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.     

Modulation of the neuronal glutamate transporter EAAT4 by two interacting proteins

Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system and is removed from the synaptic cleft by sodium-dependent glutamate transporters. To date, five distinct glutamate transporters have been cloned from animal and human tissue: GLAST (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5 (refs 1-5). GLAST and GLT-1 are localized primarily in astrocytes, whereas EAAC1 (refs 8, 9), EAAT4 (refs 9-11) and EAAT5 (ref 5) are neuronal. Studies of EAAT4 and EAAC1 indicate an extrasynaptic localization on perisynaptic membranes that are near release sites. This localization facilitates rapid glutamate binding, and may have a role in shaping the amplitude of postsynaptic responses in densely packed cerebellar terminals. We have used a yeast two-hybrid screen to identify interacting proteins that may be involved in regulating EAAT4--the glutamate transporter expressed predominately in the cerebellum--or in targeting and/or anchoring or clustering the transporter to the target site. Here we report the identification and characterization of two proteins, GTRAP41 and GTRAP48 (for glutamate transporter EAAT4 associated protein) that specifically interact with the intracellular carboxy-terminal domain of EAAT4 and modulate its glutamate transport activity.     

Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein

At least three different glucose transport systems exist in mammalian cells. These are: (1) the constitutively active, facilitative carrier characteristic of human erythrocytes, Hep G2 (ref. 2) cells and rat brain; (2) the Na-dependent active transporter of kidney and small intestine; and (3) the facilitative carrier of rat liver (B. Thorens and H. F. Lodish, personal communication). A fourth possible glucose transport system is the insulin-dependent carrier that may be specific to muscle and adipose tissue. This transporter resides primarily in an intracellular compartment in resting cells from where it translocates to the cell surface upon cellular insulin exposure. This raises the question of whether hormonal regulation of glucose transport is conferred by virtue of a tissue-specific signalling mechanism or a tissue-specific glucose transporter. Here we present data supporting the latter concept based upon a monoclonal antibody against the fat cell glucose transporter that identifies a unique, insulin-regulatable glucose transport protein in muscle and adipose tissue.     

A family of mammalian Na+-dependent L-ascorbic acid transporters.

Vitamin C (L-ascorbic acid) is essential for many enzymatic reactions, in which it serves to maintain prosthetic metal ions in their reduced forms (for example, Fe2+, Cu+), and for scavenging free radicals in order to protect tissues from oxidative damage. The facilitative sugar transporters of the GLUT type can transport the oxidized form of the vitamin, dehydroascorbic acid, but these transporters are unlikely to allow significant physiological amounts of vitamin C to be taken up in the presence of normal glucose concentrations, because the vitamin is present in plasma essentially only in its reduced form. Here we describe the isolation of two L-ascorbic acid transporters, SVCT1 and SVCT2, from rat complementary DNA libraries, as the first step in investigating the importance of L-ascorbic acid transport in regulating the supply and metabolism of vitamin C. We find that SVCT1 and SVCT2 each mediate concentrative, high-affinity L-ascorbic acid transport that is stereospecific and is driven by the Na+ electrochemical gradient. Despite their close sequence homology and similar functions, the two isoforms of the transporter are discretely distributed: SVCT1 is mainly confined to epithelial systems (intestine, kidney, liver), whereas SVCT2 serves a host of metabolically active cells and specialized tissues in the brain, eye and other organs.     

Expression cloning of a cocaine- and antidepressant-sensitive human noradrenaline transporter

At most synapses, chemical signalling is terminated by a rapid reaccumulation of neurotransmitter into presynaptic terminals. Uptake systems for the biogenic amines are the initial site of action for therapeutic antidepressants and drugs such as cocaine and the amphetamines. We have isolated a complementary DNA clone encoding a human noradrenaline transporter. The cDNA sequence predicts a protein of 617 amino acids, with 12-13 highly hydrophobic regions compatible with membrane-spanning domains. Expression of the cDNA clone in transfected HeLa cells indicates that noradrenaline transport activity is sodium-dependent and sensitive to selective noradrenaline transport inhibitors. Transporter RNA is localized to the brainstem and the adrenal gland. The predicted protein sequence demonstrates significant amino-acid identity with the Na+/gamma-aminobutyric acid transporter, thus identifying a new gene family for neurotransmitter transporter proteins. Analysis of its structure and function may lead to structure-based drug design for the treatment of human depression and could help determine whether transporter abnormalities underlie affective disorders.     

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.     

The Rab8 GTPase regulates apical protein localization in intestinal cells

A number of proteins are known to be involved in apical/basolateral transport of proteins in polarized epithelial cells. The small GTP-binding protein Rab8 was thought to regulate basolateral transport in polarized kidney epithelial cells through the AP1B-complex-mediated pathway. However, the role of Rab8 (Rab8A) in cell polarity in vivo remains unknown. Here we show that Rab8 is responsible for the localization of apical proteins in intestinal epithelial cells. We found that apical peptidases and transporters localized to lysosomes in the small intestine of Rab8-deficient mice. Their mislocalization and degradation in lysosomes led to a marked reduction in the absorption rate of nutrients in the small intestine, and ultimately to death. Ultrastructurally, a shortening of apical microvilli, an increased number of enlarged lysosomes, and microvillus inclusions in the enterocytes were also observed. One microvillus inclusion disease patient who shows an identical phenotype to Rab8-deficient mice expresses a reduced amount of RAB8 (RAB8A; NM_005370). Our results demonstrate that Rab8 is necessary for the proper localization of apical proteins and the absorption and digestion of various nutrients in the small intestine.     

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.     

Functional analysis of an archaebacterial voltage-dependent K+ channel

All living organisms use ion channels to regulate the transport of ions across cellular membranes. Certain ion channels are classed as voltage-dependent because they have a voltage-sensing structure that induces their pores to open in response to changes in the cell membrane voltage. Until recently, the voltage-dependent K+, Ca2+ and Na+ channels were regarded as a unique development of eukaryotic cells, adapted to accomplish specialized electrical signalling, as exemplified in neurons. Here we present the functional characterization of a voltage-dependent K+ (K(V)) channel from a hyperthermophilic archaebacterium from an oceanic thermal vent. This channel possesses all the functional attributes of classical neuronal K(V) channels. The conservation of function reflects structural conservation in the voltage sensor as revealed by specific, high-affinity interactions with tarantula venom toxins, which evolved to inhibit eukaryotic K(V) channels.     

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.     

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.     

Renal cytochrome P450-related arachidonate metabolite inhibits (Na+ + K+)ATPase

The function of the nephron, the anatomical unit of the kidney, is segmented; at least 12 segments have been identified that differ in their morphology, transport properties and hormonal responsiveness. The medullary portion of the thick ascending limb of the loop of Henle (mTALH) has one of the highest concentrations of (Na+ + K+)ATPase found in mammalian tissues, reflecting the importance of this nephron segment in the regulation of extracellular fluid volume, as active sodium transport is driven by (Na+ + K+)ATPase. Here, in cells derived primarily from the mTALH of the outer medulla of rabbit kidney, we have identified a cytochrome P450-dependent monooxygenase system which metabolizes arachidonic acid to two biologically active oxygenated products; one of the products inhibits (Na+ + K+)ATPase and the other relaxes blood vessels. We report that formation of these oxygenated arachidonate metabolites is stimulated by arginine vasopressin (AVP) and salmon calcitonin (SCT).     

Electrogenic glutamate uptake in glial cells is activated by intracellular potassium

Uptake of glutamate into glial cells in the CNS maintains the extracellular glutamate concentration below neurotoxic levels and helps terminate its action as a neurotransmitter. The co-transport of two sodium ions on the glutamate carrier is thought to provide the energy needed to transport glutamate into cells. We have shown recently that glutamate uptake can be detected electrically because the excess of Na+ ions transported with each glutamate anion results in a net current flow into the cell. We took advantage of the control of the environment, both inside and outside the cell, provided by whole-cell patch-clamping and now report that glutamate uptake is activated by intracellular potassium and inhibited by extracellular potassium. Our results indicate that one K+ ion is transported out of the cell each time a glutamate anion and three Na+ ions are transported in. A carrier with this stoichiometry can accumulate glutamate against a much greater concentration gradient than a carrier co-transporting one glutamate anion and two Na+ ions. Pathological rises in extracellular potassium concentration will inhibit glutamate uptake by depolarizing glial cells and by preventing the loss of K+ from the glutamate carrier. This will facilitate a rise in the extracellular glutamate concentration to neurotoxic levels and contribute to the neuronal death occurring in brain anoxia and ischaemia.     

High specificity of a phosphate transport protein determined by hydrogen bonds

Transport of the essential nutrient phosphorus--primarily in the form of orthophosphate--into cells and organelles is highly specific. This is exemplified by the uptake of phosphate or its close analogue arsenate by bacterial cells by way of a high affinity active transport system dependent on a phosphate-binding protein; this system is unable to recognize other inorganic oxyanions and is, moreover, distinct from the one for sulphate transport. The phosphate-binding protein is a member of a family of periplasmic proteins acting as initial high-affinity receptors for the osmotic shock-sensitive active transport systems or permeases for various sugars, amino acids, oligopeptides, and oxyanions. We report here the highly refined 1.7 A resolution X-ray structure of the liganded form of the phosphate-binding protein. The structure reveals the atomic features responsible for phosphate selectivity, either in monobasic or dibasic form, and the exclusion of sulphate. These features are fundamental to understanding phosphate transport systems and molecular recognition of charged substrates or ions in other biological processes.     

The glial cell glutamate uptake carrier countertransports pH-changing anions.

Uptake into glial cells helps to terminate glutamate's neurotransmitter action and to keep its extracellular concentration, [Glu]o, below neurotoxic levels. The accumulative power of the uptake carrier stems from its transport of inorganic ions such as sodium (into the cell) and potassium (out of the cell). There is controversy over whether the carrier also transports a proton (or pH-changing anion). Here we show that the carrier generates an alkalinization outside and an acidification inside glial cells, and transports anions out of the cells, suggesting that there is a carrier cycle in which two Na+ accompany each glutamate anion into the cell, while one K+ and one OH- (or HCO3-) are transported out. This stoichiometry predicts a minimum [Glu]o of 0.6 microM normally (tonically activating presynaptic autoreceptors and post-synaptic NMDA receptors), and 370 microM during brain anoxia (high enough to kill neurons). Transport of OH-/HCO3- on the uptake carrier generates significant pH changes, and may provide a mechanism for neuron-glial interaction.     

Localization of nitric oxide synthase indicating a neural role for nitric oxide.

Nitric oxide (NO), apparently identical to endothelium-derived relaxing factor in blood vessels, is also formed by cytotoxic macrophages, in adrenal gland and in brain tissue, where it mediates the stimulation by glutamate of cyclic GMP formation in the cerebellum. Stimulation of intestinal or anococcygeal nerves liberates NO, and the resultant muscle relaxation is blocked by arginine derivatives that inhibit NO synthesis. It is, however, unclear whether in brain or intestine, NO released following nerve stimulation is formed in neurons, glia, fibroblasts, muscle or blood cells, all of which occur in proximity to neurons and so could account for effects of nerve stimulation on cGMP and muscle tone. We have now localized NO synthase protein immunohistochemically in the rat using antisera to the purified enzyme. We demonstrate NO synthase in the brain to be exclusively associated with discrete neuronal populations. NO synthase is also concentrated in the neural innervation of the posterior pituitary, in autonomic nerve fibres in the retina, in cell bodies and nerve fibres in the myenteric plexus of the intestine, in adrenal medulla, and in vascular endothelial cells. These prominent neural localizations provide the first conclusive evidence for a strong association of NO with neurons.     

Basolateral KCl co-transport in a NaCl-absorbing epithelium

In NaCl-absorbing epithelia such as proximal renal tubule, small intestine and gallbladder, Na+-dependent Cl- entry across the luminal membrane is an electroneutral transport process that has been attributed to Na-Cl symport, Na-K-Cl symport, or a double (Na-H, Cl-HCO3) antiport. At the basolateral (antiluminal) membrane, Na+ extrusion can be attributed to the Na+-K+ pump, and Cl- transport could be explained in principle by electrodiffusion since the intracellular Cl- activity is higher than predicted for equilibrium distribution. However, in Necturus gallbladder, as in other epithelia, the electrodiffusional Cl- permeability of the membrane (PCl) is too low to account for the transepithelial Cl- transport rate. Because K+ is at a higher chemical potential in the cell than in the extracellular fluid, and because serosal Cl- substitutions have only small effects on membrane potential, the hypothesis of carrier-mediated electroneutral KCl co-transport was proposed. The experiments reported here were designed to test this hypothesis in Necturus gallbladder epithelium. Intracellular Cl- and K+ activities (aCli, aKi) were measured with ion-sensitive intracellular microelectrodes before, during and after ionic substitutions of the serosal (basolateral) bathing medium. The results demonstrate a Na+-independent basolateral membrane KCl symport.     

Amino-acid sequence of the beta-subunit of the (Na+ + K+)ATPase deduced from a cDNA

The sodium/potassium-dependent ATPase [(Na+ + K+)ATPase], which establishes and maintains the Na+ and K+ gradients across the plasma membrane of animal cells, consists of two subunits, alpha and beta. Complementary DNA clones encoding the catalytic (alpha) subunit of sheep kidney and Torpedo californica electroplax enzymes have previously been isolated and characterized. However, there is little information concerning the primary structure of the beta-subunit, a glycoprotein of unknown function and relative molecular mass (Mr) approximately 55,000 (ref. 3). Here we describe the isolation and characterization of a cDNA clone containing the entire coding region of the beta-subunit of the sheep kidney (Na+ + K+)ATPase. We also discuss structural aspects of the protein and present evidence for a possible evolutionary relationship with the KdpC subunit of the Escherichia coli K+-ATPase.     

Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells

The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.