Molecular Determinants for Functional Differences between Alanine-Serine-Cysteine Transporter 1 and Other Glutamate Transporter Family Members

The ASCTs (alanine, serine, and cysteine transporters) belong to the solute carrier family 1 (SLC1), which also includes the human glutamate transporters (excitatory amino acid transporters, EAATs) and the prokaryotic aspartate transporter Glt_Ph. Despite the high degree of amino acid sequence identity between family members, ASCTs function quite differently from the EAATs and Glt_Ph. The aim of this study was to mutate ASCT1 to generate a transporter with functional properties of the EAATs and Glt_Ph, to further our understanding of the structural basis for the different transport mechanisms of the SLC1 family. We have identified three key residues involved in determining differences between ASCT1, the EAATs and Glt_Ph. ASCT1 transporters containing the mutations A382T, T459R, and Q386E were expressed in Xenopus laevis oocytes, and their transport and anion channel functions were investigated. A382T and T459R altered the substrate selectivity of ASCT1 to allow the transport of acidic amino acids, particularly l-aspartate. The combination of A382T and T459R within ASCT1 generates a transporter with a similar profile to that of Glt_Ph, with preference for l-aspartate over l-glutamate. Interestingly, the amplitude of the anion conductance activated by the acidic amino acids does not correlate with rates of transport, highlighting the distinction between these two processes. Q386E impaired the ability of ASCT1 to bind acidic amino acids at pH 5.5; however, this was reversed by the additional mutation A382T. We propose that these residues differences in TM7 and TM8 combine to determine differences in substrate selectivity between members of the SLC1 family.     

The Split Personality of Glutamate Transporters: A Chloride Channel and a Transporter

Transporters and ion channels are conventionally categorised into distinct classes of membrane proteins. However, some membrane proteins have a split personality and can function as both transporters and ion channels. The excitatory amino acid transporters (EAATs) in particular, function as both glutamate transporters and chloride (Cl^?) channels. The EAATs couple the transport of glutamate to the co-transport of three Na⁺ ions and one H⁺ ion into the cell, and the counter-transport of one K⁺ ion out of the cell. The EAAT Cl^? channel is activated by the binding of glutamate and Na⁺, but is thermodynamically uncoupled from glutamate transport and involves molecular determinants distinct from those responsible for glutamate transport. Several crystal structures of an EAAT archaeal homologue, Glt_Ph, at different stages of the transport cycle, alongside numerous functional studies and molecular dynamics simulations, have provided extensive insights into the mechanism of substrate transport via these transporters. However, the molecular determinants involved in Cl^? permeation, and the mechanism by which this channel is activated are not entirely understood. Here we will discuss what is currently known about the molecular determinants involved in EAAT-mediated Cl^? permeation and the mechanisms that underlie their split personality.     

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh

Glt_Ph from Pyrococcus horikoshii is a homotrimeric Na⁺-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in Glt_Ph consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na⁺ and aspartate binding to Glt_Ph. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na⁺ binds with low affinity to the apoprotein (K_d 120 mm), with a particularly low k_on value (5.1 m⁻¹s⁻¹). At least two sodium ions bind before aspartate. The binding of Na⁺ requires a very high activation energy (E_a 106.8 kJ mol⁻¹) and consequently has a large Q₁₀ value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na⁺ concentration present. Binding of aspartate was not observed in the absence of Na⁺, whereas in the presence of high Na⁺ concentrations (above the K_d for Na⁺) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (k_on of 1.4 × 10⁵ m⁻¹s⁻¹), with low E_a and Q₁₀ values (42.6 kJ mol⁻¹ and 1.8, respectively). We conclude that Na⁺ binding is most likely the rate-limiting step for substrate binding.     

Free energy simulations of ligand binding to the aspartate transporter Glt(Ph)

Glutamate/Aspartate transporters cotransport three Na⁺ and one H⁺ ions with the substrate and countertransport one K⁺ ion. The binding sites for the substrate and two Na⁺ ions have been observed in the crystal structure of the archeal homolog Glt_Ph, while the binding site for the third Na⁺ ion has been proposed from computational studies and confirmed by experiments. Here we perform detailed free energy simulations of Glt_Ph, giving a comprehensive characterization of the substrate and ion binding sites, and calculating their binding free energies in various configurations. Our results show unequivocally that the substrate binds after the binding of two Na⁺ ions. They also shed light into Asp/Glu selectivity of Glt_Ph, which is not observed in eukaryotic glutamate transporters.     

Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters

Crystal structures of the archaeal homologue Glt_Ph have provided important insights into the molecular mechanism of transport of the excitatory neurotransmitter glutamate. Whereas mammalian glutamate transporters can translocate both glutamate and aspartate, Glt_Ph is only one capable of aspartate transport. Most of the amino acid residues that surround the aspartate substrate in the binding pocket of Glt_Ph are highly conserved. However, in the brain transporters, Thr-352 and Met-362 of the reentrant hairpin loop 2 are replaced by the smaller Ala and Thr, respectively. Therefore, we have studied the effects of T352A and M362T on binding and transport of aspartate and glutamate by Glt_Ph. Substrate-dependent intrinsic fluorescence changes were monitored in transporter constructs containing the L130W mutation. Glt_Ph-L130W/T352A exhibited an ∼15-fold higher apparent affinity for l-glutamate than the wild type transporter, and the M362T mutation resulted in an increased affinity of ∼40-fold. An even larger increase of the apparent affinity for l-glutamate, around 130-fold higher than that of wild type, was observed with the T352A/M362T double mutant. Radioactive uptake experiments show that Glt_Ph-T352A not only transports aspartate but also l-glutamate. Remarkably, Glt_Ph-M362T exhibited l-aspartate but not l-glutamate transport. The double mutant retained the ability to transport l-glutamate, but its kinetic parameters were very similar to those of Glt_Ph-T352A alone. The differential impact of mutation on binding and transport of glutamate suggests that hairpin loop 2 not only plays a role in the selection of the substrate but also in its translocation.     

The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous Glt_Ph coordinates l-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na⁺ over Li⁺. S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for l-aspartate over d-aspartate and l-glutamate, and lost their selectivity for Na⁺ over Li⁺. Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.     

Transport Rates of a Glutamate Transporter Homologue Are Influenced by the Lipid Bilayer

The aspartate transporter from Pyrococcus horikoshii (Glt_Ph) is a model for the structure of the SLC1 family of amino acid transporters. Crystal structures of Glt_Ph provide insight into mechanisms of ion coupling and substrate transport; however, structures have been solved in the absence of a lipid bilayer so they provide limited information regarding interactions that occur between the protein and lipids of the membrane. Here, we investigated the effect of the lipid environment on aspartate transport by reconstituting Glt_Ph into liposomes of defined lipid composition where the primary lipid is phosphatidylethanolamine (PE) or its methyl derivatives. We showed that the rate of aspartate transport and the transmembrane orientation of Glt_Ph were influenced by the primary lipid in the liposomes. In PE liposomes, we observed the highest transport rate and showed that 85% of the transporters were orientated right-side out, whereas in trimethyl PE liposomes, 50% of transporters were right-side out, and we observed a 4-fold reduction in transport rate. Differences in orientation can only partially explain the lipid composition effect on transport rate. Crystal structures of Glt_Ph revealed a tyrosine residue (Tyr-33) that we propose interacts with lipid headgroups during the transport cycle. Based on site-directed mutagenesis, we propose that a cation-π interaction between Tyr-33 and the lipid headgroups can influence conformational flexibility of the trimerization domain and thus the rate of transport. These results provide a specific example of how interactions between membrane lipids and membrane-bound proteins can influence function and highlight the importance of the role of the membrane in transporter function.     

Molecular Dynamics Simulations Elucidate the Mechanism of Proton Transport in the Glutamate Transporter EAAT3

The uptake of glutamate in nerve synapses is carried out by the excitatory amino acid transporters (EAATs), involving the cotransport of a proton and three Na⁺ ions and the countertransport of a K⁺ ion. In this study, we use an EAAT3 homology model to calculate the pK_a of several titratable residues around the glutamate binding site to locate the proton carrier site involved in the translocation of the substrate. After identifying E374 as the main candidate for carrying the proton, we calculate the protonation state of this residue in different conformations of EAAT3 and with different ligands bound. We find that E374 is protonated in the fully bound state, but removing the Na2 ion and the substrate reduces the pK_a of this residue and favors the release of the proton to solution. Removing the remaining Na⁺ ions again favors the protonation of E374 in both the outward- and inward-facing states, hence the proton is not released in the empty transporter. By calculating the pK_a of E374 with a K⁺ ion bound in three possible sites, we show that binding of the K⁺ ion is necessary for the release of the proton in the inward-facing state. This suggests a mechanism in which a K⁺ ion replaces one of the ligands bound to the transporter, which may explain the faster transport rates of the EAATs compared to its archaeal homologs.     

Mechanism of Transport Modulation by an Extracellular Loop in an Archaeal Excitatory Amino Acid Transporter (EAAT) Homolog

Secondary transporters in the excitatory amino acid transporter family terminate glutamatergic synaptic transmission by catalyzing Na⁺-dependent removal of glutamate from the synaptic cleft. Recent structural studies of the aspartate-specific archaeal homolog, Glt_Ph, suggest that transport is achieved by a rigid body, piston-like movement of the transport domain, which houses the substrate-binding site, between the extracellular and cytoplasmic sides of the membrane. This transport domain is connected to an immobile scaffold by three loops, one of which, the 3–4 loop (3L4), undergoes substrate-sensitive conformational change. Proteolytic cleavage of the 3L4 was found to abolish transport activity indicating an essential function for this loop in the transport mechanism. Here, we demonstrate that despite the presence of fully cleaved 3L4, Glt_Ph is still able to sample conformations relevant for transport. Optimized reconstitution conditions reveal that fully cleaved Glt_Ph retains some transport activity. Analysis of the kinetics and temperature dependence of transport accompanied by direct measurements of substrate binding reveal that this decreased transport activity is not due to alteration of the substrate binding characteristics but is caused by the significantly reduced turnover rate. By measuring solute counterflow activity and cross-link formation rates, we demonstrate that cleaving 3L4 severely and specifically compromises one or more steps contributing to the movement of the substrate-loaded transport domain between the outward- and inward-facing conformational states, sparing the equivalent step(s) during the movement of the empty transport domain. These results reveal a hitherto unknown role for the 3L4 in modulating an essential step in the transport process.     

Molecular Dynamics Simulations Elucidate the Mechanism of Proton Transport in the Glutamate Transporter EAAT3

The uptake of glutamate in nerve synapses is carried out by the excitatory amino acid transporters (EAATs), involving the cotransport of a proton and three Na⁺ ions and the countertransport of a K⁺ ion. In this study, we use an EAAT3 homology model to calculate the pK_a of several titratable residues around the glutamate binding site to locate the proton carrier site involved in the translocation of the substrate. After identifying E374 as the main candidate for carrying the proton, we calculate the protonation state of this residue in different conformations of EAAT3 and with different ligands bound. We find that E374 is protonated in the fully bound state, but removing the Na2 ion and the substrate reduces the pK_a of this residue and favors the release of the proton to solution. Removing the remaining Na⁺ ions again favors the protonation of E374 in both the outward- and inward-facing states, hence the proton is not released in the empty transporter. By calculating the pK_a of E374 with a K⁺ ion bound in three possible sites, we show that binding of the K⁺ ion is necessary for the release of the proton in the inward-facing state. This suggests a mechanism in which a K⁺ ion replaces one of the ligands bound to the transporter, which may explain the faster transport rates of the EAATs compared to its archaeal homologs.     

The discovery of slowness: low-capacity transport and slow anion channel gating by the glutamate transporter EAAT5

Excitatory amino acid transporters (EAATs) control the glutamate concentration in the synaptic cleft by glial and neuronal glutamate uptake. Uphill glutamate transport is achieved by the co-/countertransport of Na⁺ and other ions down their concentration gradients. Glutamate transporters also display an anion conductance that is activated by the binding of Na⁺ and glutamate but is not thermodynamically coupled to the transport process. Of the five known glutamate transporter subtypes, the retina-specific subtype EAAT5 has the largest conductance relative to glutamate uptake activity. Our results suggest that EAAT5 behaves as a slow-gated anion channel with little glutamate transport activity. At steady state, EAAT5 was activated by glutamate, with a K_m= 61 ± 11 μM. Binding of Na⁺ to the empty transporter is associated with a K_m = 229 ± 37 mM, and binding to the glutamate-bound form is associated with a K_m = 76 ± 40 mM. Using laser-pulse photolysis of caged glutamate, we determined the pre-steady-state kinetics of the glutamate-induced anion current of EAAT5. This was characterized by two exponential components with time constants of 30 ± 1 ms and 200 ± 15 ms, which is an order of magnitude slower than those observed in other glutamate transporters. A voltage-jump analysis of the anion currents indicates that the slow activation behavior is caused by two slow, rate-limiting steps in the transport cycle, Na⁺ binding to the empty transporter, and translocation of the fully loaded transporter. We propose a kinetic transport scheme that includes these two slow steps and can account for the experimentally observed data. Overall, our results suggest that EAAT5 may not act as a classical high-capacity glutamate transporter in the retina; rather, it may function as a slow-gated glutamate receptor and/or glutamate buffering system.     

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.     

Identification of the Third Na Site and the Sequence of Extracellular Binding Events in the Glutamate Transporter

The transport cycle in the glutamate transporter (GlT) is catalyzed by the cotransport of three Na⁺ ions. However, the positions of only two of these ions (Na1 and Na2 sites) along with the substrate have been captured in the crystal structures reported for both the outward-facing and the inward-facing states of Glt_ph. Characterizing the third ion binding site (Na3) is necessary for structure-function studies attempting to investigate the mechanism of transport in GlTs at an atomic level, particularly for the determination of the sequence of the binding events during the transport cycle. In this study, we report a series of molecular dynamics simulations performed on various bound states of Glt_ph (the apo state, as well as in the presence of Na⁺, the substrate, or both), which have been used to identify a putative Na3 site. The calculated trajectories have been used to determine the water accessibility of potential ion-binding residues in the protein, as a prerequisite for their ion binding. Combined with conformational analysis of the key regions in the protein in different bound states and several additional independent simulations in which a Na⁺ ion was randomly introduced to the interior of the transporter, we have been able to characterize a putative Na3 site and propose a plausible binding sequence for the substrate and the three Na⁺ ions to the transporter during the extracellular half of the transport cycle. The proposed Na3 site is formed by a set of highly conserved residues, namely, Asp³¹², Thr⁹², and Asn³¹⁰, along with a water molecule. Simulation of a fully bound state, including the substrate and the three Na⁺ ions, reveals a stable structure—showing closer agreement to the crystal structure when compared to previous models lacking an ion in the putative Na3 site. The proposed sequence of binding events is in agreement with recent experimental models suggesting that two Na⁺ ions bind before the substrate, and one after that. Our results, however, provide additional information about the sites involved in these binding events.     

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.     

The glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Transport mechanism, regulation by ATP and characterization of the glutamine/glutamate antiport

The glutamine/amino acid transporter solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes has been previously identified as the ASCT2 transporter. The reconstituted transporter catalyses an antiport reaction in which external glutamine and Na⁺ are cotransported in exchange with internal glutamine (or other amino acids). The glutamine-Na⁺ cotransport occurred with a 1:1 stoichiometry. The concentration of Na⁺ did not influence the Km for glutamine and vice versa. Experimental data obtained by a bi-substrate analysis of the glutamine-Na⁺ cotransport, together with previous report on the glutamine_ex/glutamine_in pseudo bi-reactant analysis, indicated that the transporter catalyses a three-substrate transport reaction with a random simultaneous mechanism. The presence of ATP in the internal compartment of the proteoliposomes led to an increase of the Vmax of the transport and to a decrease of the Km of the transporter for external Na⁺. The reconstituted glutamine/amino acid transporter was inhibited by glutamate; the inhibition was more pronounced at acidic pH. A kinetic analysis revealed that the inhibition was competitive with respect to glutamine. Glutamate was also transported in exchange with glutamine. The external Km of the transporter for glutamate (13.3 mM) was slightly higher than the internal one (8.3 mM). At acidic pH the external but not the internal Km decreased. According with the Km values, glutamate should be transported preferentially from inside to outside in exchange for external glutamine and Na⁺.     

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.     

Functional Properties of the Retinal Glutamate Transporters GLT-1c and EAAT5

In the mammalian retina, glutamate uptake is mediated by members of a family of glutamate transporters known as “excitatory amino acid transporters (EAATs).” Here we cloned and functionally characterized two retinal EAATs from mouse, the GLT-1/EAAT2 splice variant GLT-1c, and EAAT5. EAATs are glutamate transporters and anion-selective ion channels, and we used heterologous expression in mammalian cells, patch-clamp recordings and noise analysis to study and compare glutamate transport and anion channel properties of both EAAT isoforms. We found GLT-1c to be an effective glutamate transporter with high affinity for Na⁺ and glutamate that resembles original GLT-1/EAAT2 in all tested functional aspects. EAAT5 exhibits glutamate transport rates too low to be accurately measured in our experimental system, with significantly lower affinities for Na⁺ and glutamate than GLT-1c. Non-stationary noise analysis demonstrated that GLT-1c and EAAT5 also differ in single-channel current amplitudes of associated anion channels. Unitary current amplitudes of EAAT5 anion channels turned out to be approximately twice as high as single-channel amplitudes of GLT-1c. Moreover, at negative potentials open probabilities of EAAT5 anion channels were much larger than for GLT-1c. Our data illustrate unique functional properties of EAAT5, being a low-affinity and low-capacity glutamate transport system, with an anion channel optimized for anion conduction in the negative voltage range.     

Voltage-dependent processes in the electroneutral amino acid exchanger ASCT2

Neutral amino acid exchange by the alanine serine cysteine transporter (ASCT)2 was reported to be electroneutral and coupled to the cotransport of one Na⁺ ion. The cotransported sodium ion carries positive charge. Therefore, it is possible that amino acid exchange is voltage dependent. However, little information is available on the electrical properties of the ASCT2 amino acid transport process. Here, we have used a combination of experimental and computational approaches to determine the details of the amino acid exchange mechanism of ASCT2. The [Na⁺] dependence of ASCT2-associated currents indicates that the Na⁺/amino acid stoichiometry is at least 2:1, with at least one sodium ion binding to the amino acid–free apo form of the transporter. When the substrate and two Na⁺ ions are bound, the valence of the transport domain is +0.81. Consistently, voltage steps applied to ASCT2 in the fully loaded configuration elicit transient currents that decay on a millisecond time scale. Alanine concentration jumps at the extracellular side of the membrane are followed by inwardly directed transient currents, indicative of translocation of net positive charge during exchange. Molecular dynamics simulations are consistent with these results and point to a sequential binding process in which one or two modulatory Na⁺ ions bind with high affinity to the empty transporter, followed by binding of the amino acid substrate and the subsequent binding of a final Na⁺ ion. Overall, our results are consistent with voltage-dependent amino acid exchange occurring on a millisecond time scale, the kinetics of which we predict with simulations. Despite some differences, transport mechanism and interaction with Na⁺ appear to be highly conserved between ASCT2 and the other members of the solute carrier 1 family, which transport acidic amino acids.     

The 4b-4c loop of excitatory amino acid transporter 1 containing four critical residues essential for substrate transport

Abstract

In the mammalians, the 4b-4c loop of excitatory amino acid transporters (EAATs) spans more than 50 amino-acid residues that are absent in glutamate transporter homologue of Pyrococcus horikoshii (Glt_Ph). This part of insertion is unique for metazoans and indispensable to the localization of EAATs. The excitatory amino acid transporter (EAAT) 1 is one of the two glial glutamate transporters, which are responsible for efficiently clearing glutamate from the synaptic cleft to prevent neurotoxicity and cell death. Although the crystal structure of EAAT1_cryst (a human thermostable EAAT1) was resolved in 2017, the structure-function relationship of the 4b-4c loop has not been elucidated in EAAT1_cryst. To investigate the role of the 4b-4c loop, we performed alanine-scanning mutagenesis in the mutants and observed dramatically decreased transport activities in T192A, Y194A, N242A, and G245A mutants. The surface expression of T192A and Y194A mutants even decreased by more than 80%, and most of them were detained in the cytoplasm. However, when T192 and Y194 were substituted with conservative residues, the transport activities and the surface expressions of T192S and Y194F were largely recovered, and their kinetic parameters (K_m values) were comparable to the wild-type EAAT1 as well. In contrast, N242 and G245 substituted with conservative residues could not rescue the uptake function, suggesting that N242 and G245 may play irreplaceable roles in the glutamate uptake process. These results indicate that the 4b-4c loop of EAAT1 may not only affect the glutamate uptake activity, but also influence the surface localization of EAAT1 by T192 and Y194.

Communicated by Ramaswamy H. Sarma     

The Domain Interface of the Human Glutamate Transporter EAAT1 Mediates Chloride Permeation

The concentration of glutamate within the glutamatergic synapse is tightly regulated by the excitatory amino-acid transporters (EAATs). In addition to their primary role of clearing extracellular glutamate, the EAATs also possess a thermodynamically uncoupled Cl⁻ conductance. Several crystal structures of an archaeal EAAT homolog, Glt_Ph, at different stages of the transport cycle have been solved. In a recent structure, an aqueous cavity located at the interface of the transport and trimerization domains has been identified. This cavity is lined by polar residues, several of which have been implicated in Cl⁻ permeation. We hypothesize that this cavity opens during the transport cycle to form the Cl⁻ channel. Residues lining this cavity in EAAT1, including Ser-366, Leu-369, Phe-373, Arg-388, Pro-392, and Thr-396, were mutated to small hydrophobic residues. Wild-type and mutant transporters were expressed in Xenopus laevis oocytes and two-electrode voltage-clamp electrophysiology, and radiolabeled substrate uptake was used to investigate function. Significant alterations in substrate-activated Cl⁻ conductance were observed for several mutant transporters. These alterations support the hypothesis that this aqueous cavity at the interface of the transport and trimerization domains is a partially formed Cl⁻ channel, which opens to form a pore through which Cl⁻ ions pass. This study enhances our understanding as to how glutamate transporters function as both amino-acid transporters and Cl⁻ channels.