Is the glutamate residue Glu-373 the proton acceptor of the excitatory amino acid carrier 1?

Glutamate transport by the neuronal excitatory amino acid carrier (EAAC1) is accompanied by the coupled movement of one proton across the membrane. We have demonstrated previously that the cotransported proton binds to the carrier in the absence of glutamate and, thus, modulates the EAAC1 affinity for glutamate. Here, we used site-directed mutagenesis together with a rapid kinetic technique that allows one to generate sub-millisecond glutamate concentration jumps to locate possible binding sites of the glutamate transporter for the cotransported proton. One candidate for this binding site, the highly conserved glutamic acid residue Glu-373 of EAAC1, was mutated to glutamine. Our results demonstrate that the mutant transporter does not catalyze net transport of glutamate, whereas Na(+)/glutamate homoexchange is unimpaired. Furthermore, the voltage dependence of the rates of Na(+) binding and glutamate translocation are unchanged compared with the wild-type. In contrast to the wild-type, however, homoexchange of the E373Q transporter is completely pH-independent. In line with these findings the transport kinetics of the mutant EAAC1 show no deuterium isotope effect. Thus, we suggest a new transport mechanism, in which Glu-373 forms part of the binding site of EAAC1 for the cotransported proton. In this model, protonation of Glu-373 is required for Na(+)/glutamate translocation, whereas the relocation of the carrier is only possible when Glu-373 is negatively charged. Interestingly, the Glu-373-homologous amino acid residue is glutamine in the related neutral amino acid transporter alanine-serine-cysteine transporter. The function of alanine-serine-cysteine transporter is neither potassium- nor proton-dependent. Consequently, our results emphasize the general importance of glutamate and aspartate residues for proton transport across membranes.     

The anion conductance of the glutamate transporter EAAC1 depends on the direction of glutamate transport

The steady-state and pre-steady-state kinetics of glutamate transport by the neuronal glutamate transporter EAAC1 were determined under conditions of outward glutamate transport and compared to those found for the inward transport mode. In both transport modes, the glutamate-induced current is composed of two components, the coupled transport current and the uncoupled anion current, and inhibited by a specific non-transportable inhibitor. Furthermore, the glutamate-independent leak current is observed in both transport modes. Upon a glutamate concentration jump outward transport currents show a distinct transient phase that deactivates within 15 ms. The results demonstrate that the general properties of EAAC1 are symmetric, but the rates of substrate transport and anion flux are asymmetric with respect to the orientation of the substrate binding site in the membrane. Therefore, the EAAC1 anion conductance differs from normal ligand-gated ion channels in that it can be activated by glutamate and Na(+) from both sides of the membrane.     

The conserved histidine 295 does not contribute to proton cotransport by the glutamate transporter EAAC1

Transmembrane glutamate transport by the excitatory amino acid carrier (EAAC1) is coupled to the cotransport of three Na(+) ions and one proton. Previously, we suggested that the mechanism of H(+) cotransport involves protonation of the conserved glutamate residue E373. However, it was also speculated that the cotransported proton is shared in a H(+)-binding network, possibly involving the conserved histidine 295 in the sixth transmembrane domain of EAAC1. Here, we used site-directed mutagenesis together with pre-steady-state electrophysiological analysis of the mutant transporters to test the protonation state of H295 and to determine its involvement in proton transport by EAAC1. Our results show that replacement of H295 with glutamine, an amino acid residue that cannot be protonated, generates a fully functional transporter with transport kinetics that are close to those of the wild-type EAAC1. In contrast, replacement with lysine results in a transporter in which substrate binding and translocation are dramatically inhibited. Furthermore, it is demonstrated that the effect of the histidine 295 to lysine mutation on the glutamate affinity is caused by its positive charge, since wild-type-like affinity can be restored by changing the extracellular pH to 10.0, thus partially deprotonating H295K. Together, these results suggest that histidine 295 is not protonated in EAAC1 at physiological pH and, thus, does not contribute to H(+) cotransport. This conclusion is supported by data from H295C-E373C double mutant transporters which demonstrate that these residues cannot be linked by oxidation, indicating that H295 and E373 are not close in space and do not form a proton binding network. A kinetic scheme is used to quantify the results, which includes binding of the cotransported proton to E373 and binding of a modulatory, nontransported proton to the amino acid side chain in position 295.     

Cooperation of the conserved aspartate 439 and bound amino acid substrate is important for high-affinity Na+ binding to the glutamate transporter EAAC1

The neuronal glutamate transporter EAAC1 contains several conserved acidic amino acids in its transmembrane domain, which are possibly important in catalyzing transport and/or binding of co/countertransported cations. Here, we have studied the effects of neutralization by site-directed mutagenesis of three of these amino acid side chains, glutamate 373, aspartate 439, and aspartate 454, on the functional properties of the transporter. Transport was analyzed by whole-cell current recording from EAAC1-expressing mammalian cells after applying jumps in voltage, substrate, or cation concentration. Neutralization mutations in positions 373 and 454, although eliminating steady-state glutamate transport, have little effect on the kinetics and thermodynamics of Na(+) and glutamate binding, suggesting that these two positions do not constitute the sites of Na(+) and glutamate association with EAAC1. In contrast, the D439N mutation resulted in an approximately 10-fold decrease of apparent affinity of the glutamate-bound transporter form for Na(+), and an approximately 2,000-fold reduction in the rate of Na(+) binding, whereas the kinetics and thermodynamics of Na(+) binding to the glutamate-free transporter were almost unchanged compared to EAAC1(WT). Furthermore, the D439N mutation converted l-glutamate, THA, and PDC, which are activating substrates for the wild-type anion conductance, but not l-aspartate, into transient inhibitors of the EAAC1(D439) anion conductance. Activation of the anion conductance by l-glutamate was biphasic, allowing us to directly analyze binding of two of the three cotransported Na(+) ions as a function of time and [Na(+)]. The data can be explained with a model in which the D439N mutation results in a dramatic slowing of Na(+) binding and a reduced affinity of the substrate-bound EAAC1 for Na(+). We propose that the bound substrate controls the rate and the extent of Na(+) interaction with the transporter, depending on the amino acid side chain in position 439.     

Neutralization of the aspartic acid residue Asp-367, but not Asp-454, inhibits binding of Na+ to the glutamate-free form and cycling of the glutamate transporter EAAC1

Substrate transport by the plasma membrane glutamate transporter EAAC1 is coupled to cotransport of three sodium ions. One of these Na(+) ions binds to the transporter already in the absence of glutamate. Here, we have investigated the possible involvement of two conserved aspartic acid residues in transmembrane segments 7 and 8 of EAAC1, Asp-367 and Asp-454, in Na(+) cotransport. To test the effect of charge neutralization mutations in these positions on Na(+) binding to the glutamate-free transporter, we recorded the Na(+)-induced anion leak current to determine the K(m) of EAAC1 for Na(+). For EAAC1(WT), this K(m) was determined as 120 mm. When the negative charge of Asp-367 was neutralized by mutagenesis to asparagine, Na(+) activated the anion leak current with a K(m) of about 2 m, indicating dramatically impaired Na(+) binding to the mutant transporter. In contrast, the Na(+) affinity of EAAC1(D454N) was virtually unchanged compared with the wild type transporter (K(m) = 90 mm). The reduced occupancy of the Na(+) binding site of EAAC1(D367N) resulted in a dramatic reduction in glutamate affinity (K(m) = 3.6 mm, 140 mm [Na(+)]), which could be partially overcome by increasing extracellular [Na(+)]. In addition to impairing Na(+) binding, the D367N mutation slowed glutamate transport, as shown by pre-steady-state kinetic analysis of transport currents, by strongly decreasing the rate of a reaction step associated with glutamate translocation. Our data are consistent with a model in which Asp-367, but not Asp-454, is involved in coordinating the bound Na(+) in the glutamate-free transporter form.     

Thallium ions can replace both sodium and potassium ions in the glutamate transporter excitatory amino acid carrier 1

The excitatory amino acid carrier EAAC1 belongs to a family of glutamate transporters that use the electrochemical transmembrane gradients of sodium and potassium to mediate uphill transport of glutamate into the cell. While the sites of cation interaction with EAAC1 are unknown, two cation binding sites were observed in the crystal structure of the bacterial glutamate transporter homologue GltPh. Although occupied by Tl(+) in the crystal structure, these sites were proposed to be Na(+) binding sites. Therefore, we tested whether Tl(+) has the ability to replace Na(+) also in the mammalian transporters. Our data demonstrate that Tl(+) can bind to EAAC1 with high affinity and mediate a host of different functions. Tl(+) can functionally replace potassium when applied to the cytoplasm and can support glutamate transport current. When applied extracellularly, Tl(+) induces some behavior that mimics that of the Na(+)-bound transporter, such as activation of the cation-induced anion conductance and creation of a substrate binding site, but it cannot replace Na(+) in supporting glutamate transport current. Moreover, our data show a differential effect of mutations to two acidic amino acids potentially involved in cation binding (D367 and D454) on Na(+) and Tl(+) affinity. Overall, our results demonstrate that the ability of the glutamate transporters to interact with Tl(+) is conserved between GltPh and a mammalian member of the transporter family. However, in contrast to GltPh, which does not bind K(+), Tl(+) is more efficient in mimicking K(+) than Na(+) when interacting with the mammalian protein.     

A high affinity fungal nitrate carrier with two transport mechanisms

We have expressed the CRNA high affinity nitrate transporter from Emericella (Aspergillus) nidulans in Xenopus oocytes and used electrophysiology to study its properties. This method was used because there are no convenient radiolabeled substrates for the transporter. Oocytes injected with crnA mRNA showed nitrate-, nitrite-, and chlorite-dependent currents. Although the gene was originally identified by chlorate selection there was no evidence for transport of this anion. The gene selection is explained by the high affinity of the transporter for chlorite, and the fact that this ion contaminates solutions of chlorate. The pH-dependence of the anion-elicited currents was consistent with H(+)-coupled mechanism of transport. At any given voltage, currents showed hyperbolic kinetics with respect to extracellular H(+), and these data could be fitted with a Michaelis-Menten relationship. But this equation did not adequately describe transport of the anion substrates. At higher concentrations of the anion substrates and more negative membrane voltages, the currents were decreased, but this effect was independent of changes in external pH. These more complicated kinetics could be fit by an equation containing two Michaelis-Menten terms. The substrate inhibition of the currents could be explained by a transport reaction cycle that included two routes for the transfer of nitrate across the membrane, one on the empty carrier and the other proton coupled. The model predicts that the substrate inhibition of transporter current depends on the cytosolic nitrate concentration. This is the first time a high affinity nitrate transport activity has been characterized in a heterologous system and the measurements show how the properties of the CRNA transporter are modified by changes in the membrane potential, external pH, and nitrate concentration. The physiological significance of these observations is discussed.     

The glutamate transporter subtypes EAAT4 and EAATs 1-3 transport glutamate with dramatically different kinetics and voltage dependence but share a common uptake mechanism

Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-micros time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 microM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na(+)-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at -90 mV). Applying step changes to the transmembrane potential, V(m), of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a tau of approximately 15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<-40 mV) and activation at positive V(m) (>0 mV). A similar inhibitory effect at V(m) < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V(m) curve. Jumping the glutamate concentration to 100 muM generated biphasic, saturable transient transport and anion currents (K(m) approximately 5 microM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1-3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.     

Charge Compensation Mechanism of a Na+-coupled, Secondary Active Glutamate Transporter

Forward glutamate transport by the excitatory amino acid carrier EAAC1 is coupled to the inward movement of three Na(+) and one proton and the subsequent outward movement of one K(+) in a separate step. Based on indirect evidence, it was speculated that the cation binding sites bear a negative charge. However, little is known about the electrostatics of the transport process. Valences calculated using the Poisson-Boltzmann equation indicate that negative charge is transferred across the membrane when only one cation is bound. Consistently, transient currents were observed in response to voltage jumps when K(+) was the only cation on both sides of the membrane. Furthermore, rapid extracellular K(+) application to EAAC1 under single turnover conditions (K(+) inside) resulted in outward transient current. We propose a charge compensation mechanism, in which the C-terminal transport domain bears an overall negative charge of -1.23. Charge compensation, together with distribution of charge movement over many steps in the transport cycle, as well as defocusing of the membrane electric field, may be combined strategies used by Na(+)-coupled transporters to avoid prohibitive activation barriers for charge translocation.     

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.     

Early intermediates in the transport cycle of the neuronal excitatory amino acid carrier EAAC1

Electrogenic glutamate transport by the excitatory amino acid carrier 1 (EAAC1) is associated with multiple charge movements across the membrane that take place on time scales ranging from microseconds to milliseconds. The molecular nature of these charge movements is poorly understood at present and, therefore, was studied in this report in detail by using the technique of laser-pulse photolysis of caged glutamate providing a 100-micros time resolution. In the inward transport mode, the deactivation of the transient component of the glutamate-induced coupled transport current exhibits two exponential components. Similar results were obtained when restricting EAAC1 to Na(+) translocation steps by removing potassium, thus, demonstrating (1) that substrate translocation of EAAC1 is coupled to inward movement of positive charge and, therefore, electrogenic; and (2) the existence of at least two distinct intermediates in the Na(+)-binding and glutamate translocation limb of the EAAC1 transport cycle. Together with the determination of the sodium ion concentration and voltage dependence of the two-exponential charge movement and of the steady-state EAAC1 properties, we developed a kinetic model that is based on sequential binding of Na(+) and glutamate to their extracellular binding sites on EAAC1 explaining our results. In this model, at least one Na(+) ion and thereafter glutamate rapidly bind to the transporter initiating a slower, electroneutral structural change that makes EAAC1 competent for further, voltage-dependent binding of additional sodium ion(s). Once the fully loaded EAAC1 complex is formed, it can undergo a much slower, electrogenic translocation reaction to expose the substrate and ion binding sites to the cytoplasm.     

How Drugs Interact with Transporters: SGLT1 as a Model

Drugs are transported by cotransporters with widely different turnover rates. We have examined the underlying mechanism using, as a model system, glucose and indican (indoxyl-beta-D: -glucopyranoside) transport by human Na(+)/glucose cotransporter (hSGLT1). Indican is transported by hSGLT1 at 10% of the rate for glucose but with a fivefold higher apparent affinity. We expressed wild-type hSGLT1 and mutant G507C in Xenopus oocytes and used electrical and optical methods to measure the kinetics of glucose (using nonmetabolized glucose analogue alpha-methyl-D: -glucopyranoside, alphaMDG) and indican transport, alone and together. Indican behaved as a competitive inhibitor of alphaMDG transport. To examine protein conformations, we recorded SGLT1 capacitive currents (charge movements) and fluorescence changes in response to step jumps in membrane voltage, in the presence and absence of indican and/or alphaMDG. In the absence of sugar, voltage jumps elicited capacitive SGLT currents that decayed to steady state with time constants (tau) of 3-20 ms. These transient currents were abolished in saturating alphaMDG but only slightly reduced (10%) in saturating indican. SGLT1 G507C rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages. Maximal fluorescence increased approximately 150% in saturating indican but decreased approximately 50% in saturating alphaMDG. Modeling indicated that the rate-limiting step for indican transport is sugar translocation, whereas for alphaMDG it is dissociation of Na(+) from the internal binding sites. The inhibitory effects of indican on alphaMDG transport are due to its higher affinity and a 100-fold lower translocation rate. Our results indicate that competition between substrates and drugs should be taken into consideration when targeting transporters as drug delivery systems.     

Glutamate 268 regulates transport probability of the anion/proton exchanger ClC-5

The Cl(-)/H(+) exchange mediated by ClC transporters can be uncoupled by external SCN(-) and mutations of the proton glutamate, a conserved residue at the internal side of the protein. We show here for the mammalian ClC transporter ClC-5 that acidic internal pH led to a greater increase in currents upon exchanging extracellular Cl(-) for SCN(-). However, transport uncoupling, unitary current amplitudes, and the voltage dependence of the depolarization-induced activation were not altered by low pH values. Therefore, it is likely that an additional gating process regulates ClC-5 transport. Higher internal [H(+)] and the proton glutamate mutant E268H altered the ratio between ClC-5 transport and nonlinear capacitance, indicating that the gating charge movements in ClC-5 arise from incomplete transport cycles and that internal protons increase the transport probability of ClC-5. This was substantiated by site-directed sulfhydryl modification of the proton glutamate mutant E268C. The mutation exhibited small transport currents together with prominent gating charge movements. The charge restoration using a negatively charged sulfhydryl reagent reinstated also the WT phenotype. Neutralization of the charge of the gating glutamate 211 by the E211C mutation abolished the effect of internal protons, showing that the increased transport probability of ClC-5 results from protonation of this residue. S168P (a mutation that decreases the anion affinity of the central binding site) reduced also the internal pH dependence of ClC-5. These results support the idea that protonation of the gating glutamate 211 at the central anion-binding site of ClC-5 is mediated by the proton glutamate 268.     

Reaction mechanism of the reconstituted aspartate/glutamate carrier from bovine heart mitochondria

A functional model for the aspartate/glutamate carrier of the inner mitochondrial membrane was established based on a kinetic evaluation of this transporter. Antiport kinetics were measured in proteoliposomes that contained partially purified carrier protein of definite transmembrane orientation (Dierks, T. and Kr?mer, R. (1988) Biochim. Biophys. Acta 937, 122-126). Bireactant initial velocity analyses of the counterexchange reaction were carried out varying substrate concentrations both in the internal and the external compartment. The kinetic patterns obtained were inconsistent with a pong-pong mechanism; rather they demonstrated the formation of a ternary complex as a consequence of sequential binding of one internal and one external substrate molecule to the carrier. Studies on transport activity in the presence of aspartate and glutamate in the same compartment (formally treated as substrate inhibition) clearly indicated that during exchange only one form of the carrier at either membrane surface exposes its binding sites, for which the two different substrates compete. In the deenergized state (pH 6.5) both substrates were translocated at about the same rate. Aspartate/glutamate antiport became asymmetric if a membrane potential was imposed, due to the electrogenic nature of the heteroexchange resulting from proton cotransport together with glutamate. Investigation of the electrical properties of aspartate/aspartate homoexchange led to the conclusion that the translocating carrier-substrate intermediate exhibits a transmembrane symmetry with respect to the (negative) charge, which again only is conceivable assuming a ternary complex. Thus, an antiport model is outlined that shows the functional complex of the carrier with two substrate molecules bound, one at either side of the membrane. The conformational change associated with the transition of both substrate molecules across the membrane then occurs in a single step. Furthermore the model implicates a distinct proton binding site, which is derived from the different influence of H+ concentration observed on transport affinity and transport velocity, respectively, when glutamate is used as a substrate.     

Modulation of the neural glutamate transporter EAAC1 by the addicsin-interacting protein ARL6IP1

Addicsin (Arl6ip5) is a murine homologue of rat glutamate transporter-associated protein 3-18 (GTRAP3-18), a putative negative modulator of Na+-dependent neural glutamate transporter-excitatory amino acid carrier 1 (EAAC1). Here we report that ADP-ribosylation factor-like 6 interacting protein 1 (Arl6ip1) is a novel addicsin-associated partner that indirectly promotes EAAC1-mediated glutamate transport activity in a protein kinase C activity-dependent manner. Like addicsin, Arl6ip1 is expressed in numerous tissues and proved likely to be co-localized with addicsin in certain neurons in the matured brain. Arl6ip1 was not translocated from the subcellular compartments under any of the test conditions and had no association with any molecules on the plasma membrane. Immunoprecipitation assay demonstrated that Arl6ip1 bound directly to addicsin and that the hydrophobic region located at amino acids 103-117 of addicsin was crucial to the formation of the Arl6ip1-addicsin heterodimer and addicsin homodimer. Glutamate transport assay revealed that increasing the expression of Arl6ip1 in C6BU-1 cells markedly enhanced Na+-dependent EAAC1-mediated glutamate transport activity in the presence of 100 nm phorbol 12-myristate 13-acetate. Under these conditions, kinetic analyses demonstrated that EAAC1 altered glutamate transport activity by increasing its glutamate affinity but not its maximal velocity. Meanwhile, increasing expression of addicsin Y110A/L112A mutant lacking binding ability for Arl6ip1 showed no enhancement of EAAC1-mediated glutamate transport activity, regardless of phorbol 12-myristate 13-acetate activation, suggesting that association between addicsin and Arl6ip1 causes altered EAAC1-mediated glutamate transport activity. Our findings suggest that Arl6ip1 is a novel addicsin-associated partner that promotes EAAC1-mediated glutamate transport activity by decreasing the number of addicsin molecules available for interaction with EAAC1.     

Cysteine Transport through Excitatory Amino Acid Transporter 3 (EAAT3)

Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1–5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1–3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.     

Transport of L-[14C]cystine and L-[14C]cysteine by subtypes of high affinity glutamate transporters over-expressed in HEK cells

Transport of L-cystine across the cell membrane is essential for synthesis of the major cellular antioxidant, glutathione (gamma-glutamylcysteinylglycine). In this study, uptake of L-[14C]cystine by three of the high affinity sodium-dependent mammalian glutamate transporters (GLT1, GLAST and EAAC1) individually expressed in HEK cells has been determined. All three transporters display saturable uptake of L-[14C]cystine with Michaelis affinity (K(m)) constants in the range of 20-110 microM. L-glutamate and L-homocysteate are potent inhibitors of sodium-dependent L-[14C]cystine uptake in HEK(GLAST), HEK(GLT1) and HEK(EAAC1) cells. Reduction of L-[14C]cystine to L-[14C]cysteine in the presence of 1mM cysteinylglycine increases the uptake rate in HEK(GLT1), HEK(GLAST) and HEK(EAAC1) cells, but only a small proportion (<10%) of L-[14C]cysteine uptake in HEK(GLT1) and HEK(GLAST) cells occurs by the high affinity glutamate transporters. The majority (>90%) of L-[14C]cysteine transport in these cells is mediated by the ASC transport system. In HEK(EAAC1) cells, on the other hand, L-[14C]cysteine is transported equally by the ASC and EAAC1 transporters. L-homocysteine inhibits L-[14C]cysteine transport in both HEK(GLAST) and HEK(GLT1) cells, but not in HEK(EAAC1) cells. It is concluded that the quantity of L-[14C]cyst(e)ine taken up by individual high affinity sodium-dependent glutamate transporters is determined both by the extracellular concentration of amino acids, such as glutamate and homocysteine, and by the extracellular redox potential, which will control the oxidation state of L-cystine.     

Cu(II) inhibition of the proton translocation machinery of the influenza A virus M2 protein

The homotetrameric M2 integral membrane protein of influenza virus forms a proton-selective ion channel. An essential histidine residue (His-37) in the M2 transmembrane domain is believed to play an important role in the conduction mechanism of this channel. Also, this residue is believed to form hydrogen-bonded interactions with the ammonium group of the anti-viral compound, amantadine. A molecular model of this channel suggests that the imidazole side chains of His-37 from symmetry-related monomers of the homotetrameric pore converge to form a coordination site for transition metals. Thus, membrane currents of oocytes of Xenopus laevis expressing the M2 protein were recorded when the solution bathing the oocytes contained various transition metals. Membrane currents were strongly and reversibly inhibited by Cu2+ with biphasic reaction kinetics. The biphasic inhibition curves may be explained by a two-site model involving a fast-binding peripheral site with low specificity for divalent metal ions, as well as a high affinity site (Kdiss approximately 2 microM) that lies deep within the pore and shows rather slow-binding kinetics (kon = 18.6 +/- 0.9 M-1 s-1). The pH dependence of the interaction with the high affinity Cu2+-binding site parallels the pH dependence of inhibition by amantadine, which has previously been ascribed to protonation of His-37. The voltage dependence of the inhibition at the high affinity site indicates that the binding site lies within the transmembrane region of the pore. Furthermore, the inhibition by Cu2+ could be prevented by prior application of the reversible blocker of M2 channel activity, BL-1743, providing further support for the location of the site within the pore region of M2. Finally, substitutions of His-37 by alanine or glycine eliminated the high affinity site and resulted in membrane currents that were only partially inhibited at millimolar concentrations of Cu2+. Binding of Cu2+ to the high affinity site resulted in an approximately equal inhibition of both inward and outward currents. The wild-type protein showed very high specificity for Cu2+ and was only partially inhibited by 1 mM Ni2+, Pt2+, and Zn2+. These data are discussed in terms of the functional role of His-37 in the mechanism of proton translocation through the channel.     

Functional properties of the Arabidopsis peptide transporters AtPTR1 and AtPTR5

The Arabidopsis di- and tripeptide transporters AtPTR1 and AtPTR5 were expressed in Xenopus laevis oocytes, and their selectivity and kinetic properties were determined by voltage clamping and by radioactive uptake. Dipeptide transport by AtPTR1 and AtPTR5 was found to be electrogenic and dependent on protons but not sodium. In the absence of dipeptides, both transporters showed proton-dependent leak currents that were inhibited by Phe-Ala (AtPTR5) and Phe-Ala, Trp-Ala, and Phe-Phe (AtPTR1). Phe-Ala was shown to reduce leak currents by binding to the substrate-binding site with a high apparent affinity. Inhibition of leak currents was only observed when the aromatic amino acids were present at the N-terminal position. AtPTR1 and AtPTR5 transport activity was voltage-dependent, and currents increased supralinearly with more negative membrane potentials and did not saturate. The voltage dependence of the apparent affinities differed between Ala-Ala, Ala-Lys, and Ala-Asp and was not conserved between the two transporters. The apparent affinity of AtPTR1 for these dipeptides was pH-dependent and decreased with decreasing proton concentration. In contrast to most proton-coupled transporters characterized so far, -I(max) increased at high pH, indicating that regulation of the transporter by pH overrides the importance of protons as co-substrate.     

Effect of nitrite-anions on Ca2+ transport into the sarcolemma of myometrium

The influence of nitrite-anions physiological concentration on Ca2+ input into vesicles was investigated when using the "outside-out" vesicles of myometrial plasmalemma and 45Ca2+. It was established that nitrite-anions increased Ca(2+)-permeability of plasmalemma and increased the affinity of cation-transport system. The effects are probably connected with reversible modification of glutamate residues that bound and transported Ca2+ within the membrane. These findings showed that nitrite-anions are competitive activators of the passive calcium transport. On the other hand the decrease of Ca2+ affinity for the transport system under transmembrane proton scattering by the membrane, by rapid dissipation of transmembrane delta pH. It may be possible that the dissipation of transmembrane proton gradient changed the conformation of calcium transport system that calls the difference of kinetic mechanism of NO2- action in case of delta pH = 0 and delta pH = 1.5 on vesicle membranes.