RESUMEN
The system N transporter SN1 has been proposed to mediate the efflux of glutamine from cells required to sustain the urea cycle and the glutamine-glutamate cycle that regenerates glutamate and gamma-aminobutyric acid (GABA) for synaptic release. We now show that SN1 also mediates an ionic conductance activated by glutamine, and this conductance is selective for H(+). Although SN1 couples amino acid uptake to H(+) exchange, the glutamine-gated H(+) conductance is not stoichiometrically coupled to transport. Protons thus permeate SN1 both coupled to and uncoupled from amino acid flux, providing novel mechanisms to regulate the transfer of glutamine between cells.
Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Animales , Astrocitos/metabolismo , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Activación del Canal Iónico , Protones , Xenopus , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Glutamate transporters are essential for terminating synaptic excitation and for maintaining extracellular glutamate concentrations below neurotoxic levels. These transporters also mediate a thermodynamically uncoupled chloride flux that is activated by two of the molecules that they transport - sodium and glutamate. Five eukaryotic glutamate transporters have been cloned and identified. They exhibit approximately 50% identity and this homology is even greater in the carboxyl terminal half, which is predicted to have an unusual topology. Determination of the topology shows that the carboxyl terminal part of the molecule contains several transmembrane domains that are separated by at least two re-entrant loops. In these structural elements, we have identified several conserved amino acid residues that play crucial roles in the interaction with the transporter substrates sodium, potassium and glutamate.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG/química , Animales , Arginina/química , Sitios de Unión , Secuencia Conservada , Ácido Glutámico/química , Humanos , Modelos Biológicos , Modelos Químicos , Familia de Multigenes , Potasio/química , Unión Proteica , Sodio/química , Relación Estructura-ActividadRESUMEN
The sodium-dependent neutral amino acid transporter type 2 (ASCT2) was recently identified as a cell surface receptor for endogenously inherited retroviruses of cats, baboons, and humans as well as for horizontally transmitted type-D simian retroviruses. By functional cloning, we obtained 10 full-length 2.9-kilobase pair (kbp) cDNAs and two smaller identical 2.1-kbp cDNAs that conferred susceptibility to these viruses. Compared with the 2.9-kbp cDNA, the 2.1-kbp cDNA contains exonic deletions in its 3' noncoding region and a 627-bp 5' truncation that eliminates sequences encoding the amino-terminal portion of the full-length ASCT2 protein. Although expression of the truncated mRNA caused enhanced amino acid transport and viral receptor activities, the AUG codon nearest to its 5' end is flanked by nucleotides that are incompatible with translational initiation and the next in-frame AUG codon is far downstream toward the end of the protein coding sequence. Interestingly, the 5' region of the truncated ASCT2 mRNA contains a closely linked series of CUG(Leu) and GUG(Val) codons in optimal consensus contexts for translational initiation. By deletion and site-directed mutagenesis, cell-free translation, and analyses of epitope-tagged ASCT2 proteins synthesized intracellularly, we determined that the truncated mRNA encodes multiple ASCT2 isoforms with distinct amino termini that are translationally initiated by a leaky scanning mechanism at these CUG and GUG codons. Although the full-length ASCT2 mRNA contains a 5'-situated AUG initiation codon, a significant degree of leaky scanning also occurred in its translation. ASCT2 isoforms with relatively short truncations were active in both amino acid transport and viral reception, whereas an isoform with a 79-amino acid truncation that lacked the first transmembrane sequence was active only in viral reception. We conclude that ASCT2 isoforms with truncated amino termini are synthesized in mammalian cells by a leaky scanning mechanism that employs multiple alternative CUG and GUG initiation codons.
Asunto(s)
Proteínas Portadoras/metabolismo , Codón , Biosíntesis de Proteínas , Receptores Virales/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Cartilla de ADN , ADN Complementario , Humanos , Ratones , Datos de Secuencia Molecular , Conformación Proteica , ARN Mensajero/genética , Receptores Virales/química , Receptores Virales/genética , Retroviridae/metabolismoRESUMEN
Glutamate transporters from the central nervous system play a crucial role in the clearance of the transmitter from the synaptic cleft. Glutamate is cotransported with sodium ions, and the electrogenic translocation cycle is completed by countertransport of potassium. Mutants that cannot interact with potassium are only capable of catalyzing electroneutral exchange. Here we identify a residue involved in controlling substrate recognition in the neuronal transporter EAAC-1 that transports acidic amino acids as well as cysteine. When arginine 447, a residue conserved in all glutamate transporters, is replaced by cysteine, transport of glutamate or aspartate is abolished, but sodium-dependent cysteine transport is left intact. Analysis of other substitution mutants shows that the replacement of arginine rather than the introduced cysteine is responsible for the observed phenotype. In further contrast to wild type, acidic amino acids are unable to inhibit cysteine transport in R447C-EAAC-1, indicating that the selectivity change is manifested at the binding step. Electrophysiological analysis shows that in the mutant cysteine, transport has become electroneutral, and its interaction with the countertransported potassium is impaired. Thus arginine 447 plays a pivotal role in the sequential interaction of acidic amino acids and potassium with the transporter and, thereby, constitutes one of the molecular determinants of coupling their fluxes.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG , Arginina/metabolismo , Proteínas Portadoras/metabolismo , Glutamatos/metabolismo , Neuronas/metabolismo , Simportadores , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Xenopus laevisRESUMEN
Freeze-fracture electron microscopy was used to study the structure of a human neuronal glutamate transporter (EAAT3). EAAT3 was expressed in Xenopus laevis oocytes, and its function was correlated with the total number of transporters in the plasma membrane of the same cells. Function was assayed as the maximum charge moved in response to a series of transmembrane voltage pulses. The number of transporters in the plasma membrane was determined from the density of a distinct 10-nm freeze-fracture particle, which appeared in the protoplasmic face only after EAAT3 expression. The linear correlation between EAAT3 maximum carrier-mediated charge and the total number of the 10-nm particles suggested that this particle represented functional EAAT3 in the plasma membrane. The cross-sectional area of EAAT3 in the plasma membrane (48 +/- 5 nm(2)) predicted 35 +/- 3 transmembrane alpha-helices in the transporter complex. This information along with secondary structure models (6-10 transmembrane alpha-helices) suggested an oligomeric state for EAAT3. EAAT3 particles were pentagonal in shape in which five domains could be identified. They exhibited fivefold symmetry because they appeared as equilateral pentagons and the angle at the vertices was 110 degrees. Each domain appeared to contribute to an extracellular mass that projects approximately 3 nm into the extracellular space. Projections from all five domains taper toward an axis passing through the center of the pentagon, giving the transporter complex the appearance of a penton-based pyramid. The pentameric structure of EAAT3 offers new insights into its function as both a glutamate transporter and a glutamate-gated chloride channel.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG , Proteínas Portadoras/biosíntesis , Ácido Glutámico/metabolismo , Neuronas/metabolismo , Simportadores , Animales , Proteínas Portadoras/genética , Transportador 3 de Aminoácidos Excitadores , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Humanos , Xenopus laevisRESUMEN
The addition of methyl tert-butyl ether (MTBE) to gasoline has resulted in public uncertainty regarding the continued reliance on biological processes for gasoline remediation. Despite this concern, researchers have shown that MTBE can be effectively degraded in the laboratory under aerobic conditions using pure and mixed cultures with half-lives ranging from 0.04 to 29 days. Ex-situ aerobic fixed-film and aerobic suspended growth bioreactor studies have demonstrated decreases in MTBE concentrations of 83% and 96% with hydraulic residence times of 0.3 hrs and 3 days, respectively. In microcosm and field studies, aerobic biodegradation half-lives range from 2 to 693 days. These half-lives have been shown to decrease with increasing dissolved oxygen concentrations and, in some cases, with the addition of exogenous MTBE-degraders. MTBE concentrations have also been observed to decrease under anaerobic conditions; however, these rates are not as well defined. Several detailed field case studies describing the use of ex-situ reactors, natural attenuation, and bioaugmentation are presented in this paper and demonstrate the potential for successful remediation of MTBE-contaminated aquifers. In conclusion, a substantial amount of literature is available which demonstrates that the in-situ biodegradation of MTBE is contingent on achieving aerobic conditions in the contaminated aquifer.
Asunto(s)
Éteres Metílicos/metabolismo , Bacterias Aerobias/metabolismo , Biodegradación AmbientalRESUMEN
The kinetic properties of the excitatory amino acid transporter EAAT2 were studied using rapid applications of L-glutamate to outside-out patches excised from transfected human embryonic kidney 293 cells. In the presence of the highly permeant anion SCN(-), pulses of glutamate rapidly activated transient anion channel currents mediated by the transporter. In the presence of the impermeant anion gluconate, glutamate pulses activated smaller currents predicted to result from stoichiometric flux of cotransported ions. Both anion and stoichiometric currents displayed similar kinetics, suggesting that anion channel gating and stoichiometric charge movements are linked to early transitions in the transport cycle. Transporter-mediated anion currents were recorded with ion and glutamate gradients favoring either unidirectional influx or exchange. Analysis of deactivation and recovery kinetics in these two conditions suggests that, after binding, translocation of substrate is more likely than unbinding under physiological conditions. The kinetic properties of EAAT2, the dominant glutamate transporter in brain astrocytes, distinguish it as an efficient sink for synaptically released glutamate.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Astrocitos/fisiología , Ácido Glutámico/metabolismo , Receptores de Neurotransmisores/fisiología , Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Sistema de Transporte de Aminoácidos X-AG , Astrocitos/efectos de los fármacos , Línea Celular , Transportador 2 de Aminoácidos Excitadores , Ácido Glutámico/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Humanos , Bombas Iónicas/efectos de los fármacos , Bombas Iónicas/fisiología , Riñón/citología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Receptores de Neurotransmisores/efectos de los fármacos , Transfección/genéticaRESUMEN
Within the mammalian central nervous system, the efficient removal of L-glutamate from the extracellular space by excitatory amino acid transporters (EAATs) has been postulated to contribute to signal termination, the recycling of transmitter, and the maintenance of L-glutamate at concentrations below those that are excitotoxic. The development of potent and selective inhibitors of the EAATs has contributed greatly to the understanding of the functional roles of these transporters. In the present study, we use a library of conformationally constrained glutamate analogs to address two key issues: the differentiation of substrates from nontransportable inhibitors and the comparison of the pharmacological profile of synaptosomal uptake with those of the individual EAAT clones. We demonstrate that the process of transporter-mediated heteroexchange can be exploited in synaptosomes to rapidly distinguish transportable from nontransportable inhibitors. Using this approach, we demonstrate that 2,4-methanopyrrolidine-2,4-dicarboxylate, cis-1-aminocyclobutane-1,3-dicarboxylate, and L-trans-2, 4-pyrrolidine dicarboxylate act as substrates for the rat forebrain synaptosomal glutamate uptake system. In contrast, L-anti-endo-3, 4-methanopyrrolidine-3,4-dicarboxylate, L-trans-2,3-pyrrolidine dicarboxylate, and dihydrokainate proved to be competitive inhibitors of D-[(3)H]aspartate uptake that exhibited little or no activity as substrates. When these same compounds were characterized for substrate activity by recording currents in voltage-clamped Xenopus laevis oocytes expressing the human transporter clones EAAT1, EAAT2, or EAAT3, it was found that the pharmacological profile of the synaptosomal system exhibited the greatest similarity with the EAAT2 subtype, a transporter believed to be expressed primarily on glial cells.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG , Proteínas Portadoras/antagonistas & inhibidores , Ácido Glutámico/farmacología , Simportadores , Sinaptosomas/efectos de los fármacos , Animales , Ácido Aspártico/metabolismo , Unión Competitiva , Transporte Biológico , Transportador 1 de Aminoácidos Excitadores , Transportador 2 de Aminoácidos Excitadores , Transportador 3 de Aminoácidos Excitadores , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Ácido Glutámico/análogos & derivados , Técnicas In Vitro , Modelos Moleculares , Neuroglía/metabolismo , Oocitos/metabolismo , Prosencéfalo/metabolismo , Isoformas de Proteínas/metabolismo , Ratas , Receptores de Neurotransmisores/química , Receptores de Neurotransmisores/metabolismo , Sinaptosomas/metabolismo , Tritio , Xenopus laevisRESUMEN
The acidic amino acid L-glutamate acts as both a primary excitatory neurotransmitter and a potential neurotoxin within the mammalian central nervous system. Functionally juxtaposed between these neurophysiological and pathological actions are an assorted group of integral membrane transporter proteins that rapidly and efficiently sequester glutamate into cellular and subcellular compartments. While multiple systems exist that are capable of mediating the uptake of L-glutamate, the high-affinity, sodium-dependent transporters have emerged as the most prominent players in the CNS with respect to terminating the excitatory signal, recycling the transmitter, and regulating extracellular levels of glutamate below those which could induce excitotoxic pathology. The focus of the present review is on the pharmacological specificity of these sodium-dependent transporters and, more specifically, on the competitive inhibitors that have been used to delineate the chemical requirements for binding and translocation. Analogues of glutamate that are conformationally constrained as a consequence of either the addition of substituents to the carbon backbone of glutamate or aspartate (e.g., beta-hydroxyaspartate or methylglutamate derivatives) or the incorporation of ring systems (e.g., (carboxycyclopropyl)glycines, aminocyclobutane dicarboxylates, or pyrrolidine dicarboxylates), have been especially valuable in these efforts. In this review, a particular emphasis is placed on the identification of analogues that exhibit preferential activity among the recently cloned transporter subtypes and on the differentiation of substrates from non-transportable inhibitors.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Sistema Nervioso Central/metabolismo , Ácido Glutámico/análogos & derivados , Sodio/metabolismo , Sistema de Transporte de Aminoácidos X-AG , Animales , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacología , Unión Competitiva , Transporte Biológico , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Humanos , Neurotransmisores/metabolismoRESUMEN
Like the CCR5 chemokine receptors of humans and rhesus macaques, the very homologous (approximately 98-99% identical) CCR5 of African green monkeys (AGMs) avidly binds beta-chemokines and functions as a coreceptor for simian immunodeficiency viruses. However, AGM CCR5 is a weak coreceptor for tested macrophage-tropic (R5) isolates of human immunodeficiency virus type 1 (HIV-1). Correspondingly, gp120 envelope glycoproteins derived from R5 isolates of HIV-1 bind poorly to AGM CCR5. We focused on a unique extracellular amino acid substitution at the juncture of transmembrane helix 4 (TM4) and extracellular loop 2 (ECL2) (Arg for Gly at amino acid 163 (G163R)) as the likely source of the weak R5 gp120 binding and HIV-1 coreceptor properties of AGM CCR5. Accordingly, a G163R mutant of human CCR5 was severely attenuated in its ability to bind R5 gp120s and to mediate infection by R5 HIV-1 isolates. Conversely, the R163G mutant of AGM CCR5 was substantially strengthened as a coreceptor for HIV-1 and had improved R5 gp120 binding affinity relative to the wild-type AGM CCR5. These substitutions at amino acid position 163 had no effect on chemokine binding or signal transduction, suggesting the absence of structural alterations. The 2D7 monoclonal antibody has been reported to bind to ECL2 and to block HIV-1 binding and infection. Whereas 2D7 antibody binding to CCR5 was unaffected by the G163R mutation, it was prevented by a conservative ECL2 substitution (K171R), shared between rhesus and AGM CCR5s. Thus, it appears that the 2D7 antibody binds to an epitope that includes Lys-171 and may block HIV-1 infection mediated by CCR5 by occluding an HIV-1-binding site in the vicinity of Gly-163. In summary, our results identify a site for gp120 interaction that is critical for R5 isolates of HIV-1 in the central core of human CCR5, and we propose that this site collaborates with a previously identified region in the CCR5 amino terminus to enable gp120 binding and HIV-1 infections.
Asunto(s)
Glicina/metabolismo , VIH-1/fisiología , Fusión de Membrana/fisiología , Receptores CCR5/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Sitios de Unión de Anticuerpos , Unión Competitiva , Línea Celular , Chlorocebus aethiops , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/patogenicidad , Humanos , Macaca mulatta , Datos de Secuencia Molecular , Receptores CCR5/química , Receptores CCR5/inmunología , Homología de Secuencia de Aminoácido , Transducción de Señal , Especificidad de la EspecieRESUMEN
Glutamate transport is a primary mechanism for the synaptic inactivation of glutamate. Excitatory amino acid transporter 4 (EAAT4) is a novel glutamate transporter with properties of a ligand-gated chloride channel that was recently cloned from human brain. Here we report the cloning of rat EAAT4 (rEAAT4) cDNA from rat cerebellum. The nucleotide sequence of rEAAT4 was 88% identical to the human sequence, and the predicted peptide was 89% identical to the human protein. The transport activity encoded by rEAAT4 has high affinity for L-glutamate. In Xenopus laevis oocytes expressing rEAAT4, L-glutamate and other transporter substrates elicited a current predominantly carried by chloride ions. Like human EAAT4, the rEAAT4 mRNA was largely restricted to cerebellar Purkinje cells; the rEAAT4 protein was localized to Purkinje cell somas and dendrites.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG , Canales de Cloruro/genética , Células de Purkinje/química , Células de Purkinje/fisiología , Receptores de Glutamato/genética , Simportadores , Animales , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Clonación Molecular , ADN Complementario , Estimulación Eléctrica , Electrofisiología , Transportador 4 de Aminoácidos Excitadores , Expresión Génica/fisiología , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Ácido Glutámico/farmacología , Activación del Canal Iónico/efectos de los fármacos , Ligandos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Oocitos/fisiología , Ratas , Receptores de Glutamato/metabolismo , Homología de Secuencia de Aminoácido , Xenopus laevisAsunto(s)
Sistema de Transporte de Aminoácidos X-AG , Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Oocitos/fisiología , Canales de Potasio/química , Canales de Potasio/fisiología , Conformación Proteica , Simportadores , Animales , Proteínas Portadoras/genética , Membrana Celular/fisiología , Clonación Molecular/métodos , Femenino , Fluoresceínas , Colorantes Fluorescentes , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Glutamatos/metabolismo , Humanos , Cinética , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Canales de Potasio/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Canales de Potasio de la Superfamilia Shaker , Espectrometría de Fluorescencia/instrumentación , Espectrometría de Fluorescencia/métodos , Xenopus laevisRESUMEN
The behavior of a Cl- channel associated with a glutamate transporter was studied using intracellular and patch recording techniques in Xenopus oocytes injected with human EAAT1 cRNA. Channels could be activated by application of glutamate to either face of excised membrane patches. The channel exhibited strong selectivity for amphipathic anions and had a minimum pore diameter of approximately 5A. Glutamate flux exhibited a much greater temperature dependence than Cl- flux. Stationary and nonstationary noise analysis was consistent with a sub-femtosiemen Cl- conductance and a maximum channel Po << 1. The glutamate binding rate was similar to estimates for receptor binding. After glutamate binding, channels activated rapidly followed by a relaxation phase. Differences in the macroscopic kinetics of channels activated by concentration jumps of L-glutamate or D-aspartate were correlated with differences in uptake kinetics, indicating a close correspondence of channel gating to state transitions in the transporter cycle.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Sistema de Transporte de Aminoácidos X-AG , Animales , Aniones/farmacocinética , Ácido Aspártico/farmacología , Transporte Biológico/genética , Química Encefálica/fisiología , Clonación Molecular , Conductividad Eléctrica , Ácido Glutámico/farmacocinética , Humanos , Activación del Canal Iónico/fisiología , Cinética , Potenciales de la Membrana/fisiología , Oocitos/fisiología , Técnicas de Placa-Clamp , Potasio/farmacocinética , Protones , Sodio/farmacocinética , XenopusRESUMEN
Signal transductions by the dual-function CXCR4 and CCR5 chemokine receptors/HIV type 1 (HIV-1) coreceptors were electrophysiologically monitored in Xenopus laevis oocytes that also coexpressed the viral receptor CD4 and a G protein-coupled inward-rectifying K+ channel (Kir 3.1). Large Kir 3.1-dependent currents generated in response to the corresponding chemokines (SDF-1alpha for CXCR4 and MIP-1alpha; MIP-1beta and RANTES for CCR5) were blocked by pertussis toxin, suggesting involvement of inhibitory guanine nucleotide-binding proteins. Prolonged exposures to chemokines caused substantial but incomplete desensitization of responses with time constants of 5-7 min and recovery time constants of 12-19 min. CXCR4 and CCR5 exhibited heterologous desensitization in this oocyte system, suggesting possible inhibition of a common downstream step in their signaling pathways. In contrast to chemokines, perfusion with monomeric or oligomeric preparations of the glycoprotein of Mr 120, 000 (gp120) derived from several isolates of HIV-1 did not activate signaling by CXCR4 or CCR5 regardless of CD4 coexpression. However, adsorption of the gp120 from a T-cell-tropic virus resulted in CD4-dependent antagonism of CXCR4 response to SDF-1alpha, whereas gp120 from macrophage-tropic viruses caused CD4-dependent antagonism of CCR5 response to MIP-1alpha. These antagonisms could be partially overcome by high concentrations of chemokines and were specific for coreceptors of the corresponding HIV-1 isolates, suggesting that they resulted from direct interactions of gp120-CD4 complexes with coreceptors and that they did not involve the desensitization pathway. These results indicate that monomeric or oligomeric gp120s specifically antagonize CXCR4 and CCR5 signaling in response to chemokines, but they do not exclude the possibility that gp120s might also function as weak agonists in some cells. The gp120-mediated disruption of CXCR4 and CCR5 signaling may contribute to AIDS pathogenesis.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/farmacología , Receptores CCR5/fisiología , Receptores CXCR4/fisiología , Transducción de Señal/efectos de los fármacos , Animales , Antígenos CD4/fisiología , Quimiocinas/farmacología , Electrofisiología , Femenino , Humanos , Transducción de Señal/fisiología , Xenopus laevisRESUMEN
Glutamate is the major excitatory neurotransmitter in the vertebrate retina. Native glutamate transporters have been well characterized in several retinal neurons, particularly from the salamander retina. We have cloned five distinct glutamate transporters from the salamander retina and examined their localization and functional properties: sEAAT1, sEEAAT2A, sEAAT2B, sEAAT5A and sEAAT5B. sEAAT1 is a homologue of the glutamate transporter EAAT1 (GLAST), sEAAT2A and sEAAT2B are homologues of EAAT2 (GLT-1) and sEAAT5A and sEAAT5B are homologues of the recently cloned human retinal glutamate transporter EAAT5. Localization was determined by immunocytochemical techniques using antibodies directed at portions of the highly divergent carboxy terminal. Glutamate transporters were found in glial, photoreceptor, bipolar, amacrine and ganglion cells. The pharmacology and ionic dependence were determined by two-electrode voltage clamp recordings from Xenopus laevis oocytes which had previously been injected with one of the glutamate transporter mRNAs. Each of the transporters behaved in a manner consistent with a glutamate transporter and there were some distinguishing characteristics which make it possible to link the function in native cells with the behavior of the cloned transporters in this study.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/análisis , Glutamatos/análisis , Retina/química , Secuencia de Aminoácidos , Sistema de Transporte de Aminoácidos X-AG , Animales , Transporte Biológico Activo , Proteínas Portadoras/análisis , Proteínas Portadoras/química , Clonación Molecular , Aminoácidos Excitadores/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Glutamatos/metabolismo , Potenciales de la Membrana , Oocitos/fisiología , Urodelos , XenopusRESUMEN
The excitatory amino acid transporter EAAT4 is expressed predominantly in Purkinje neurons in the rat cerebellum (1-3), and it participates in postsynaptic reuptake of glutamate released at the climbing fiber synapse (4). Transporter-mediated currents in Purkinje neurons are increased more than 3-fold by arachidonic acid, a second messenger that is liberated following depolarization-induced Ca2+ activation of phospholipase A2 (5). In this study we demonstrate that application of arachidonic acid to oocytes expressing rat EAAT4 increased glutamate-induced currents to a similar extent. However, arachidonic acid did not cause an increase in the rate of glutamate transport or in the chloride current associated with glutamate transport but rather activated a proton-selective conductance. These data reveal a novel action of arachidonate on a glutamate transporter and suggest a mechanism by which synaptic activity may decrease intracellular pH in neurons where this transporter is localized.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG , Ácido Araquidónico/farmacología , Protones , Células de Purkinje/efectos de los fármacos , Receptores de Glutamato/metabolismo , Simportadores , Animales , Transportador 4 de Aminoácidos Excitadores , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Células de Purkinje/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , XenopusRESUMEN
Glutamate transporters remove this transmitter from the extracellular space by cotransport with three sodium ions and a proton. The cycle is completed by translocation of a potassium ion in the opposite direction. Recently we have identified two adjacent amino acid residues of the glutamate transporter GLT-1 that influence potassium coupling. Using the scanning cysteine accessibility method we have now explored the highly conserved region surrounding them. Replacement of each of the five consecutive residues 396-400 by cysteine abolished transport activity but at several other positions the substitution is tolerated. One residue, tyrosine 403, was identified where cysteine substitution renders the transporter sensitive to modification by positively charged methanethiosulfonate derivates in a sodium-protectable fashion. In the presence of sodium, the nontransported glutamate analogue dihydrokainate potentiated the covalent modification, presumably by binding to the glutamate site and locking the protein in a conformation in which tyrosine 403 is accessible from the external bulk medium. In contrast, transported substrates significantly slowed the reaction, suggesting that during the transport cycle residue 403 becomes occluded. On the other hand, transportable substrates are not able to protect Y403C transporters against N-ethylmaleimide, which is highly permeant but unable to modify cysteine residues buried within membrane proteins. These results indicate that tyrosine 403 is alternately accessible from either side of the membrane, consistent with its role as structural determinant of the potassium binding site.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Potasio/metabolismo , Conformación Proteica , Transportadoras de Casetes de Unión a ATP/genética , Sistema de Transporte de Aminoácidos X-AG , Aminoácidos/farmacología , Ácido Aspártico/farmacocinética , Sitios de Unión/genética , Transporte Biológico/fisiología , Secuencia Conservada/fisiología , Cisteína/metabolismo , Etilmaleimida/farmacología , Células HeLa , Humanos , Ácido Kaínico/análogos & derivados , Ácido Kaínico/farmacología , Mesilatos/farmacología , Mutagénesis Sitio-Dirigida , Sodio/farmacología , Reactivos de Sulfhidrilo/farmacologíaRESUMEN
The rapid re-uptake of extracellular glutamate mediated by a family of high-affinity glutamate transporter proteins is essential to continued glutamatergic signaling and neuronal viability, but the contributions of individual transporter subtypes toward cellular physiology are poorly understood. Because the physiology of glutamate transport in the salamander retina has been well described, we have examined the expression and function of glutamate transporter subtypes in this preparation. cDNAs encoding five distinct salamander excitatory amino acid transporter (sEAAT) subtypes were isolated, and their molecular properties and distributions of expression were compared. We report evidence that at least four distinct sEAAT subtypes are expressed in glial (Müller) cells. In addition, four of the five transporter subtypes are localized in neurons throughout the retina. The brightest immunostaining was seen in the synaptic regions of the inner and outer plexiform layers and in the outer nuclear layer. Using electrophysiological measurements in the Xenopus oocyte expression system, we also examined the pharmacology and ionic dependence of the four expressing transporter subtypes that make it possible to distinguish, on the basis of functional behavior, among the various subtypes. Although no simple correlation between transporter subtype and retinal cell physiology can be made, the diverse population of sEAAT transporter subtypes with unique localization and functional properties indicates that glutamate transporters play a wide variety of roles in retinal function and are likely to underlie both the uptake of glutamate by Müller cells and the glutamate-elicited chloride conductance involved in signal transduction by photoreceptors and bipolar cells.