Stimulation of EAAC1 in C6 glioma cells by store-operated calcium influx

This study investigated how modulation of intracellular calcium alters the functional activity of the EAAC1 glutamate transporter in C6 glioma cells. Pre-incubation of C6 glioma cells with the endoplasmic reticulum Ca²⁺ ATP pump inhibitor, thapsigargin (10 μM) produced a time-dependent increase in the V_max for d-[³H]aspartate transport that reached a maximum at 15 min (143% of control; P < 0.001) that was accompanied by increased plasma membrane expression of EAAC1 and was blocked by inhibition of protein kinase C. Pre-incubation of C6 glioma cells with phorbol myristate-3-acetate (100 nM for 20 min) also caused a significant increase in the V_max of sodium-dependent d-[³H]aspartate transport (190% of control; P < 0.01). In contrast, in the absence of extracellular calcium, thapsigargin caused a significant inhibition in d-[³H]aspartate transport that was not mediated by protein kinase C. Blockade of store-operated calcium channels with 2-aminoethoxydiphenyl borate (50 μM) or SKF 96365 (10 μM) caused a net inhibition of d-[³H]aspartate uptake. Co-incubation of C6 glioma cells with both thapsigargin and 2-aminoethoxydiphenyl borate (but not SKF 96365) prevented the increase in d-[³H]aspartate transport that was observed in the presence of thapsigargin alone. Furthermore, 2-aminoethoxydiphenyl borate, but not SKF 96365, reduced the increase in intracellular calcium that occurred following pre-incubation of the cells with thapsigargin. It is concluded that, in C6 glioma cells, stimulation of EAAC1-mediated glutamate transport by thapsigargin is dependent on entry of calcium via the NSCC-1 subtype of store operated calcium channel and is mediated by protein kinase C. In contrast, in the absence of store operated calcium entry, thapsigargin inhibits transport.     

Activity of Ionotropic Glutamate Receptors in Retinal Cells: Effect of Ascorbate/Fe2+-Induced Oxidative Stress

Abstract: The effect of oxidative stress induced by the oxidant pair ascorbate/Fe²⁺ on the activity of ionotropic glutamate receptors was studied in cultured chick retina cells. The release of [³H]GABA and the increase of the intracellular free Na⁺ concentration ([Na⁺]_i), evoked by glutamate receptor agonists, were used as functional assays for the activity of the receptors. The results show that the maximal release of [³H]GABA evoked by kainate (KA; ～20% of the total) or AMPA (～11% of the total) was not different in control and peroxidized cells, whereas the EC₅₀ values determined for peroxidized cells (33.6 ± 1.7 and 8.0 ± 2.0 µM for KA and AMPA, respectively) were significantly lower than those determined under control conditions (54.1 ± 6.6 and 13.0 ± 2.2 µM for KA and AMPA, respectively). The maximal release of [³H]GABA evoked by NMDA under K⁺ depolarization was significantly higher in peroxidized cells (7.5 ± 0.5% of the total) as compared with control cells (4.0 ± 0.2% of the total), and the effect of oxidative stress was significantly reduced by a phospholipase A₂ inhibitor or by fatty acid-free bovine serum albumin. The change in the intracellular [Na⁺]_i evoked by saturating concentrations of NMDA under depolarizing conditions was significantly higher in peroxidized cells (8.9 ± 0.6 mM) than in control cells (5.9 ± 1.0 mM). KA, used at a subsaturating concentration (35 µM), evoked significantly greater increases of the [Na⁺]_i in peroxidized cells (11.8 ± 1.7 mM) than in control cells (7.1 ± 0.8 mM). A saturating concentration (150 µM) of this agonist triggered similar increases of the [Na⁺]_i in control and peroxidized cells. Accordingly, the maximal number of binding sites for (+)-5-[³H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate ([³H]MK-801) was increased after peroxidation, whereas the maximal number of binding sites for [³H]KA was not affected by oxidative stress. These data suggest that under oxidative stress the activity of the ionotropic glutamate receptors is increased, with the NMDA receptor being the most affected by peroxidation.     

Mechanism of Cation Binding to the Glutamate Transporter EAAC1 Probed with Mutation of the Conserved Amino Acid Residue Thr101

The glutamate transporter excitatory amino acid carrier 1 (EAAC1) catalyzes the co-transport of three Na⁺ ions, one H⁺ ion, and one glutamate molecule into the cell, in exchange for one K⁺ ion. Na⁺ binding to the glutamate-free form of the transporter generates a high affinity binding site for glutamate and is thus required for transport. Moreover, sodium binding to the transporters induces a basal anion conductance, which is further activated by glutamate. Here, we used the [Na⁺] dependence of this conductance as a read-out of Na⁺ binding to the substrate-free transporter to study the impact of a highly conserved amino acid residue, Thr¹⁰¹, in transmembrane domain 3. The apparent affinity of substrate-free EAAC1 for Na⁺ was dramatically decreased by the T101A but not by the T101S mutation. Interestingly, in further contrast to EAAC1_WT, in the T101A mutant this [Na⁺] dependence was biphasic. This behavior can be explained by assuming that the binding of two Na⁺ ions prior to glutamate binding is required to generate a high affinity substrate binding site. In contrast to the dramatic effect of the T101A mutation on Na⁺ binding, other properties of the transporter, such as its ability to transport glutamate, were impaired but not eliminated. Our results are consistent with the existence of a cation binding site deeply buried in the membrane and involving interactions with the side chain oxygens of Thr¹⁰¹ and Asp³⁶⁷. A theoretical valence screening approach confirms that the predicted site of cation interaction has the potential to be a novel, so far undetected sodium binding site.     

High-affinity binding ofl-glutamate to chick retinal membranes

Binding ofl-[³H]glutamate to membranes from whole chick retina and from subcellular fractions enriched with photoreceptor terminals (P₁), or terminals from the inner plexiform layer (P₂) was studied. Na⁺-dependent and Na⁺-independent binding to these membranes was demonstrated. Na⁺-independent binding was stereospecific. Kinetic analysis of the binding process indicated a single high-affinity system (K _B=0.55 M) with a capacity of approximately 20 pmoles/mg protein in all the membrane fractions. [³H]Glutamate binding to P₁ and P₂ fractions was effectively displaced by several structural analogues of glutamate. Glutamate diethyl-ester appreciably displaced binding, whereas kainic acid did not displace bound glutamate. Data indicate the binding of [³H]glutamate to physiologically relevant receptors in the chick retina.     

Sodium-Dependent Efflux of [3H]GABA from Synaptosomes Probably Related to Mitochondrial Calcium Mobilization

It has been suggested that mitochondria might modify transmitter release through the control of intracellular Ca²⁺levels. Treatments known to inhibit Ca²⁺retention by mitochondria lead to an increased transmitter liberation in the absence of external Ca²⁺, both at the frog neuromuscular junction and from isolated nerve endings. Sodium ions stimulate Ca²⁺efflux from mitochondria isolated from excitable tissues. In the present study, the effect of increasing internal Na⁺ levels on [³H]γ-aminobutyric acid ([³H]GABa) release from isolated nerve endings is reported. Results show that the efflux of [³H]GABA from prelabeled synaptosomes is stimulated by ouabain, veratrine, gramicidin D, and K⁺-free medium, which increase the internal sodium concentration. This effect was not observed when Na⁺ was omitted from the incubation medium and it was independent of external Ca²⁺, the experiments having been performed in a Ca²⁺-free, EGTA-containing medium. Since preincubation of synaptosomes with 2,4-diaminobutyric acid did not prevent the stimulatory effect of increased internal Na⁺ levels on [³H]GABA efflux, it appears to be unrelated to an enhanced activity of the outward carrier-mediated GABA transport. These results suggest that the augmented release of [³H]GABA may be due to an increased Ca²⁺efflux from mitochondria eiicited by the accumulation of Na⁺ at the nerve endings. Sandoval M. E. Sodium-dependent efflux of [³H]GABA from synaptosomes probably related to mitochondrial calcium mobilization. J. Neurochem. 35 , 915–921 (1980).     

Cerebral Alanine Transport and Alanine Aminotransferase Reaction: Alanine as a Source of Neuronal Glutamate

Abstract: Alanine transport and the role of alanine amino-transferase in the synthesis and consumption of glutamate were investigated in the preparation of rat brain synaptosomes. Alanine was accumulated rapidly via both the high-and low-affinity uptake systems. The high-affinity transport was dependent on the sodium concentration gradient and membrane electrical potential, which suggests a cotransport with Na⁺. Rapid accumulation of the Na⁺-alanine complex by synaptosomes stimulated activity of the Na⁺/K⁺ pump and increased energy utilization; this, in turn, activated the ATP-producing pathways, glycolysis and oxidative phosphorylation. Accumulation of Na⁺ also caused a small depolarization of the plasma membrane, a rise in [Ca²⁺]₁, and a release of glutamate. Intra-synaptosomal metabolism of alanine via alanine aminotransferase, as estimated from measurements of N fluxes from labeled precursors, was much slower than the rate of alanine uptake, even in the presence of added oxoacids. The velocity of [¹⁵N]alanine formation from [¹⁵N]glutamine was seven to eight times higher than the rate of [¹⁵N]glutamate generation from [¹⁵N]alanine. It is concluded that (a) overloading of nerve endings with alanine could be deleterious to neuronal function because it increases release of glutamate; (b) the activity of synaptosomal alanine aminotransferase is much slower than that of glutaminase and hence unlikely to play a major role in maintaining [glutamate] during neuronal activity; and (c) alanine aminotransferase might serve as a source of glutamate during recovery from ischemia/hypoxia when the alanine concentration rises and that of glutamate falls.     

COUPLED TRANSPORT OF GLUTAMATE AND SODIUM IN A CEREBELLAR NERVE CELL LINE

The cerebellar nerve cell line ε¹ has a very effective active transport system for glutamate. Glutamate uptake is dependent on extracellular Na⁺ and furthermore, ²²Na⁺ uptake is stimulated by glutamate, indicating that glutamate uptake and Na⁺ uptake are coupled. Two molecules of Na ⁺ are transported for each molecule of glutamate. The K_m for glutamate is found to be 5 × 10^?5M in both the glutamate uptake assay and the ²²Na⁺ uptake assay, providing additional evidence for glutamate-Na⁺ coupling. Pre-incubation with ouabain, which inhibits the Na⁺-K⁺ ATPase, results in a gradual inhibition of glutamate uptake due to the deterioration of the Na⁺ gradient. Tetrodotoxin, however, has no effect on glutamate-induced ²²Na⁺ uptake, showing that this Na⁺ flux does not occur via voltage-dependent Na⁺ channels. Studies on the specificity of the ε¹ glutamate transport system show that it is distinct from systems that transport alanine and glycine. l -Glutamate, d -aspartate, l -cysteate, and l -cysteine sulfinate are able to utilize the transport system efficiently. d -Glutamate, l -homocysteate, N-methyl-d , l -aspartate, and kainic acid are very poor substrates for the glutamate transport system, and in addition do not stimulate ²²Na⁺ uptake. These data allow us to distinguish the glutamate transport system from the glutamate receptor which is known to mediate depolarization in response to all nine of the above compounds. Thus, ε¹ does not have an excitatory glutamate receptor.     

Workshop 7: 2

Glutamine, the preferred precursor for neurotransmitter glutamate, is likely to be the principal substrate for the neuronal System A transporter SAT1 in vivo. By measuring currents associated with SAT1 expression in Xenopus oocytes, we found that SAT1 mediates transport of small, neutral, aliphatic amino acids including glutamine, alanine and the System A‐specific analogue 2‐(methylamino) isobutyrate, each with K_0.5 of 0.3–0.5 mm . Amino acid transport is driven by the Na⁺ electrochemical gradient. Kinetic data indicates that Na⁺/cotransport comprises the ordered binding first of Na⁺ (a voltage‐dependent step), then alanine, then simultaneous translocation. Li⁺ (but not H⁺) can substitute for Na⁺ but results in reduced V_max. In the absence of amino acid, SAT1 mediates a cation leak with selectivity Na⁺, Li⁺, H⁺, K⁺. The temperature‐dependence of the leak current (E_a = 17 ± 3 kcal/mol) is consistent with carrier‐mediated Na⁺ uniport activity (cf 13 ± 2 kcal/mol for Na⁺/alanine cotransport) but the leak does not saturate at physiological [Na⁺], suggesting channel activity. Despite a Na⁺ Hill coefficient of 1, we obtained Na⁺/amino acid coupling coefficients greater than 1 from simultaneous measurement of charge and [³H]alanine or [³H]glutamine uptake. Interpretation of these data is model‐dependent and consistent with either (1) an all‐carrier model in which Na⁺/amino acid cotransport is thermodynamically coupled 2 : 1, cotransport is preferred over Na⁺ uniport, and in which there is little cooperativity between Na⁺ binding events, or (2) 1 : 1 coupling in parallel with an always‐on Na⁺ channel activity. In either scenario, the presence of SAT1 at the plasma membrane and resultant Na⁺ fluxes will place a significant energy burden on the cell.     

Activation of Na/H Exchange in Erythrocytes of Frog Rana ridibunda

Transport of Na⁺ in isolated erythrocytes of the frog Rana ridibunda was studied using radioactive isotope ^{22 22}Na. Treatment of erythrocytes with -adrenergic agonist isoproterenol (ISP) or with a combination of ISP and phosphodiesterase blocker 3-isobutyl-methyl-xanthine (IBMX) did not affect the Na⁺ transport into the cells. These data indicated that cAMP-dependent protein kinase A did not participate in regulation of the Na⁺ transport into the frog erythrocytes. Incubation of erythrocytes with protein kinase C activator phorbol ester (PMA, 0.15 µM) led to a pronounced increase of ^{22 22}Na accumulation and intracellular Na⁺ concentration. These changes of the Na⁺ transport into the cells were completely blocked in the presence of 50 µM ethyl-isopropyl-amiloride (EIPA), a selective blocker of the NHE1-isoform of Na⁺/H⁺ exchanger. Hence, PMA produced activation of Na⁺/H⁺ exchange in frog erythrocytes. The unidirectional Na⁺ influx into erythrocytes amounted, on average, to 0.99 ± 0.12 and 147 ± 9 mmol/l cells/h for control and PMA-treated cells, respectively. The EIPA concentration producing a 50% inhibition of the PMA-induced Na⁺ influx (IC₅₀) was 0.28 µM. A high sensitivity of the frog Na/H exchanger to EIPA indicates its similarity with the mammalian NHE1 isoform. The obtained data for the first time clearly indicate an important role of PKC in Na/H exchange regulation in the frog red blood cells.     

Na+ requirement for glutamate-dependent sugar transport byFusobacterium nucleatum ATCC 10953

Resting cells ofFusobacterium nucleatum ATCC 10953, when provided with glutamic acid (Na⁺ salt) as fermentable energy source, rapidly accumulated [¹⁴C]glucose, from the medium. Sugar accumulation was not observed when Na⁺ glutamate was replaced by ammonium glutamate. However, addition of Na⁺ (chloride) to the latter system elicited uptake of [¹⁴C]glucose by the organism. Of other monovalent cations tested, only Li⁺ was found to be slightly stimulatory, but K⁺, Rb⁺, and Cs⁺ ions were ineffective. For determination of the role(s) of Na⁺ in sugar accumulation, the transport of [¹⁴C]glucose and [¹⁴C]glutamic acid by the cells was studied independently, with lysine as an alternate (and Na⁺-independent) energy source. In the presence of lysine, cells ofF. nucleatum 10953 accumulated [¹⁴C]glucose from a Na⁺-free medium, but, in contrast, uptake and fermentation of [¹⁴C]glutamic acid was Na⁺-dependent. The glucose transport system is Na⁺-independent. However, our data indicate dual role(s) for Na⁺ in the transport and intracellular metabolism of glutamic acid. The Na⁺-dependent glutamate fermentation pathway provides the necessary energy for active transport of glucose by the resting cell.     

Pentylenetetrazol modulates redox system by inducing addicsin translocation from endoplasmic reticulum to plasma membrane in NG108-15 cells

Addicsin (Arl6ip5) is a multifunctional physiological and pathophysiological regulator that exerts its effects by readily forming homo- and hetero-complexes with various functional factors. In particular, addicsin acts as a negative modulator of neural glutamate transporter excitatory amino acid carrier 1 (EAAC1) and participates in the regulation of intracellular glutathione (GSH) content by negatively modulating EAAC1-mediated cysteine and glutamate uptake. Addicsin is considered to play a crucial role in the onset of neurodegenerative diseases including epilepsy. However, the molecular dynamics of addicsin remains largely unknown. Here, we report the dynamics of addicsin in NG108-15 cells upon exposure to pentylenetetrazol (PTZ), a representative epileptogenic agent acting on the gamma-Aminobutyric acid A (GABA_A) receptor. Fluorescent immunostaining analysis demonstrated that addicsin drastically changed its localization from the endoplasmic reticulum (ER) to the plasma membrane within 1 h of PTZ exposure in a dose-dependent manner. Moreover, addicsin was co-localized with the plasma membrane markers EAAC1 and Na⁺/K⁺ ATPase alpha-3 upon PTZ stimulation. This translocation was significantly inhibited by a non-competitive GABA_A receptor antagonist, picrotoxin, but not by a competitive GABA_A receptor antagonist, bicuculline. Furthermore, lactate dehydrogenase (LDH) assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay showed that PTZ-induced addicsin translocation was accompanied by a decrease of radical-scavenging activity and an increase of cytotoxicity in a PTZ dose-dependent manner. These findings suggest that PTZ induces the translocation of addicsin from the ER to the plasma membrane and modulates the redox system by regulating EAAC1-mediated GSH synthesis, which leads to the activation of cell death signaling.     

Pertussis toxin-sensitive modulation of glutamate transport by endothelin-1 type A receptors in glioma cells

Endothelin-1 (ET-1) is a 21 amino acids peptide that exerts several biological activities through interaction with specific G-protein coupled receptors. Increased ET-1 expression is frequently associated with pathological situations involving alterations in glutamate levels. In the present study, a brief exposure to ET-1 was found to increase aspartate uptake in C6 glioma cells, which endogenously express the neuronal glutamate transporter EAAC1 (pEC₅₀ of 9.89). The stimulatory effect of ET-1 mediated by ET_A receptors corresponds to a 62% increase in the V_max with no modification of the affinity for the substrate. While protein kinase C activity is known to participate in the regulation of EAAC1, the effect of ET-1 on the glutamate uptake was found to be independent of this kinase activation. In contrast, the inactivation of G_o/i type G-protein dependent signaling with pertussis toxin was found to impair ET-1-mediated regulation of EAAC1. An examination of the cell surface expression of EAAC1 by protein biotinylation studies or by confocal analysis of immuno-fluorescence staining demonstrated that ET-1 stimulates EAAC1 translocation to the cell surface. Hence, the disruption of the cytoskeleton with cytochalasin D prevented ET-1-stimulated aspartate uptake. Together, the data presented in the current study suggest that ET-1 participates in the acute regulation of glutamate transport in glioma cells. Considering the documented role of glutamate excitotoxicity in the development of brain tumors, endothelinergic system constitutes a putative target for the pharmacological control of glutamate transmission at the vicinity of glioma cells.     

Metabolic and ionoregulatory responses of the Amazonian cichlid, <Emphasis Type="Italic">Astronotus ocellatus</Emphasis>, to severe hypoxia

We examined the metabolic and ionoregulatory responses of the Amazonian cichlid, Astronotus ocellatus, to 20 h exposure to severe hypoxia (0.37 ± 0.19 mg O₂/l; 4.6% air saturation) or 8 h severe hypoxia followed by 12 h recovery in normoxic water. During 20 h exposure to hypoxia, white muscle [ATP] was maintained at normoxic levels primarily through a 20% decrease in [creatine phosphate] (CrP) and an activation of glycolysis yielding lactate accumulation. Muscle lactate accumulation maintained cytoplasmic redox state ([NAD⁺]/[NADH]) and was associated with an inactivation of the mitochondrial enzyme pyruvate dehydrogenase (PDH). The inactivation of PDH was not associated with significant changes in cytoplasmic allosteric modulators ([ADP_free], redox state, or [pyruvate]). Hypoxia exposure caused a ∼65% decrease in gill Na⁺/K⁺ ATPase activity, which was not matched by changes in Na⁺/K⁺ ATPase α-subunit protein abundance indicating post-translational modification of Na⁺/K⁺ ATPase was responsible for the decrease in activity. Despite decreases in gill Na⁺/K⁺ ATPase activity, plasma [Na⁺] increased, but this increase was possibly due to a significant hemoconcentration and fluid shift out of the extracellular space. Hypoxia caused an increase in Na⁺/K⁺ ATPase α-subunit mRNA abundance pointing to either reduced mRNA degradation during exposure to hypoxia or enhanced expression of Na⁺/K⁺ ATPase α-subunit relative to other genes.     

Characterization of glutamate transport system in hydrophobic protein (H protein) of Bacillus subtilis

Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na⁺ gradient across the membrane was imposed. The maximum effect of Na⁺ on the transport was achieved at a concentration of about 40 mM, while the apparent K_m for Na⁺ was approximately 8 mM. On the other hand, K_m for glutamate in the presence of 50 mM Na⁺ was about 8 μM. Increasing the concentration of Na⁺ resulted in a decrease in K_m for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na⁺ ionophore).Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K⁺-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent K_m for glutamate was approximately 25 μM.These results indicate that two kinds of glutamate transport system were present in H protein: one is Na⁺ dependent and the other is H⁺ dependent.     

Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding

Summary Loop diuretic-sensitive (Na⁺,K⁺,Cl^–)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na⁺,K⁺) gradien-driven³⁶Cl fluxes weree half-maximally inhibited by 0.14 m bumetanide and 44 m furosimide. While⁸⁶Rb uptake rates showed hyperbolic dependencies on Na⁺ and K⁺ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl^– concentration, Hill coefficient 1.8, indicating a 1 Na⁺: 1 K⁺:2 Cl^– stoichiometry of ion transport.The interaction of putative (Na⁺, K⁺, Cl^–)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [³H]-bumetanide. The requirement for the simulataneous presence of Na⁺,K⁺, and Cl^–, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na⁺, K⁺, Cl^–)-cotransporter. [³H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K _D1=0.13 m;B _max1 =6.4 pmol/mg of protein) and low-affinity (K _D2=34 m;B _max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl^– (K ₁=60mm), whereas low-affinity sites seem to be unaffected by Cl^–. We have shown that only high-affinity [³H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.     

Distinct transport activity of tetraethylammonium from l-carnitine in rat renal brush-border membranes

We investigated the contribution of the Na⁺/l-carnitine cotransporter in the transport of tetraethylammonium (TEA) by rat renal brush-border membrane vesicles. The transient uphill transport of l-carnitine was observed in the presence of a Na⁺ gradient. The uptake of l-carnitine was of high affinity (K_m=21 μM) and pH dependent. Various compounds such as TEA, cephaloridine, and p-chloromercuribenzene sulfonate (PCMBS) had potent inhibitory effects for l-carnitine uptake. Therefore, we confirmed the Na⁺/l-carnitine cotransport activity in rat renal brush-border membranes. Levofloxacin and PCMBS showed different inhibitory effects for TEA and l-carnitine uptake. The presence of an outward H⁺ gradient induced a marked stimulation of TEA uptake, whereas it induced no stimulation of l-carnitine uptake. Furthermore, unlabeled TEA preloaded in the vesicles markedly enhanced [¹⁴C]TEA uptake, but unlabeled l-carnitine did not stimulate [¹⁴C]TEA uptake. These results suggest that transport of TEA across brush-border membranes is independent of the Na⁺/l-carnitine cotransport activity, and organic cation secretion across brush-border membranes is predominantly mediated by the H⁺/organic cation antiporter.     

Differential effects of metal ligands on synaptic membrane glutamate binding and uptake systems

The high affinity, Na⁺-independentl-[³H]glutamate binding process in synaptic membranes and in the purified binding protein was shown to be inhibited to an almost equal extent by the metal ligands NaN₃, KCN, ando-phenanthroline, and by 2,4,5-trihydroxyphenylalanine (6-OH DOPA). The high affinity, Na⁺-dependent glutamate transport activity in these membranes was almost totally insensitive to NaN₃,o-phenanthroline, KCN, and 6-OH DOPA. These agents, especially 6-OH DOPA, may be useful tools in achieving a discrimination between putative physiologic receptors and uptake carrier sites forl-glutamate in synaptic membranes. The sensitivity of the glutamate binding sites to the effects of the metal ligands may be correlated to the presence of an iron-sulfur center in the purified glutamate binding protein. Some of the characteristics of this metallic center were explored by optical and paramagnetic resonance spectroscopic techniques and are described in this study.This research was supported by grants DAAG29-79-C-0156 from the Army Research Office and AA 04732 from NIAAA.     

Identification of a novel sugar-H+ symport protein, FucP, for transport of L-fucose into Escherichia coli

l -Fucose (6-deoxy-l -galactose) is used as sole carbon source by many microorganisms, and its transport into Escherichia coli is mediated by An l -fucose-H⁺ symport activity, in order to determine the nature of a putative transporter encoded by the E. coli fucP gene and Identify its protein product it was cloned downstream of the inducible T7 RNA polymerase and lambda O_l P_l promoters, induction of the T7 promoter resulted in the expression of [¹⁴C]-l -fucose uptake activity and the concomitant expression of a [³⁵S]-Met-labelled 32 kDa protein at levels too tow for detection by staining with Coomassie briiiiant blue or for protein sequencing, induction of the lambda O_l P_l promoter caused the appearance of l -fucose-H₊ symport activity and of a Coomassie brilliant blue-stained 32 kDa membrane protein expressed at high levels sufficient for identification as FucP by N-terminal protein sequencing. The FucP protein is, therefore, a sugar-H₊ symporter different in amino acid sequence from any other known transporter. These and other results illustrate the general unpredictability of cloning strategies for attempting the amplified expression of membrane transport proteins.     

Transport in halobacterium halobium: Light-induced cation-gradients,amino acid transport kinetics,and properties of transport carriers

Cell envelope vesicles prepared from H. halobium contain bacteriorhodopsin and upon illumination protons are ejected. Coupled to the proton motive force is the efflux of Na⁺. Measurements of ²²Na flux, exterior pH change, and membrane potential, ΔΨ (with the dye 3,3′-dipentyloxadicarbocyanine) indicate that the means of Na⁺ transport is sodium/proton exchange. The kinetics of the pH changes and other evidence suggests that the antiport is electrogenic (H⁺/Na⁺ > 1). The resulting large chemical gradient for Na⁺ (outside > inside), as well as the membrane potential, will drive the transport of 18 amino acids. The 19th, glutamate, is unique in that its accumulation is indifferent to ΔΨ: this amino acid is transported only when a chemical gradient for Na⁺ is present. Thus, when more and more NaCl is included in the vesicles glutamate transport proceeds with longer and longer lags. After illumination the gradient of H⁺ collapses within 1 min, while the large Na⁺ gradient and glutamate transporting activity persists for 10–15 min, indicating that proton motive force is not necessary for transport. A chemical gradient of Na⁺, arranged by suspending vesicles loaded with KCl in NaCl, drives glutamate transport in the dark without other sources of energy, with V_max and K_m comparable to light-induced transport. These and other lines of evidence suggest that the transport of glutamate is facilitated by symport with Na⁺, in an electrically neutral fashion, so that only the chemical component of the Na⁺ gradient is a driving force. The transport of all amino acids but glutamate is bidirectional. Actively driven efflux can be obtained with reversed Na⁺ gradients (inside > outside), and passive efflux is considerably enhanced by intravesicle Na⁺. These results suggest that the transport carriers are functionally symmetrical. On the other hand, noncompetitive inhibition of transport by cysteine (a specific inhibitor of several of the carriers) is only obtained from the vesicle exterior and only for influx: these results suggest that in some respects the carriers are asymmetrical. A protein fraction which binds glutamate has been found in cholate-solubilized H. halobium membranes, with an apparent molecular weight of 50,000. When this fraction (but not the others eluted from an Agarose column) is reconstituted with soybean lipids to yield lipoprotein vesicles, facilitated transport activity is regained. Neither binding nor reconstituted transport depend on the presence of Na⁺. The kinetics of the transport and of the competitive inhibition by glutamate analogs suggest that the protein fraction responsible is derived from the intact transport system.     

Ionic Requirements for the Specific Binding of [3H]GBR 12783 to a Site Associated with the Dopamine Uptake Carrier

At 0°C, when Na⁺ was the only cation present in the incubation medium, increasing the Na⁺ concentration from 3 to 10 mM enhanced the affinity of [³H]l-[2-(di-phenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([³H]GBR 12783) for the specific binding site present in rat striatal membranes without affecting the 5_max. For higher Na⁺ concentrations, specific binding values plateaued and then slightly decreased at 130 mM Na⁺. In a 10 mM Na⁺ medium, the K_D and the B_max were, respectively, 0.23 nM and 12.9 pmol/mg of protein. In the presence of 0.4 nM [³H]GBR 12783, the half-maximal specific binding occurred at 5 mM Na⁺. A similar Na⁺ dependence was observed at 20°C. Scatchard plots indicated that K⁺, Ca²⁺, Mg²⁺, and Tris⁺ acted like competitive inhibitors of the specific binding of [³H]GBR 12783. The inhibitory potency of various cations (K⁺, Ca²⁺, Mg²⁺, Tris⁺, Li⁺ and choline) was enhanced when the Na⁺ concentration was decreased from 130 to 10 mM. In a 10 mM Na⁺ medium, the rank order of inhibitory potency was Ca²⁺ (0.13 mM) > Mg²⁺ > Tris⁺ > K⁺ (15 mM). The requirement for Na⁺ was rather specific, because none of the other cations acted as a substitute for Na⁺. No anionic requirement was found: Cl^-, Br^-, and F^- were equipotent. These results suggest that low Na⁺ concentrations are required for maximal binding; higher Na⁺ concentrations protect the specific binding site against the inhibitory effect of other cations.