Transient kinetics reveal the mechanism of competitive inhibition of the neutral amino acid transporter ASCT2.

ASCT2 (alanine serine cysteine transporter 2), a member of the solute carrier 1 family, mediates Na+-dependent exchange of small neutral amino acids across cell membranes. ASCT2 was shown to be highly expressed in tumor cells, making it a promising target for anticancer therapies. In this study, we explored the binding mechanism of the high-affinity competitive inhibitor L-cis hydroxyproline biphenyl ester (Lc-BPE) with ASCT2, using electrophysiological and rapid kinetic methods. Our investigations reveal that Lc-BPE binding requires one or two Na+ ions initially bound to the apo-transporter with high affinity, with Na1 site occupancy being more critical for inhibitor binding. In contrast to the amino acid substrate bound form, the final, third Na+ ion cannot bind, due to distortion of its binding site (Na2), thus preventing the formation of a translocation-competent complex. Based on the rapid kinetic analysis, the application of Lc-BPE generated outward transient currents, indicating that despite its net neutral nature, the binding of Lc-BPE in ASCT2 is weakly electrogenic, most likely because of asymmetric charge distribution within the amino acid moiety of the inhibitor. The preincubation with Lc-BPE also led to a decrease of the turnover rate of substrate exchange and a delay in the activation of substrate-induced anion current, indicating relatively slow Lc-BPE dissociation kinetics. Overall, our results provide new insight into the mechanism of binding of a prototypical competitive inhibitor to the ASCT transporters.

Rational design of ASCT2 inhibitors using an integrated experimental-computational approach.

ASCT2 (SLC1A5) is a sodium-dependent neutral amino acid transporter that controls amino acid homeostasis in peripheral tissues. In cancer, ASCT2 is up-regulated where it modulates intracellular glutamine levels, fueling cell proliferation. Nutrient deprivation via ASCT2 inhibition provides a potential strategy for cancer therapy. Here, we rationally designed stereospecific inhibitors exploiting specific subpockets in the substrate binding site using computational modeling and cryo-electron microscopy (cryo-EM). The final structures combined with molecular dynamics simulations reveal multiple pharmacologically relevant conformations in the ASCT2 binding site as well as a previously unknown mechanism of stereospecific inhibition. Furthermore, this integrated analysis guided the design of a series of unique ASCT2 inhibitors. Our results provide a framework for future development of cancer therapeutics targeting nutrient transport via ASCT2, as well as demonstrate the utility of combining computational modeling and cryo-EM for solute carrier ligand discovery.

Pre-Steady-State Kinetics and Reverse Transport in Rat Glutamate Transporter EAAC1 with an Immobilized Transport Domain.

Plasma membrane glutamate transporters move glutamate across the cell membrane in a process that is thought to involve elevator-like movement of the transport domain relative to the static trimerization domain. Conformational changes associated with this elevator-like movement have been blocked by covalent crosslinking of cysteine pairs inserted strategically in several positions in the transporter structure, resulting in inhibition of steady-state transport activity. However, it is not known how these crosslinking restraints affect other partial reactions of the transporter that were identified based on pre-steady-state kinetic analysis. Here, we re-examine two different introduced cysteine pairs in the rat glutamate transporter EAAC1 recombinantely expressed in HEK293 cells, W440C/K268C and K64C/V419C, with respect to the molecular mechanism of their impairment of transporter function. Pre-steady-state kinetic studies of glutamate-induced partial reactions were performed using laser photolysis of caged glutamate to achieve sub-millisecond time resolution. Crosslinking of both cysteine pairs abolished steady-state transport current, as well as the majority of pre-steady-state glutamate-induced charge movements, in both forward and reverse transport mode, suggesting that it is not only the elevator-like movement associated with translocation, but also other transporter partial reactions that are inhibited. In contrast, sodium binding to the empty transporter, and glutamate-induced anion conductance were still intact after the W440C/K268C crosslink. Our results add to the previous mechanistic view of how covalent restraints of the transporter affect function and structural changes linked to individual steps in the transport cycle.

Interaction of the neutral amino acid transporter ASCT2 with basic amino acids.

Glutamine transport across cell membranes is performed by a variety of transporters, including the alanine serine cysteine transporter 2 (ASCT2). The substrate-binding site of ASCT2 was proposed to be specific for small amino acids with neutral side chains, excluding basic substrates such as lysine. A series of competitive inhibitors of ASCT2 with low µM affinity were developed previously, on the basis of the 2,4-diaminobutyric acid (DAB) scaffold with a potential positive charge in the side chain. Therefore, we tested whether basic amino acids with side chains shorter than lysine can interact with the ASCT2 binding site. Molecular docking of L-1,3-diaminopropionic acid (L-DAP) and L-DAB suggested that these compounds bind to ASCT2. Consistent with this prediction, L-DAP and L-DAB, but not ornithine, lysine or D-DAP, elicited currents when applied to ASCT2-expressing cells. The currents were carried by anions and showed the hallmark properties of ASCT2 currents induced by transported substrates. The L-DAP response could be eliminated by a competitive ASCT2 inhibitor, suggesting that binding occurs at the substrate binding site. The KM for L-DAP was weakly voltage dependent. Furthermore, the pH dependence of the L-DAP response showed that the compound can bind in several protonation states. Together, these results suggest that the ASCT2 binding site is able to recognize L-amino acids with short, basic side chains, such as the L-DAP derivative ß-N-methylamino-l-Alanine (BMAA), a well-studied neurotoxin. Our results expand the substrate specificity of ASCT2 to include amino acid substrates with positively charged side chains.

A K+/Na+ co-binding state: Simultaneous versus competitive binding of K+ and Na+ to glutamate transporters.

Plasma membrane-associated glutamate transporters play a key role in signaling by the major excitatory neurotransmitter glutamate. Uphill glutamate uptake into cells is energetically driven by coupling to co-transport of three Na+ ions. In exchange, one K+ ion is counter-transported. Currently accepted transport mechanisms assume that Na+ and K+ effects are exclusive, resulting from competition of these cations at the binding level. Here, we used electrophysiological analysis to test the effects of K+ and Na+ on neuronal glutamate transporter excitatory amino acid carrier 1 (EAAC1; the rat homologue of human excitatory amino acid transporter 3 (EAAT3)). Unexpectedly, extracellular K+ application to EAAC1 induced anion current, but only in the presence of Na+ This result could be explained with a K+/Na+ co-binding state in which the two cations simultaneously bind to the transporter. We obtained further evidence for this co-binding state, and its anion conductance, by analyzing transient currents when Na+ was exchanged for K+ and effects of the [K+]/[Na+] ratio on glutamate affinity. Interestingly, we observed the K+/Na+ co-binding state not only in EAAC1 but also in the subtypes EAAT1 and -2, which, unlike EAAC1, conducted anions in response to K+ only. We incorporated these experimental findings in a revised transport mechanism, including the K+/Na+ co-binding state and the ability of K+ to activate anion current. Overall, these results suggest that differentiation between Na+ and K+ does not occur at the binding level but is conferred by coupling of cation binding to conformational changes. These findings have implications also for other exchangers.

A Quantitative Model for BicD2/Cargo Interactions.

Dynein adaptor proteins such as Bicaudal D2 (BicD2) are integral components of the dynein transport machinery, as they recognize cargoes for cell cycle-specific transport and link them to the motor complex. Human BicD2 switches from selecting secretory and Golgi-derived vesicles for transport in G1 and S phase (by recognizing Rab6GTP), to selecting the nucleus for transport in G2 phase (by recognizing nuclear pore protein Nup358), but the molecular mechanisms governing this switch are elusive. Here, we have developed a quantitative model for BicD2/cargo interactions that integrates affinities, oligomeric states, and cellular concentrations of the reactants. BicD2 and cargo form predominantly 2:2 complexes. Furthermore, the affinity of BicD2 toward its cargo Nup358 is higher than that toward Rab6GTP. Based on our calculations, an estimated 1000 BicD2 molecules per cell would be recruited to the nucleus through Nup358 in the absence of regulation. Notably, RanGTP is a negative regulator of the Nup358/BicD2 interaction that weakens the affinity by a factor of 10 and may play a role in averting dynein recruitment to the nucleus outside of the G2 phase. However, our quantitative model predicts that an additional negative regulator remains to be identified. In the absence of negative regulation, the affinity of Nup358 would likely be sufficient to recruit BicD2 to the nucleus in G2 phase. Our quantitative model makes testable predictions of how cellular transport events are orchestrated. These transport processes are important for brain development, cell cycle control, signaling, and neurotransmission at synapses.

Electrogenic Steps Associated with Substrate Binding to the Neuronal Glutamate Transporter EAAC1.

Glutamate transporters actively take up glutamate into the cell, driven by the co-transport of sodium ions down their transmembrane concentration gradient. It was proposed that glutamate binds to its binding site and is subsequently transported across the membrane in the negatively charged form. With the glutamate binding site being located partially within the membrane domain, the possibility has to be considered that glutamate binding is dependent on the transmembrane potential and, thus, is electrogenic. Experiments presented in this report test this possibility. Rapid application of glutamate to the wild-type glutamate transporter subtype EAAC1 (excitatory amino acid carrier 1) through photo-release from caged glutamate generated a transient inward current, as expected for the electrogenic inward movement of co-transported Na(+) In contrast, glutamate application to a transporter with the mutation A334E induced transient outward current, consistent with movement of negatively charged glutamate into its binding site within the dielectric of the membrane. These results are in agreement with electrostatic calculations, predicting a valence for glutamate binding of -0.27. Control experiments further validate and rule out other possible explanations for the transient outward current. Electrogenic glutamate binding can be isolated in the mutant glutamate transporter because reactions, such as glutamate translocation and/or Na(+) binding to the glutamate-bound state, are inhibited by the A334E substitution. Electrogenic glutamate binding has to be considered together with other voltage-dependent partial reactions to cooperatively determine the voltage dependence of steady-state glutamate uptake and glutamate buffering at the synapse.

Distinct Membrane Disruption Pathways Are Induced by 40-Residue ß-Amyloid Peptides.

Cellular membrane disruption induced by ß-amyloid (Aß) peptides has been considered one of the major pathological mechanisms for Alzheimer disease. Mechanistic studies of the membrane disruption process at a high-resolution level, on the other hand, are hindered by the co-existence of multiple possible pathways, even in simplified model systems such as the phospholipid liposome. Therefore, separation of these pathways is crucial to achieve an in-depth understanding of the Aß-induced membrane disruption process. This study, which utilized a combination of multiple biophysical techniques, shows that the peptide-to-lipid (P:L) molar ratio is an important factor that regulates the selection of dominant membrane disruption pathways in the presence of 40-residue Aß peptides in liposomes. Three distinct pathways (fibrillation with membrane content leakage, vesicle fusion, and lipid uptake through a temporarily stable ionic channel) become dominant in model liposome systems under specific conditions. These individual systems are characterized by both the initial states of Aß peptides and the P:L molar ratio. Our results demonstrated the possibility to generate simplified Aß-membrane model systems with a homogeneous membrane disruption pathway, which will benefit high-resolution mechanistic studies in the future. Fundamentally, the possibility of pathway selection controlled by P:L suggests that the driving forces for Aß aggregation and Aß-membrane interactions may be similar at the molecular level.

Structure activity relationships of benzylproline-derived inhibitors of the glutamine transporter ASCT2.

The glutamine transporter ASCT2 has been identified as a promising target to inhibit rapid growth of cancer cells. However, ASCT2 pharmacology is not well established. In this report, we performed a systematic structure activity analysis of a series of substituted benzylproline derivatives. Substitutions on the phenyl ring resulted in compounds with characteristics of ASCT2 inhibitors. Apparent binding affinity increased with increasing hydrophobicity of the side chain. In contrast, interaction of the ASCT2 binding site with specific positions on the phenyl ring was not observed. The most potent compound inhibits the ASCT2 anion conductance with a Ki of 3µM, which is in the same range as that of more bulky and higher molecular weight inhibitors recently reported by others. The experimental results are consistent with computational analysis based on docking of the inhibitors against an ASCT2 homology model. The benzylproline scaffold provides a valuable tool for further improving binding potency of future ASCT2 inhibitors.

Ligand Discovery for the Alanine-Serine-Cysteine Transporter (ASCT2, SLC1A5) from Homology Modeling and Virtual Screening.

The Alanine-Serine-Cysteine transporter ASCT2 (SLC1A5) is a membrane protein that transports neutral amino acids into cells in exchange for outward movement of intracellular amino acids. ASCT2 is highly expressed in peripheral tissues such as the lung and intestines where it contributes to the homeostasis of intracellular concentrations of neutral amino acids. ASCT2 also plays an important role in the development of a variety of cancers such as melanoma by transporting amino acid nutrients such as glutamine into the proliferating tumors. Therefore, ASCT2 is a key drug target with potentially great pharmacological importance. Here, we identify seven ASCT2 ligands by computational modeling and experimental testing. In particular, we construct homology models based on crystallographic structures of the aspartate transporter GltPh in two different conformations. Optimization of the models' binding sites for protein-ligand complementarity reveals new putative pockets that can be targeted via structure-based drug design. Virtual screening of drugs, metabolites, fragments-like, and lead-like molecules from the ZINC database, followed by experimental testing of 14 top hits with functional measurements using electrophysiological methods reveals seven ligands, including five activators and two inhibitors. For example, aminooxetane-3-carboxylate is a more efficient activator than any other known ASCT2 natural or unnatural substrate. Furthermore, two of the hits inhibited ASCT2 mediated glutamine uptake and proliferation of a melanoma cancer cell line. Our results improve our understanding of how substrate specificity is determined in amino acid transporters, as well as provide novel scaffolds for developing chemical tools targeting ASCT2, an emerging therapeutic target for cancer and neurological disorders.

Protonation-Driven Membrane Insertion of a pH-Low Insertion Peptide.

The pH-low insertion peptide (pHLIP) inserts into membranes and forms a transmembrane (TM) α-helix in response to slight acidity, and has shown great potential for cancer diagnosis and treatment. As a lead, pHLIP is challenging to optimize because the mechanism of its pH-dependent membrane interactions is not completely understood. Within pHLIP there are multiple D/E residues which could sense the pH change, the particular role played by each of them in the protonation-driven insertion process is not clear. The precise location of the TM helix within the pHLIP sequence is also unknown. In this work, solid-state NMR spectroscopy is used to address these central questions. Tracing backbone conformations revealed that the TM helix spans from A10 to D33 with a break at T19 to P20. Residue-specific pKa values of D31, D33, D25, and D14 were determined to be 6.5, 6.3, 6.1, and 5.8, respectively, and define the sequence of protonations which lead to insertion. Furthermore, possible intermediate states which disrupt membranes at pH 6.4 were proposed based on tryptophan fluorescence quenching and NMR data.

SLC1 glutamate transporters.

The plasma membrane transporters for the neurotransmitter glutamate belong to the solute carrier 1 family. They are secondary active transporters, taking up glutamate into the cell against a substantial concentration gradient. The driving force for concentrative uptake is provided by the cotransport of Na(+) ions and the countertransport of one K(+) in a step independent of the glutamate translocation step. Due to eletrogenicity of transport, the transmembrane potential can also act as a driving force. Glutamate transporters are expressed in many tissues, but are of particular importance in the brain, where they contribute to the termination of excitatory neurotransmission. Glutamate transporters can also run in reverse, resulting in glutamate release from cells. Due to these important physiological functions, glutamate transporter expression and, therefore, the transport rate, are tightly regulated. This review summarizes recent literature on the functional and biophysical properties, structure-function relationships, regulation, physiological significance, and pharmacology of glutamate transporters. Particular emphasis is on the insight from rapid kinetic and electrophysiological studies, transcriptional regulation of transporter expression, and reverse transport and its importance for pathophysiological glutamate release under ischemic conditions.

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.

Design and Characterization of Prodrug-like Inhibitors for Preventing Glutamate Efflux through Reverse Transport.

Glutamate transporters are responsible for active transport of the major excitatory neurotransmitter glutamate across the cell membrane, regulating the extracellular glutamate concentration in the mammalian brain. Extracellular glutamate levels in the brain are usually in the submicromolar range but can increase by exocytosis, inhibition of cellular uptake, or through glutamate release by reverse transport, as well as other mechanisms, which can lead to neurodegeneration and neuronal cell death. Such conditions can be encountered upon energy deprivation during an ischemic stroke. Here, we developed acetoxymethyl (AM) ester prodrug-like derivatives of excitatory amino acid transporter (EAAT) inhibitors that permeate the cell membrane and are activated, most likely through hydrolysis by endogenous cellular esterases, to form the active EAAT inhibitor. Upon increase in external K+ concentration, the inhibitors block glutamate efflux by EAAT reverse transport. Using a novel high-affinity fluorescent prodrug-like inhibitor, dl-threo-9-anthracene-methoxy-aspartate (TAOA) AM ester, we demonstrate that the precursor rapidly accumulates inside cells. Electrophysiological methods and fluorescence assays utilizing the iGluSnFR external glutamate sensor were used to demonstrate the efficacy of AM ester-protected inhibitors in inhibiting K+-mediated glutamate release. Together, our results provide evidence for a novel method to potentially prevent glutamate release by reverse transport under pathophysiological conditions in a model cell system, as well as in human astrocytes, while leaving glutamate uptake under physiological conditions operational. This method could have wide-ranging applications in the prevention of glutamate-induced neuronal cell death.

Conserved allosteric inhibition mechanism in SLC1 transporters.

Excitatory amino acid transporter 1 (EAAT1) is a glutamate transporter belonging to the SLC1 family of solute carriers. It plays a key role in the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined in complex with UCPH-101, apotent, non-competitive inhibitor of EAAT1. Alanine serine cysteine transporter 2 (ASCT2) is a neutral amino acid transporter, which regulates pools of amino acids such as glutamine between intracellular and extracellular compartments . ASCT2 also belongs to the SLC1 family and shares 58% sequence similarity with EAAT1. However, allosteric modulation of ASCT2 via non-competitive inhibitors is unknown. Here, we explore the UCPH-101 inhibitory mechanisms of EAAT1 and ASCT2 by using rapid kinetic experiments. Our results show that UCPH-101 slows substrate translocation rather than substrate or Na+ binding, confirming a non-competitive inhibitory mechanism, but only partially inhibits wild-type ASCT2. Guided by computational modeling using ligand docking and molecular dynamics simulations, we selected two residues involved in UCPH-101/EAAT1 interaction, which were mutated in ASCT2 (F136Y, I237M, F136Y/I237M) in the corresponding positions. We show that in the F136Y/I237M double-mutant transporter, 100% of the inhibitory effect of UCPH-101 could be restored, and the apparent affinity was increased (Ki = 4.3 µM), much closer to the EAAT1 value of 0.6 µM. Finally, we identify a novel non-competitive ASCT2 inhibitor, through virtual screening and experimental testing against the allosteric site, further supporting its localization. Together, these data indicate that the mechanism of allosteric modulation is conserved between EAAT1 and ASCT2. Due to the difference in binding site residues between ASCT2 and EAAT1, these results raise the possibility that more potent, and potentially selective ASCT2 allosteric inhibitors can be designed .

Mechanism of inhibition of the glutamate transporter EAAC1 by the conformationally constrained glutamate analogue (+)-HIP-B.

Glutamate transporters play an important role in the regulation of extracellular glutamate concentrations in the mammalian brain and are, thus, promising targets for therapeutics. Despite this importance, the development of pharmacological tools has mainly focused on the synthesis of competitive inhibitors, which are amino acid analogues that bind to the substrate binding site. In this report, we describe the characterization of the mechanism of glutamate transporter inhibition by a constrained, cyclic glutamate analogue, (+)-3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid [(+)-(3aS,6S,6aS)-HIP-B]. Our results show that (+)-HIP-B is a nontransportable amino acid that inhibits glutamate transporter function in a mixed mechanism. Although (+)-HIP-B inhibits the glutamate-associated anion conductance, it has no effect on the leak anion conductance, in contrast to competitive inhibitors. Furthermore, (+)-HIP-B is unable to alleviate the effect of the competitive inhibitor dl-threo-ß-benzyloxyaspartic acid (TBOA), which binds to the substrate binding site. (+)-HIP-B is more potent in inhibiting forward transport compared to reverse transport. In a mutant transporter, which is activated by glutamine, but not glutamate, (+)-HIP-B still acts as an inhibitor, although this mutant transporter is insensitive to TBOA. Finally, we analyzed the effect of (+)-HIP-B on the pre-steady-state kinetics of the glutamate transporter. The results can be explained with a mixed mechanism at a site that may be distinct from the substrate binding site, with a preference for the inward-facing configuration of the transporter and slow inhibitor binding. (+)-HIP-B may represent a new paradigm of glutamate transporter inhibition that is based on targeting of a regulatory site.

Defining substrate and blocker activity of alanine-serine-cysteine transporter 2 (ASCT2) Ligands with Novel Serine Analogs.

The neutral amino acid transporter alanine-serine-cysteine transporter 2 (ASCT2) belongs to the solute carrier 1 (SLC1) family of solute transporters and transports small, neutral amino acids across the membrane, including the physiologically important and ubiquitous amino acid glutamine. Our understanding of the involvement of ASCT2 in the physiological processes involving glutamine is hampered by a lack of understanding of its pharmacology and the absence of high-affinity inhibitors. In this study, we combined an in silico docking approach with experimental investigation of binding parameters to develop new ASCT2 inhibitors and substrates, a series of serine esters, and to determine structural parameters that govern their functional effects. The series of compounds was synthesized using standard methods and exhibited a range of properties, from inhibitors to partial substrates and full substrates. Our results suggest that amino acid derivatives with small side-chain volume and low side-chain hydrophobicity interact strongly with the closed-loop form of the binding site, in which re-entrant loop 2, the presumed extracellular gate for the substrate binding site, is closed off. However, these derivatives bind weakly to the open-loop form (external gate open to the extracellular side), acting as transported substrates. In contrast, inhibitors bind preferentially to the open-loop form. An aromatic residue in the side chain is required for high-affinity interaction. One of the compounds, the l-serine ester serine biphenyl-4-carboxylate reversibly inhibits ASCT2 function with an apparent affinity of 30 µM.

A conserved aspartate residue located at the extracellular end of the binding pocket controls cation interactions in brain glutamate transporters.

In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na(+) ions, followed by countertransport of K(+). Recent studies, based on several crystal structures of the archeal homologue Glt(Ph), indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na(+) binding, but functional and computational studies suggest some candidate sites. In the Glt(Ph) structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of Glt(Ph). Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na(+) and K(+), respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters.

Hetero-oligomerization of neuronal glutamate transporters.

Excitatory amino acid transporters (EAATs) mediate the uptake of glutamate into neuronal and glial cells of the mammalian central nervous system. Two transporters expressed primarily in glia, EAAT1 and EAAT2, are crucial for glutamate homeostasis in the adult mammalian brain. Three neuronal transporters (EAAT3, EAAT4, and EAAT5) appear to have additional functions in regulating and processing cellular excitability. EAATs are assembled as trimers, and the existence of multiple isoforms raises the question of whether certain isoforms can form hetero-oligomers. Co-expression and pulldown experiments of various glutamate transporters showed that EAAT3 and EAAT4, but neither EAAT1 and EAAT2, nor EAAT2 and EAAT3 are capable of co-assembling into heterotrimers. To study the functional consequences of hetero-oligomerization, we co-expressed EAAT3 and the serine-dependent mutant R501C EAAT4 in HEK293 cells and Xenopus laevis oocytes and studied glutamate/serine transport and anion conduction using electrophysiological methods. Individual subunits transport glutamate independently of each other. Apparent substrate affinities are not affected by hetero-oligomerization. However, polarized localization in Madin-Darby canine kidney cells was different for homo- and hetero-oligomers. EAAT3 inserts exclusively into apical membranes of Madin-Darby canine kidney cells when expressed alone. Co-expression with EAAT4 results in additional appearance of basolateral EAAT3. Our results demonstrate the existence of heterotrimeric glutamate transporters and provide novel information about the physiological impact of EAAT oligomerization.