Modulation of the neuronal glutamate transporter EAAC1 by the interacting protein GTRAP3-18

Excitatory amino-acid carrier 1 (EAAC1) is a high-affinity Na+-dependent L-glutamate/D,L-aspartate cell-membrane transport protein. It is expressed in brain as well as several non-nervous tissues. In brain, EAAC1 is the primary neuronal glutamate transporter. It has a polarized distribution in cells and mainly functions perisynaptically to transport glutamate from the extracellular environment. In the kidney it is involved in renal acidic amino-acid re-absorption and amino-acid metabolism. Here we describe the identification and characterization of an EAAC1-associated protein, GTRAP3-18. Like EAAC1, GTRAP3-18 is expressed in numerous tissues. It localizes to the cell membrane and cytoplasm, and specifically interacts with carboxy-terminal intracellular domain of EAAC1. Increasing the expression of GTRAP3-18 in cells reduces EAAC1-mediated glutamate transport by lowering substrate affinity. The expression of GTRAP3-18 can be upregulated by retinoic acid, which results in a specific reduction of EAAC1-mediated glutamate transport. These studies show that glutamate transport proteins can be regulated potently and that GTRAP can modulate the transport functions ascribed to EAAC1. GTRAP3-18 may be important in regulating the metabolic function of EAAC1.     

高氧对新生小鼠视网膜谷氨酸释放的影响

为研究新生小鼠高氧状态下视网膜谷氨酸摩尔质量浓度的变化及其机制,探讨谷氨酸在高氧视网膜损伤中的作用,实验组将新生小鼠置于体积分数为95%的高氧环境中造成视网膜损伤,对照组将新生小鼠置于正常空气中,HE染色观察视网膜损伤情况,多功能酶标仪检测视网膜谷氨酸(Glu)摩尔质量浓度变化,逆转录聚合酶链反应(RT-PCR)检测视网膜谷氨酸转运体(GLAST)与谷氨酰胺合成酶(GS)mRNA表达情况.结果发现,与空气对照组比较,高氧处理组视网膜受损,视网膜Glu摩尔质量浓度增加,GLAST和GS mRNA表达下降（P<0.05）.高氧可导致新生小鼠视网膜的谷氨酸增高,其机制与GLAST和GS mRNA表达下降有关.谷氨酸增加可能是高氧导致视网膜损伤的原因之一.     

跑台运动通过增强星形胶质细胞Glu的摄取转运功能改善PD模型大鼠运动功能

本文选取雄性SD大鼠,随机分为4组:假手术组(Control)、假手术运动组(Control+Ex)、PD组(PD)和PD运动组(PD+Ex).于大鼠内侧前脑束注射6-羟基多巴(6-OHDA)建立单侧损伤PD模型,术后24h实施运动干预.在手术后第1周、第2周和第4周皮下注射阿朴吗啡(APO)评价PD模型可靠性.4周训练结束后进行圆筒与网格行为学测试,并利用高效液相色谱技术(HPLC)检测纹状体Glu浓度;采用免疫组织化学技术观察纹状体GFAP和GLT-1的表达水平.APO旋转行为测试和圆筒及网格行为测试结果显示,PD+Ex组大鼠行为功能较PD组显著改善(P0.05).PD+Ex组大鼠较PD组纹状体Glu浓度下降(P0.01),GLT-1表达显著上调,而GFAP的表达显著下调(P0.05).4周跑台运动干预可加速星形胶质细胞对Glu的摄取转运能力,降低纹状体Glu浓度,改善PD模型大鼠行为功能障碍.推测运动促进PD模型大鼠皮层-纹状体Glu能通路的突触可塑性可能也与星形胶质细胞对Glu的摄取转运功能有关.     

Structure of a glutamate transporter homologue from Pyrococcus horikoshii

Glutamate transporters are integral membrane proteins that catalyse the concentrative uptake of glutamate from the synapse to intracellular spaces by harnessing pre-existing ion gradients. In the central nervous system glutamate transporters are essential for normal development and function, and are implicated in stroke, epilepsy and neurodegenerative diseases. Here we present the crystal structure of a eukaryotic glutamate transporter homologue from Pyrococcus horikoshii. The transporter is a bowl-shaped trimer with a solvent-filled extracellular basin extending halfway across the membrane bilayer. At the bottom of the basin are three independent binding sites, each cradled by two helical hairpins, reaching from opposite sides of the membrane. We propose that transport of glutamate is achieved by movements of the hairpins that allow alternating access to either side of the membrane.     

Structure and mechanism of the S component of a bacterial ECF transporter

The energy-coupling factor (ECF) transporters, responsible for vitamin uptake in prokaryotes, are a unique family of membrane transporters. Each ECF transporter contains a membrane-embedded, substrate-binding protein (known as the S component), an energy-coupling module that comprises two ATP-binding proteins (known as the A and A' components) and a transmembrane protein (known as the T component). The structure and transport mechanism of the ECF family remain unknown. Here we report the crystal structure of RibU, the S component of the ECF-type riboflavin transporter from Staphylococcus aureus at 3.6-? resolution. RibU contains six transmembrane segments, adopts a previously unreported transporter fold and contains a riboflavin molecule bound to the L1 loop and the periplasmic portion of transmembrane segments 4-6. Structural analysis reveals the essential ligand-binding residues, identifies the putative transport path and, with sequence alignment, uncovers conserved structural features and suggests potential mechanisms of action among the ECF transporters.     

A monosaccharide transporter is localized to sieve plate and plasmodesmal channel in developing apple fruit

Two major classes of plant sugar transporters, sucrose and monosaccharide transporters, may be localized to tonoplast or plasma membrane. The monosaccharide transporters may also be localized in plastid. However, whether these transporters reside in other subcellular compartments remains unclear. We recently detected in apple fruit a 52 kD plasma membrane-localized monosaccharide transporter, and showed that this transporter may be functional in phloem unloading in the fruit. In this paper, we report that this monosaccharide transporter is also localized to sieve plate and plasmodesmal channel in apple fruit. The amount of this sieve plate- and plasmodesma-associated transporter changes during fruit development. This amount of the transporter expression may be altered in the phloem sieve elements but not in the parenchyma cells by a photoassimilate deficiency applied by the shoot girdling treatment, suggesting that the monosaccharide transporter of the special sub-cellular localization may be of biological significance.     

Mechanism of chloride interaction with neurotransmitter:sodium symporters

Neurotransmitter:sodium symporters (NSS) have a critical role in regulating neurotransmission and are targets for psychostimulants, anti-depressants and other drugs. Whereas the non-homologous glutamate transporters mediate chloride conductance, in the eukaryotic NSS chloride is transported together with the neurotransmitter. In contrast, transport by the bacterial NSS family members LeuT, Tyt1 and TnaT is chloride independent. The crystal structure of LeuT reveals an occluded binding pocket containing leucine and two sodium ions, and is highly relevant for the neurotransmitter transporters. However, the precise role of chloride in neurotransmitter transport and the location of its binding site remain elusive. Here we show that introduction of a negatively charged amino acid at or near one of the two putative sodium-binding sites of the GABA (gamma-aminobutyric acid) transporter GAT-1 from rat brain (also called SLC6A1) renders both net flux and exchange of GABA largely chloride independent. In contrast to wild-type GAT-1, a marked stimulation of the rate of net flux, but not of exchange, was observed when the internal pH was lowered. Equivalent mutations introduced in the mouse GABA transporter GAT4 (SLC6A11) and the human dopamine transporter DAT (SLC6A3) also result in chloride-independent transport, whereas the reciprocal mutations in LeuT and Tyt1 render substrate binding and/or uptake by these bacterial NSS chloride dependent. Our data indicate that the negative charge, provided either by chloride or by the transporter itself, is required during binding and translocation of the neurotransmitter, probably to counterbalance the charge of the co-transported sodium ions.     

Expression cloning and cDNA sequencing of the Na+/glucose co-transporter

Organic substrates (sugars, amino acids, carboxylic acids and neutrotransmitters) are actively transported into eukaryotic cells by Na+ co-transport. Some of the transport proteins have been identified--for example, intestinal brush border Na+/glucose and Na+/proline transporters and the brain Na+/CI-/GABA transporter--and progress has been made in locating their active sites and probing their conformational states. The archetypical Na+-driven transporter is the intestinal brush border Na+/glucose co-transporter (see ref. 8), and a defect in the co-transporter is the origin of the congenital glucose-galactose malabsorption syndrome. Here we describe cloning of this co-transporter by a method new to membrane proteins. We have sequenced the cloned DNA and have found no homology between the Na+/glucose co-transporter and either the mammalian facilitated glucose carrier or the bacterial sugar transport proteins. This suggests that the mammalian Na+-driven transporter has no evolutionary relationship to the other sugar transporters.     

Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4

Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns, and members of the pro-inflammatory interleukin-1 receptor (IL-1R) family, share homologies in their cytoplasmic domains called Toll/IL-1R/plant R gene homology (TIR) domains. Intracellular signalling mechanisms mediated by TIRs are similar, with MyD88 (refs 5-8) and TRAF6 (refs 9, 10) having critical roles. Signal transduction between MyD88 and TRAF6 is known to involve the serine-threonine kinase IL-1 receptor-associated kinase 1 (IRAK-1) and two homologous proteins, IRAK-2 (ref. 12) and IRAK-M. However, the physiological functions of the IRAK molecules remain unclear, and gene-targeting studies have shown that IRAK-1 is only partially required for IL-1R and TLR signalling. Here we show by gene-targeting that IRAK-4, an IRAK molecule closely related to the Drosophila Pelle protein, is indispensable for the responses of animals and cultured cells to IL-1 and ligands that stimulate various TLRs. IRAK-4-deficient animals are completely resistant to a lethal dose of lipopolysaccharide (LPS). In addition, animals lacking IRAK-4 are severely impaired in their responses to viral and bacterial challenges. Our results indicate that IRAK-4 has an essential role in innate immunity.     

pH-sensitive activation of the intracellular-pH regulation system in squid axons by ATP-gamma-S

The regulation of intracellular pH (pHi) is essential for normal cell function, and controlled changes in pHi may play a central role in cell activation. Sodium-dependent Cl-HCO3 exchange is the dominant mechanism of pHi regulation in the invertebrate cells examined, and also occurs in mammalian cells. The transporter extrudes acid from the cell by exchanging extracellular Na+ and HCO3- (ref. 9) (or a related species) for intracellular Cl- (refs 3, 4). It is blocked by the stilbene derivatives DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulphonate, ref. 10) and SITS (4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonate, ref. 3), and has a stoichiometry of two intracellular H+ neutralized for each Na+ taken up and each Cl- extruded by the axon. Because the inwardly-directed Na+ concentration gradient is sufficiently large to energize both the HCO3- influx and Cl- efflux, this electroneutral exchanger could be a classic secondary active transporter, thermodynamically independent of ATP hydrolysis. However, at least in the squid axon, the exchanger has an absolute requirement for ATP (ref. 3). Thus, a major unresolved issue is whether this Na-dependent Cl-HCO3 exchanger stoichiometrically hydrolyses ATP (the pump hypothesis), or whether ATP activates the transporter by a mechanism such as phosphorylation or simple binding (the activation hypothesis). We have now explored the role of ATP in pHi regulation by dialysing axons with the ATP analogue ATP-gamma-S. In many systems, ATP-gamma-S is an acceptable substrate for protein kinases, whereas the resulting thiophosphorylated proteins are not as readily hydrolysed by phosphatases as are phosphorylated proteins. Our results rule out the pump hypothesis, and show that the basis of the axon's ATP requirement is the pH-dependent activation (by, for instance, phosphorylation or ATP binding) of the exchanger itself, or of an essential activator.     

Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Synaptic vesicles are loaded with neurotransmitter by means of specific vesicular transporters. Here we show that expression of BNPI, a vesicle-bound transporter associated with sodium-dependent phosphate transport, results in glutamate uptake by intracellular vesicles. Substrate specificity and energy dependence are very similar to glutamate uptake by synaptic vesicles. Stimulation of exocytosis--fusion of the vesicles with the cell membrane and release of their contents--resulted in quantal release of glutamate from BNPI-expressing cells. Furthermore, we expressed BNPI in neurons containing GABA (gamma-aminobutyric acid) and maintained them as cultures of single, isolated neurons that form synapses to themselves. After stimulation of these neurons, a component of the postsynaptic current is mediated by glutamate as it is blocked by a combination of the glutamate receptor antagonists, but is insensitive to a GABA(A) receptor antagonist. We conclude that BNPI functions as vesicular glutamate transporter and that expression of BNPI suffices to define a glutamatergic phenotype in neurons.     

Segregation of pathways leading from area V2 to areas V4 and V5 of macaque monkey visual cortex

V5 and V4 are areas of macaque monkey prestriate visual cortex that are specialized for involvement in different aspects of visual perception, namely motion for V5 (refs 1-4) and colour vision, with other possible functions, for V4 (refs 2, 5-9). Thus, it is unlikely that they should be fed the same information for further processing, yet both receive a strong input from patches of the upper layers of V2 (refs 10, 11), the area immediately adjoining the primary visual cortex, V1. V2, however, seems to comprise functionally distinct subregions, which can be revealed by staining the tissue for the mitochondrial enzyme cytochrome oxidase. Here we report that V4 and V5 are connected with separate cytochrome oxidase-defined subregions of V2, suggesting that cortical pathways dealing with motion and colour perception are segregated in their passage through V2, and reinforcing evidence for functional specialization in the visual cortex.     

Location of MHC-encoded transporters in the endoplasmic reticulum and cis-Golgi.

Immune recognition of intracellular proteins is mediated by major histocompatibility complex (MHC) class I molecules that present short peptides to cytotoxic T cells. Evidence suggests that peptides arise by cleavage of proteins in the cytoplasm and are transported by a signal-independent mechanism into a pre-Golgi region of the cell, where they take part in the assembly of class I heavy chains with beta 2-microglobulin (reviewed in refs 5-7). Analysis of cells that have defects in class I molecule assembly and antigen presentation has shown that this phenotype can result from mutations in either of the two ABC transporter genes located in the class II region of the MHC. This suggested that the protein complex encoded by these two genes transports peptides from the cytosol into the endoplasmic reticulum. Here we report additional evidence by showing that the transporter complex is located in the endoplasmic reticulum membrane and is probably oriented with its ATP-binding domains in the cytosol.     

Cyclic nucleotides control a system which regulates Ca2+ sensitivity of platelet secretion

Cellular responses to extracellular signals are mediated by changes in the intracellular concentrations of one or more second messengers. In platelets, inhibitory agonists increase intracellular cyclic-3',5'-AMP [( cyclic AMP]i (refs 2, 3] whereas excitatory agonists increase [Ca2+]i and/or [1,2-diacylglycerol]i (refs 4-9), and in some cases decrease [cyclic AMP]i (refs 10, 11). Both activation and inhibition of platelet responses have been attributed to an increase in [cyclic-3',5'-GMP]i (refs 8, 12). The activity of protein kinase C, which is associated with the platelet secretory response, is increased by both 1,2-diacylglycerol and Ca2+ (refs 4, 7, 8). The role of cyclic AMP may involve either inhibition of Ca2+ mobilization to the cytosol or stimulation of intracellular Ca2+ uptake, and in addition inhibition of 1,2-diacylglycerol formation. The relationship between cyclic-3',5'-GMP (cyclic GMP) and other second messengers in platelet activation has not been defined. Using platelets made permeable by exposure to an intense electric field, we demonstrate here modulation of the Ca2+ sensitivity of platelet secretion by thrombin, and by 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleyl-2- acetylglycerol ( OAG ), both potent activators of protein kinase C. The effect of thrombin is selectively modified by cyclic GMP and cyclic AMP. The response to OAG and TPA is also modulated by cyclic AMP but to a much lesser extent.     

Cell-specific ATP7A transport sustains copper-dependent tyrosinase activity in melanosomes

Copper is a cofactor for many cellular enzymes and transporters. It can be loaded onto secreted and endomembrane cuproproteins by translocation from the cytosol into membrane-bound organelles by ATP7A or ATP7B transporters, the genes for which are mutated in the copper imbalance syndromes Menkes disease and Wilson disease, respectively. Endomembrane cuproproteins are thought to incorporate copper stably on transit through the trans-Golgi network, in which ATP7A accumulates by dynamic cycling through early endocytic compartments. Here we show that the pigment-cell-specific cuproenzyme tyrosinase acquires copper only transiently and inefficiently within the trans-Golgi network of mouse melanocytes. To catalyse melanin synthesis, tyrosinase is subsequently reloaded with copper within specialized organelles called melanosomes. Copper is supplied to melanosomes by ATP7A, a cohort of which localizes to melanosomes in a biogenesis of lysosome-related organelles complex-1 (BLOC-1)-dependent manner. These results indicate that cell-type-specific localization of a metal transporter is required to sustain metallation of an endomembrane cuproenzyme, providing a mechanism for exquisite spatial control of metalloenzyme activity. Moreover, because BLOC-1 subunits are mutated in subtypes of the genetic disease Hermansky-Pudlak syndrome, these results also show that defects in copper transporter localization contribute to hypopigmentation, and hence perhaps other systemic defects, in Hermansky-Pudlak syndrome.     

Glutamate release in severe brain ischaemia is mainly by reversed uptake

The release of glutamate during brain anoxia or ischaemia triggers the death of neurons, causing mental or physical handicap. The mechanism of glutamate release is controversial, however. Four release mechanisms have been postulated: vesicular release dependent on external calcium or Ca2+ released from intracellular stores; release through swelling-activated anion channels; an indomethacin-sensitive process in astrocytes; and reversed operation of glutamate transporters. Here we have mimicked severe ischaemia in hippocampal slices and monitored glutamate release as a receptor-gated current in the CA1 pyramidal cells that are killed preferentially in ischaemic hippocampus. Using blockers of the different release mechanisms, we demonstrate that glutamate release is largely by reversed operation of neuronal glutamate transporters, and that it plays a key role in generating the anoxic depolarization that abolishes information processing in the central nervous system a few minutes after the start of ischaemia. A mathematical model incorporating K+ channels, reversible uptake carriers and NMDA (N-methyl-D-aspartate) receptor channels reproduces the main features of the response to ischaemia. Thus, transporter-mediated glutamate homeostasis fails dramatically in ischaemia: instead of removing extracellular glutamate to protect neurons, transporters release glutamate, triggering neuronal death.