar segments, in what appears to be the dorsal nucleus of Clarke, and in mid laminae III-IV. In contrast, VGLUT2 was present in numerous NPs at all analyzed spinal segments, except the lateral aspects of the ventral horns, especially at the lumbar enlargement, where it was virtually absent. VGLUT3 was detected in a discrete number of NPs in laminae III-IV of the dorsal horn. Axotomy resulted in a moderate decrease in the number of DRG NPs expressing VGLUT3, whereas VGLUT1 and VGLUT2 were unaffected. Likewise, the percentage of NPs expressing VGLUT transcripts remained unaltered after hindpaw inflammation, both in DRGs and the spinal cord. Altogether, these results confirm previous descriptions on VGLUTs expression in adult mice DRGs, with the exception of VGLUT1, whose protein expression was detected in a lower percentage of mouse DRG NPs. A detailed account on the location of neurons expressing VGLUTs transcripts in the adult mouse spinal cord is also presented. Finally, the lack of change in the number of neurons expressing VGLUT1 and VGLUT2 transcripts after axotomy, as compared to data on protein expression, suggests translational rather than transcriptional regulation of VGLUTs after injury.


Asunto(s)
Ganglios Espinales/metabolismo , Neuronas/metabolismo , Médula Espinal/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/análisis , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animales , Axotomía , Miembro Posterior , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/metabolismo , Nervio Ciático/lesiones , Proteína 1 de Transporte Vesicular de Glutamato/análisis , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/análisis , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Proteínas de Transporte Vesicular de Glutamato/análisis
6.
Annu Rev Pharmacol Toxicol ; 39: 431-56, 1999.
Artículo en Inglés | MEDLINE | ID: mdl-10331091

RESUMEN

As the most predominant excitatory neurotransmitter, glutamate has the potential to influence the function of most neuronal circuits in the central nervous system. To limit receptor activation during signaling and prevent the overstimulation of glutamate receptors that can trigger excitotoxic mechanisms and cell death, extracellular concentrations of excitatory amino acids are tightly controlled by transport systems on both neurons and glial cells. L-Glutamate is a potent neurotoxin, and the inadequate clearance of excitatory amino acids may contribute to the neurodegeneration seen in a variety of conditions, including epilepsy, ischemia, and amyotrophic lateral sclerosis. To establish the contributions of carrier systems to the etiology of neurological disorders, and to consider their potential utility as therapeutic targets, a detailed understanding of transporter function and pharmacology is required. This review summarizes current knowledge of the structural and functional diversity of excitatory amino acid transporters and explores how they might serve as targets for drug design.


Asunto(s)
Proteínas Portadoras/metabolismo , Aminoácidos Excitadores/metabolismo , Sistemas de Transporte de Aminoácidos , Animales , Transporte Biológico/fisiología , Proteínas Portadoras/genética , Ácido Glutámico/metabolismo , Humanos , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades del Sistema Nervioso/metabolismo , Transducción de Señal/fisiología
7.
Proc Natl Acad Sci U S A ; 98(26): 15324-9, 2001 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-11752470

RESUMEN

Excitatory amino acid transporters (EAATs) buffer and remove synaptically released L-glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. Here, we demonstrate that modification of a cysteine substituted within a C-terminal domain of EAAT1 abolishes transport in both the forward and reverse directions without affecting activation of the anion conductance. EC(50)s for L-glutamate and sodium are significantly lower after modification, consistent with kinetic models of the transport cycle that link anion channel gating to an early step in substrate translocation. Also, decreasing the pH from 7.5 to 6.5 decreases the EC(50) for L-glutamate to activate the anion conductance, without affecting the EC(50) for the entire transport cycle. These findings demonstrate for the first time a structural separation of transport and the uncoupled anion flux. Moreover, they shed light on some controversial aspects of the EAAT transport cycle, including the kinetics of proton binding and anion conductance activation.


Asunto(s)
Transportador 1 de Aminoácidos Excitadores/metabolismo , Compuestos de Sulfhidrilo/química , Aniones , Transporte Biológico , Transportador 1 de Aminoácidos Excitadores/química , Humanos , Activación del Canal Iónico , Cinética , Conformación Proteica
8.
J Neurochem ; 79(2): 297-302, 2001 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11677257

RESUMEN

D,L-threo-beta-Benzyloxyaspartate (D,L-TBOA), an analog of threo-beta-hydroxyaspartate (THA) possessing a bulky substituent, is a potent non-transportable blocker for the excitatory amino acid transporters, EAAT1, 2 and 3, while L-threo-beta-methoxyaspartate (L-TMOA) is a blocker for EAAT2, but a substrate for EAAT1 and EAAT3. To characterize the actions of these THA analogs and the function of EAAT4 and EAAT5, we performed electrophysiological analyses in EAAT4 or EAAT5 expressed on Xenopus oocytes. In EAAT4-expressing oocytes, D,L-TBOA acted as a non-transportable blocker, while L-TMOA like D,L-THA was a competitive substrate. In contrast, D,L-THA, D,L-TBOA and L-TMOA all strongly attenuated the glutamate-induced currents generated by EAAT5. Among them, L-TMOA showed the most potent inhibitory action. Moreover, D,L-THA, D,L-TBOA and L-TMOA themselves elicited outward currents at negative potentials and remained inward at positive potentials suggesting that D,L-TBOA and L-TMOA, as well as D,L-THA, not only act as non-transportable blockers, but also block the EAAT5 leak currents. These results indicate that EAATs 4 and 5 show different sensitivities to THA analogs although they share properties of a glutamate-gated chloride channel.


Asunto(s)
Sistema de Transporte de Aminoácidos X-AG , Ácido Aspártico/farmacología , Proteínas Portadoras/fisiología , Receptores de Glutamato/fisiología , Simportadores , Animales , Ácido Aspártico/análogos & derivados , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/efectos de los fármacos , Electrofisiología , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Oocitos , Receptores de Glutamato/efectos de los fármacos , Xenopus
9.
Recept Channels ; 6(1): 51-64, 1998.
Artículo en Inglés | MEDLINE | ID: mdl-9664622

RESUMEN

Sodium-dependent glutamate transporters influence neurotransmission in the central nervous system by removing synaptically released glutamate from the extracellular space and by maintaining extracellular glutamate concentrations below neurotoxic levels. In insects, glutamate also serves as the neurotransmitter at the neuromuscular junction, but the mechanism for neurotransmitter clearance at this synapse has not well-established. Here we report that cloning and characterization of a sodium-dependent glutamate transporter, dEAAT, from Drosophila melanogaster. The 479 amino acid dEAAT gene product is 40-50% homologous to mammalian members of this carrier family. A 3.3 kilobase (kb) transcript for dEAAT was detected in adult fly heads and to a lesser extent in bodies by Northern-blot analysis and was also localized to neurons in the central nervous system by in situ hybridization. The transport activity observed following express of dEAAT in Xenopus oocytes or COS-7 cells shows a high affinity for L-glutamate, L-aspartate and D-aspartate, an absolute dependence on external sodium ions, and considerable stereoselectivity for the transport of L-glutamate over D-glutamate. As has been observed for the human carriers, EAAT 4 and EAAT 5, a significant component of the current activated by L-glutamate application to dEAAT-expressing oocytes appears to arise from the activation of a chloride channel associated with the carrier.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , ADN Complementario/genética , Drosophila melanogaster/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Sistema de Transporte de Aminoácidos X-AG , Animales , Secuencia de Bases , Células COS , Canales de Cloruro/metabolismo , Cartilla de ADN/genética , ADN Complementario/aislamiento & purificación , Drosophila melanogaster/metabolismo , Femenino , Expresión Génica , Genes de Insecto , Humanos , Hibridación in Situ , Técnicas In Vitro , Datos de Secuencia Molecular , Neuronas/metabolismo , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Xenopus
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