and Outward-Facing Substrate Binding Sites of the Prokaryotic

Nov 18, 2016 - Characterization of the Inward- and Outward-Facing Substrate. Binding Sites of the Prokaryotic Aspartate Transporter, GltPh. Benjamin C...
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Characterization of the Inward and Outward Facing Substrate Binding Sites of the Prokaryotic Aspartate Transporter, Glt Ph

Benjamin C McIlwain, Robert J. Vandenberg, and Renae M. Ryan Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00795 • Publication Date (Web): 18 Nov 2016 Downloaded from http://pubs.acs.org on November 24, 2016

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Characterization of the Inward and Outward Facing Substrate Binding Sites of the Prokaryotic Aspartate Transporter, GltPh Benjamin C. McIlwain†, Robert J. Vandenberg and Renae M. Ryan*.

Transporter Biology Group, Discipline of Pharmacology, Sydney Medical School, University of Sydney, Sydney, Australia.

KEYWORDS Glutamate transporter; GltPh; EAAT; liposome reconstitution; aspartate transporter; amino acid transporter; SLC1A family, sided inhibition

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ABSTRACT

Crystal structures of the prokaryotic aspartate transporter GltPh have provided important insights into the mechanism of amino acid transport by GltPh and related eukaryotic members of the glutamate transporter family (SLC1A family). Identification of inhibitors of GltPh can provide valuable tools to understand the molecular basis for substrate and inhibitor specificity and selectivity of SLC1A members, but at present few inhibitors of GltPh have been identified. We have screened a collection of commercially available aspartate analogues and identified new transportable and non-transportable GltPh inhibitors. We have explored the inhibition profile of GltPh by utilizing a thiol-modification assay that isolates sided populations of the transporters reconstituted in liposomes, to determine if any aspartate analogues display preference for either the inwardly- or outwardly-directed binding sites. Here, we have characterized several new inhibitors of GltPh and identified three β-carbon substituted molecules that display a strong preference for the outwardly-directed binding site of GltPh.

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The aspartate transporter from Pyrococcus horikoshii, GltPh, is a prokaryotic homologue of the SLC1A family of transporters, which includes the Na+-dependent excitatory amino acid transporters (EAATs) and neutral amino acid transporters (ASCTs) (1). L-Aspartate transport by GltPh is coupled to the co-transport of 3 Na+ ions and is highly-selective for aspartate, with high nanomolar affinity (2-4). GltPh is homo-trimeric, with each monomer capable of transporting aspartate and also supporting a thermodynamically uncoupled chloride conductance (2, 5, 6). Several crystal structures of GltPh have been determined and provide a model for understanding the mechanism of transport and the molecular basis for substrate and inhibitor recognition for all members of this family (Fig. 1 A-C) (2, 6-8). The available crystal structures provide insights into the size, shape and chemical nature of the outward facing substrate binding site. The substrate binding site is formed by the tips of two re-entrant loops (HP1 and HP2) and unwound regions of TM7 and TM8 (Fig. 1 D, E). The α-carboxylate of the substrate aspartate binds to N401 and T398 (TM8), as well as S278 (HP2). The β-carboxylate interacts with T314 (TM7), R397 (TM8), and G359 (HP2). Finally, the amine group of the aspartate molecule binds to D394 and T398 (TM8), V355 (HP2), as well as R276 (HP1) (Fig 1E). Two of the three Na+ ions required for transport are thought to bind prior to substrate and then the third Na+ ion helps to close the extracellular gate (HP2) after which the transport domain undergoes an elevator movement to deliver substrate to the inside of the cell (2, 7, 9, 10). The crystal structure of GltPh in the presence of the nontransportable blocker DL-threo-β-benzyloxyasparte (TBOA) revealed that the aspartate moiety of TBOA binds to a similar site as L-aspartate, but the bulky benzyl group of the inhibitor prevents HP2 from closing down, preventing the binding of the third Na+ ion and transport (Fig. 1A). TBOA forms additional contacts with M311 (TM7) (Fig. 1D) (2). Although much has been gleaned from the crystal structures of GltPh, the conformational changes required for substrate release into the cell and the structure of the inward-facing 3 ACS Paragon Plus Environment

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substrate binding site are unknown. The structural symmetry between HP2 and HP1 suggests that HP1 may open up when the transport domain is facing the inside of the cell to a similar extent as HP2 and thus act as the intracellular gate, but molecular dynamics studies have explored the mechanisms of substrate release in GltPh and the findings suggest this may not be the case. Huang and Tajkhorshid found that HP1 displayed relative rigidity in the absence of substrate and proposed that, unlike HP2, it does not act as an intracellular gate (11). Simulations of the inward-occluded structure of GltPh suggest that ‘intracellular gate’ opening may be due to concerted, subtle movements of both HP2 and HP1 (12), or that movement of HP2 away from TM8 could permit aspartate un-binding (13). In addition, an apo structure of GltPh observes helical unwinding of HP2 when the transporter is inward-facing, and may represent the leaving complex (14). Reconstitution of purified GltPh protein into liposomes offers a useful tool for studying the function of this recombinant membrane protein. However, an artifact of the reconstitution process is that transporters are inserted into the membrane in a mixed orientation (4). That is, transporters are either oriented right-side out (RSO) where the binding site is facing the extraliposomal environment, or inside-out (ISO) where the binding site is facing inside the liposome (4, 15). While this mixed orientation limits detailed kinetic analysis of the transport process, the ability to isolate the two conformations offers a unique opportunity to probe the sidedness of transport, as well as the inhibition profile for the intracellular and extracellular binding sites (16). GltPh is highly selective for aspartate, while the EAATs can transport both glutamate and aspartate with similar affinity. In addition, a range of glutamate- and aspartate-based compounds have been demonstrated to inhibit the human glutamate transporters (17-20) while only two competitive inhibitors of GltPh have been identified (2, 21). Here, we employ GltPh reconstituted in liposomes to screen a range of aspartate analogues to determine what 4 ACS Paragon Plus Environment

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modifications of the aspartate moiety are tolerated in the substrate binding site of GltPh and to identify novel biochemical and crystallographic tools. We also utilize a thiol-based assay to examine the ability of the aspartate-based compounds to preferentially bind to the inward- or outward-facing substrate binding site of GltPh. A compound that displays preference for the inward-facing state could be used to capture the unknown inward-open state of GltPh. For this purpose, we investigated three modifications of the aspartate moiety (Fig. 2A, B), namely N-modification of the aspartate primary amine; backbone modifications at the α- or β-carbon position and α- or β-carboxyl modification. Our results reveal that N-modified aspartate analogues and carboxylate-modified analogues display modest to high affinity for both the inward and outward facing substrate binding site, whilst three of the β-carbon modified analogues bind with high affinity and reveal a striking preference for the outwardfacing substrate binding site of GltPh. In addition, we describe a new system for characterizing the properties of the inward- and outward-facing substrate binding sites of GltPh. This system can be used to find a high affinity inhibitor of the inward-facing binding site that may capture GltPh in the inward-facing state which would further our understanding of whether substrate release is due to opening of HP1, HP2 or a combination of both.

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Experimental Procedures Chemicals- DL-TBOA (1), L-TFB-TBOA (2) and L-threo-3-hydroxyaspartate (10) were obtained from Tocris (Ellisville, MO). N-acetyl-L-aspartate (3), N-carbobenzyloxy-Laspartate (4), N-benzoyl-D-aspartate (5), N-(2,4-dinitrophenyl)-L-aspartate (6), β-benzylester-L-aspartate (7), β-hydroxamate-L-aspartate (8), aspartame (9), DL-threo-β-methylaspartate (11), DL-α-methyl-aspartate (12), and tris(2-carboxyethyl)phosphine (TCEP) were obtained from Sigma-Aldrich (St. Louis, MO). 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET) and was obtained from Toronto Research Chemicals (Toronto, ON). The chemical structures of the inhibitor compounds used in this study are shown in Fig. 2. Protein purification- All mutations were made using the Q5 site-directed mutagenesis kit (New England Bioscience) and cysteine substitutions were introduced into a cysteine-less GltPh mutant (C321S), which is fully active (7). GltPh was expressed and purified as previously described (4). Briefly, membranes containing His-GltPh were isolated, solubilised with n-dodecyl-β-D-maltopyranoside (C12M, Anatrace) and protein was purified using NiNTA resin (Qiagen). The histidine tag was subsequently removed by digestion with thrombin (10 U/mg protein) and the protein further purified on a size-exclusion column where the detergent was exchanged to n-decyl-β-D-maltopyranoside (C10M, Anatrace). Pure protein was reconstituted into liposomes as previously described (4). E. coli polar lipid extract was combined with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids) at a ratio of 3:1. Lipids were mixed, dried under nitrogen and resuspended in internal buffer (100 mM KCl, 20 mM HEPES-Tris pH 7.5) by sonication using a cylindrical sonicator (Laboratory Supplies Co.). The lipid suspension was frozen in liquid nitrogen and thawed at least six times. Liposomes were formed by extrusion through 400 nm polycarbonate membranes (Avanti Polar Lipids) and were treated with Triton X-100 at a ratio of 0.5 % w/w. Protein was added at 2.5 µg protein/ mg lipid. The protein/ lipid 6 ACS Paragon Plus Environment

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mixture was left at room temperature for 30 minutes before detergent was removed using SM2 Biobeads (BioRad). The proteoliposome mixture was incubated with gentle agitation, with four consecutive batches of Biobeads (120 mg/ml). Liposomes were concentrated by centrifugation at 150,000 g for 30 minutes, resuspended at 100 mg lipid/ ml and either used immediately or flash-frozen in liquid nitrogen and stored at -80 °C. Transport assays- Transport function of GltPh was measured by L-[3H]aspartate radiolabelled uptake. To initiate the transport reaction, 7 µL of liposomes (50 mg/ml) were diluted ~130-fold into 900 µL of uptake buffer (100 mM NaCl, 20 mM HEPES-Tris pH 7.5, 1 µM valinomycin) containing 100 nM L-[3H]aspartate (13.1 Ci/mmol; Perkin Elmer) at 30 °C. At the time points indicated a 200 µL aliquot was removed from the assay buffer and immediately diluted in 2 mL of ice-cold quench buffer (100 mM LiCl, 20 mM HEPES-Tris pH 7.5), followed by immediate filtration over nitrocellulose filters (0.22 µm pore size, Millipore). Each reaction contained 1.9 µg of protein. The filters were washed with quench buffer (2 mL) under vacuum; filters were combined with 3 mL scintillation fluid and assayed for radioactivity using a Trilux beta counter (Perkin Elmer). Initial rates of transport represent the amount of L-[3H]aspartate transport over the linear portion of the time course derived from four time points (Fig. 3A; GltPh, 1 minute; A364C, 2 minutes). Inhibition experiments were performed by adding liposomes to uptake buffer containing increasing concentrations of the inhibitor. For inhibition curves, the data is normalised to the rate of uptake in the absence of inhibitor. The control (no inhibitor) L-[3H]aspartate uptake rate for both the ISO and RSO conditions are detailed in the Figure Legend for each inhibition curve and represent variability between reconstitutions. The background amount of uptake was measured by diluting liposomes containing GltPh or A364C into an uptake buffer without Na+ (100 mM KCl).

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We performed thiol-modification of GltPh (containing the A364C, C321S mutations) in liposomes, as previously described (15, 16). Thiol-modification of A364C was carried out by treating liposomes with 1 mM MTSET (2-(trimethylammonium) ethyl methanethiosulfonate) for 5 minutes at room temperature. A 7 µL volume of 2 mM MTSET (final concentration 1 mM) was incubated with 7 µL of liposomes (100 mg/mL). Following this 5 minute incubation, radiolabelled uptake was performed in the same manner as described above. MTSET is membrane impermeable and this treatment completely inactivates the RSO transporters leaving the ISO transporters functional. To isolate the RSO GltPh transporters, liposomes were loaded with 1mM MTSET via buffer exchange. Briefly, liposomes were resuspended with an internal buffer containing 1 mM MTSET (100 mM KCl, 20 mM HEPES-Tris pH 7.5, 1 mM MTSET), and subjected to freeze/thaw cycles using liquid nitrogen and a 30 °C water bath. Liposomes were then pelleted by centrifugation, excess buffer removed, and again resuspended in the MTSET-containing internal buffer. This process was repeated a total of 3 times to ensure buffer exchange. Liposomes were then extruded through a 400 nm membrane, centrifuged and resuspended to 50 mg/mL. To initiate the transport reaction, 7 µL of liposomes were diluted into an uptake buffer containing 20 mM tris(2-carboxyethyl)phosphine (TCEP). TCEP is also membrane impermeant and reduces MTSET modified cysteine residues on the external surface restoring the activity of the RSO transporters, but leaving the ISO transporters inactive. The rate of transport is approximately 50% of untreated liposomes, which demonstrates that the reduction of A364C by excess TCEP is immediate (15). Counter-flow experiments- Compounds that exhibited an IC50 below 10µM were selected to further investigate in a counter-flow assay to determine if they were transportable or nontransportable inhibitors. Liposomes were loaded with 100 mM NaCl, 20 mM HEPES-Tris pH 7.5, 100 µM unlabeled compound (L-aspartate, β-benzyl-ester-L-aspartate (7),

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β8

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hydroxamate-L-aspartate (8), L-threo-3-hydroxyaspartate (10), and DL-threo-β-methylaspartate (11)) and diluted into uptake buffer containing 100 mM NaCl, 20 mM HEPES-Tris pH 7.5 and 100 nM 3H-L-aspartate as described previously (15, 22). Under these conditions Na+-dependent electroneutral exchange will only occur for compounds that can be transported by GltPh. Background levels were determined using liposomes loaded with 100 mM KCl. Data analysis- Analysis of kinetic data was conducted using GraphPad Prism for Windows 5.0 (GraphPad Software, La Jolla, CA). Data values represent the mean ± S.E.M of experiments performed in triplicate. Initial rate of uptake (IR) as a function of substrate concentration ([S]) was fit by least-squares analysis to Equation 1, where IRmax represents the maximal rate, EC50 is the concentration of substrate that generates a half-maximal rate, and [S] is the concentration of substrate. Eq. 1 IR/IRmax = [S]/(EC50 + [S] Concentration-dependent inhibition of 3H-L-aspartate uptake by aspartate analogues was fit to Equation 2, where [B] represents the concentration of the blocker, and IC50 is the concentration of blocker that generates half-maximal inhibition. Eq. 2 IR/IRmax = 1 – ([B]/(IC50 + [B])) As the apparent affinity of aspartate for the RSO and ISO is different (15) Ki values were calculated from IC50 values using equation 3 (23), where the IC50 is derived from Equation 2, [Asp] is the concentration of [3H]Aspartate used in the assays (100 nM) and EC50 is the value for aspartate transport derived from Equation 1 for the RSO or ISO. Ki values are reported in Table 1. Eq. 3 Ki = IC50/(1 + [Asp]/EC50)

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Results Inhibition profile of GltPh- Proteo-liposomes containing GltPh accumulate L-[3H]aspartate in the presence of an inwardly-directed Na+ gradient (Fig. 3A). To investigate the action of inhibitors we performed competition experiments, measuring uptake of L-[3H]aspartate into liposomes containing GltPh in the presence of an excess of a range of inhibitors. Initially we examined the well-characterized, potent EAAT inhibitors, DL-TBOA (1) and L-TFB-TBOA (2), which also inhibit aspartate transport by GltPh by approximately ~70% at 100-fold excess (10 µM; Fig. 3B). Next, we extended our screen to a collection of commercially available, aspartate-based compounds to determine the inhibition profile for GltPh. Figure 3 C-E shows the remaining L-[3H]aspartate transport following competition against these aspartate-based compounds. When the transport assays were performed in the presence of excess N-modified aspartate based compounds (compounds 3 - 6), we observed very little inhibition at 100-fold excess (data not shown). A 1000-fold excess of inhibitor (100 µM) was required to observe any inhibition of aspartate transport, which reflects a markedly reduced affinity of the Nmodified compounds for GltPh. N-carbobenzyloxy-L-aspartate (4) and N-(2,4-dinitrophenyl)L-aspartate (6) both inhibited L-[3H]aspartate transport by approximately 50% at 100 µM. Nacetyl-L-aspartate (3) and N-benzoyl-D-aspartate (5) were less potent inhibitors of aspartate transport by GltPh (Fig. 3C). We extended the investigation to determine which modifications to β- and α-carboxylate were tolerated, namely β-benzyl-ester-L-aspartate (7), β-hydroxamate-L-aspartate (8) and aspartame (9) (Fig. 3D). β-benzyl-ester-L-aspartate (7) offers modest inhibition of L[3H]aspartate transport by GltPh, of approximately 30% at 100-fold excess. This is in marked contrast to β-hydroxamate-L-aspartate (8) which at the same concentration (10 µM) nearly abolishes aspartate transport by GltPh. Aspartame (9), which is an aspartate and phenylalanine

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joined at the aspartate α-carboxylate, was a weak inhibitor of GltPh at 100-fold excess (10 µM). To complete the preliminary exploration of the aspartate binding site, we investigated whether modifications of the aspartate carbon backbone were permitted. Specifically, we looked at modifications at the α-carbon position (DL-α-methyl-aspartate, compound 12) and β-carbon position (L-threo-3-hydroxyaspartate and DL-threo-β-methyl-aspartate, compounds 10 and 11, respectively) (Fig. 3E). Inhibition of transport by the β-carbon modified aspartate analogues is far more robust. Indeed, to observe significant reduction of L-[3H]aspartate transport, a 100-fold excess of inhibitor was sufficient (in contrast to the N-modified aspartate molecules which required 1000-fold excess). In contrast, α-carbon modification offers no significant inhibition of transport rates at 100-fold excess (10 µM). MTSET-modified A364C allows interrogation of the outward- and inward-facing binding sites of GltPh- Reconstitution of GltPh into liposomes results in a random orientation of the transporters in the membrane and thus the inhibition profiles described above are derived from a mixed population of transporters. We have developed an assay that utilizes a single alanine for cysteine mutation (A364C) in a cysteine-free background (GltPhC321S). In both GltPh and the human glutamate transporter EAAT1, a cysteine residue introduced at position 364 is only accessible from the extracellular side of the membrane and when it is modified by the thiol reactive reagent MTSET, substrate transport is inhibited (15, 24, 25). In a previous study, we have shown that incubation with 5 mM MTSET for 5 minutes on both sides of the liposome membrane results in complete inhibition of aspartate transport by GltPhA364C (15). When MTSET is applied to the outside of the liposome, transport is inhibited by ~50%, while application of MTSET to only the inside of the liposome also results in a ~50% reduction in transport. These results confirm that GltPh reconstituted into liposomes results in 11 ACS Paragon Plus Environment

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a mixed orientation, where 50% of the transporters facing the right-side out (RSO) and 50% of the transporters are inverted with the inward-facing binding site facing the outside of the liposome (ISO) (Fig. 4A). We have also shown that the apparent affinity for aspartate is 3.6fold higher for the outwardly-directed (RSO) binding site than for the inwardly-directed (ISO) binding site (15). In this study, we have utilized this thiol-based assay to study the ability of a range of aspartate analogues to bind to the ISO and RSO binding sites of GltPh. First, we needed to confirm that the A364C mutation does not affect the function of the transporter or the ability of the aspartate analogues to bind to the transporter. The results in Figure 3 show that the GltPh-A364C transporter displays similar levels of L-[3H]aspartate uptake (Fig. 3A) and has a similar inhibition profile to WT GltPh in the presence of the 12 aspartate analogues tested (Fig. 3B-D). Thus, the A364C mutation does not affect aspartate transport or the ability of the test compounds to bind and is suitable to characterize the potency of the compounds for inhibition of aspartate transport when binding to the RSO or ISO facing GltPh. Our first investigation of the sided inhibition of GltPh was for the potent EAAT inhibitors, DL-TBOA and TFB-TBOA. To determine if these compounds inhibited the ISO and RSO substrate-binding sites equally, or if they demonstrated a preference for the RSO in a similar manner to L-aspartate (15) we assayed 3H-Laspartate uptake at a range of inhibitor concentrations (Fig. 4C, D). Interestingly, both DL-TBOA and L-TFB-TBOA displayed a significant (~130-fold) preference for the RSO over the ISO, despite having different overall Ki’s at GltPh (Fig. 4C, D; Table 1). Specifically, DL-TBOA inhibited aspartate transport by GltPh with a Ki of 66 ± 29 µM and 0.50 ± 0.14 µM for the ISO and RSO, respectively, while TFB-TBOA was more potent with a Ki of 17 ± 5 µM and 0.13 ± 0.03 µM for the ISO and RSO, respectively. These results demonstrate that the RSO and the ISO substrate binding

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sites of GltPh are different and that this thiol-based assay can be used to determine the apparent affinity of inhibitors at these two different binding sites. N-modified aspartate compounds are poor inhibitors of GltPh. The next group of compounds examined was the N-modified aspartate analogues (compounds 3-6). Ki’s were determined for the RSO and ISO facing binding site of GltPh (Fig. 5; Table 1). Interestingly, all of the N-modified compounds tested exhibited low affinity for GltPh with Ki values ranging from ~50 - 350 µM (Fig. 5B-E and Table 1) suggesting that modification at this position is not well tolerated. Two of the N-modified aspartate analogues demonstrated a slight preference for the RSO binding site; N-carbobenzyloxy-L-aspartate (4) (7.6-fold) and N-acetyl-L-aspartate (3) (2.7-fold) (Fig. 5D and Table 1). α- and β -carboxylate modified aspartate analogues- Next we investigated compounds that were modified at the α- and β- carboxylate (Fig. 6A). Aspartame (9), an α-carboxylate modified analogue, is a low affinity inhibitor of GltPh with Ki’s of 132 ± 72 µM and 108 ± 77 µM for the ISO and RSO binding sites respectively (Fig. 6D). Although α-carboxylate modification is poorly tolerated, changes to the β-carboxylate appear to be well tolerated. βbenzyl-ester-L-aspartate (7) and β-hydroxamate-L-aspartate (8) remain high affinity inhibitors of GltPh. β-benzyl-ester-L-aspartate does not discriminate between the two binding sites and inhibits aspartate transport via GltPh with a Ki of 1.2 ± 0.2 µM (ISO) and 1.6 ± 0.4 µM (RSO) (Fig. 6B). While the smaller β-hydroxamate-L-aspartate results in a high affinity inhibitor with a Ki of 53 ± 2 nM (ISO) and 20 ± 8 nM (RSO) and exhibits a slight (2.6-fold) preference for the RSO binding site (Fig. 6C). β-carbon modification of aspartate selects for the outwardly-directed binding siteAmino acid residues in the binding pocket of GltPh (Fig. 1E) make no specific interactions with the carbon backbone of the bound aspartate substrate and thus, we hypothesized that 13 ACS Paragon Plus Environment

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modifications along the carbon backbone might be better tolerated than the N-modified compounds (Fig. 7). L-threo-3-hydroxyaspartate (10) displays high affinity for GltPh, with an approximate 113-fold preference for the RSO (Ki; 6 ± 2 nM) over the ISO (Ki; 700 ± 122 nM) (Fig. 7B). This preference is similar to the preference of the much bulkier compounds TBOA and TFB-TBOA for the RSO binding site (Fig. 4C, D). In addition, to our knowledge this is the highest affinity inhibitor that has been described for GltPh. In contrast, DL-threo-βmethyl-aspartate (11), where the hydroxyl group is replaced with a methyl group, demonstrates only a slight (1.9-fold) preference for RSO, displaying significantly lower Ki‘s of 3.21 ± 0.82 µM (ISO) and 1.69 ± 0.56 µM (RSO) (Fig. 7C). Finally, we investigated the inhibition profile of DL-α-methyl-aspartate (12) for both the ISO and RSO. Modification of the α-methyl is poorly tolerated and there is no marked difference between the Ki’s measured for the two transporter populations with the RSO (84 ± 26 µM) and ISO (111 ± 55 µM) being similar (Fig. 7D). Counter-flow experiments distinguish transportable versus non-transportable compounds- To determine if select, high affinity compounds were non-transportable or transportable blockers, a counter-flow assay was established. The distinction between the two types of inhibitors arises from whether a molecule binds to the substrate binding site but is not transported, or whether a compound is transported rather than aspartate. TBOA and TFBTBOA are known to be non-transportable inhibitors of the human glutamate transporters (26, 27) but we wanted to determine if the other high affinity (Ki‘s 1; preference of the RSO and