Identification of PS1/gamma-secretase and glutamate transporter GLT-1 interaction sites.

The recently discovered interaction between Presenilin 1 (PS1), a catalytic subunit of γ-secretase responsible for generating amyloid-ß peptides, and GLT-1, a major glutamate transporter in the brain (EAAT2), provides a mechanistic link between these two key factors involved in Alzheimer's disease (AD) pathology. Modulating this interaction can be crucial to understand the consequence of such crosstalk in AD context and beyond. However, the interaction sites between these two proteins are unknown. Herein, we utilized an alanine scanning approach coupled with FRET-based fluorescence lifetime imaging microscopy to identify the interaction sites between PS1 and GLT-1 in their native environment within intact cells. We found that GLT-1 residues at position 276 to 279 (TM5) and PS1 residues at position 249 to 252 (TM6) are crucial for GLT-1-PS1 interaction. These results have been cross validated using AlphaFold Multimer prediction. To further investigate whether this interaction of endogenously expressed GLT-1 and PS1 can be prevented in primary neurons, we designed PS1/GLT-1 cell-permeable peptides (CPPs) targeting the PS1 or GLT-1 binding site. We used HIV TAT domain to allow for cell penetration which was assayed in neurons. First, we assessed the toxicity and penetration of CPPs by confocal microscopy. Next, to ensure the efficiency of CPPs, we monitored the modulation of GLT-1-PS1 interaction in intact neurons by fluorescence lifetime imaging microscopy. We saw significantly less interaction between PS1 and GLT-1 with both CPPs. Our study establishes a new tool to study the functional aspect of GLT-1-PS1 interaction and its relevance in normal physiology and AD models.

Structural insights into inhibitory mechanism of human excitatory amino acid transporter EAAT2.

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2.

A complex relative motion between hairpin loop 2 and transmembrane domain 5 in the glutamate transporter GLT-1.

Excitatory amino acid transporter 2, also known as glial glutamate transporter type 1 (GLT-1), plays an important role in maintaining suitable synaptic glutamate concentrations. Reentrant helical hairpin loop (HP) 2, as the extracellular gate, has been shown to participate in the binding of substrate and ions. Several residues in transmembrane domain (TM) 5 have been shown to be involved in the construction of the transport pathway. However, the spatial relationship between HP2 and TM5 during the recycling of glutamate has not yet been clarified. We introduced cysteine residue pairs in HP2 and TM5 of cysteine-less-GLT-1 by using site-directed mutagenesis in order to assess the proximity of HP2 and TM5. A significant decrease in substrate uptake was seen in the I283C/S443C and S287C/S443C mutants when the oxidative cross-linking agent copper(II) (1,10-phenanthroline)3 (CuPh) was used. The inhibitory effect of CuPh on the transport activity of the S287/S443C mutant was increased after the application of glutamate or potassium. In contrast, an apparent protection of the transport activity of the I283C/S443C mutant was observed after glutamate or potassium addition. The membrane-impermeable sulfhydryl reagent (2-trimethylammonium) methanethiosulfonate (MTSET) was used to detect the aqueous permeability of each single mutant. The aqueous permeability of the I283C mutant was identical to that of the S443C mutant. The sensitivity of I283C and S443C to MTSET was attenuated by glutamate and potassium. All these data indicate that there is a complex relative motion between TM5 and HP2 during the transport cycle.

Proximity of transmembrane segments 5 and 8 of the glutamate transporter GLT-1 inferred from paired cysteine mutagenesis.

BACKGROUND: GLT-1 is a glial glutamate transporter which maintains low synaptic concentrations of the excitatory neurotransmitter enabling efficient synaptic transmission. Based on the crystal structure of the bacterial homologue Glt(Ph), it has been proposed that the reentrant loop HP2, which connects transmembrane domains (TM) 7 and 8, moves to open and close access to the binding pocket from the extracellular medium. However the conformation change between TM5 and TM8 during the transport cycle is not clear yet. We used paired cysteine mutagenesis in conjunction with treatments with Copper(II)(1,10-Phenanthroline)(3) (CuPh), to verify the predicted proximity of residues located at these structural elements of GLT-1. METHODOLOGY/PRINCIPAL FINDINGS: To assess the proximity of transmembrane domain (TM) 5 relative to TM8 during transport by the glial glutamate transporter GLT-1/EAAT2, cysteine pairs were introduced at the extracellular ends of these structural elements. A complete inhibition of transport by Copper(II)(1,10-Phenanthroline)(3) is observed in the double mutants I295C/I463C and G297C/I463C, but not in the corresponding single mutants. Glutamate and potassium, both expected to increase the proportion of inward-facing transporters, significantly protected against the inhibition of transport activity of I295C/I463C and G297C/I463C by CuPh. Transport by the double mutants I295C/I463C and G297C/I463C also was inhibited by Cd(2+). CONCLUSIONS/SIGNIFICANCE: Our results suggest that TM5 (Ile-295, Gly-297) is in close proximity to TM8 (Ile-463) in the mammalian transporter, and that the spatial relationship between these domains is altered during the transport cycle.

Ubiquitination-mediated internalization and degradation of the astroglial glutamate transporter, GLT-1.

Sodium-dependent glutamate uptake is essential for limiting excitotoxicity, and dysregulation of this process has been implicated in a wide array of neurological disorders. The majority of forebrain glutamate uptake is mediated by the astroglial glutamate transporter, GLT-1. We and others have shown that this transporter undergoes endocytosis and degradation in response to activation of protein kinase C (PKC), however, the mechanisms involved remain unclear. In the current study, transfected C6 glioma cells or primary cortical cultures were used to show that PKC activation results in incorporation of ubiquitin into GLT-1 immunoprecipitates. Mutation of all 11 lysine residues in the amino and carboxyl-terminal domains to arginine (11R) abolished this signal. Selective mutation of the seven lysine residues in the carboxyl terminus (C7K-R) did not eliminate ubiquitination, but it completely blocked PKC-dependent internalization and degradation. Two families of variants of GLT-1 were prepared with various lysine residues mutated to arginine. Analyses of these constructs indicated that redundant lysine residues in the carboxyl terminus were sufficient for the appearance of ubiquitinated product and degradation of GLT-1. Together these data define a novel mechanism by which the predominant forebrain glutamate transporter can be rapidly targeted for degradation.

A caspase-3-cleaved fragment of the glial glutamate transporter EAAT2 is sumoylated and targeted to promyelocytic leukemia nuclear bodies in mutant SOD1-linked amyotrophic lateral sclerosis.

EAAT2 (excitatory amino acid transporter 2) is a high affinity, Na+-dependent glutamate transporter of glial origin that is essential for the clearance of synaptically released glutamate and prevention of excitotoxicity. During the course of human amyotrophic lateral sclerosis (ALS) and in a transgenic mutant SOD1 mouse model of the disease, expression and activity of EAAT2 is remarkably reduced. We previously showed that some of the mutant SOD1 proteins exposed to oxidative stress inhibit EAAT2 by triggering caspase-3 cleavage of EAAT2 at a single defined locus. This gives rise to two fragments that we termed truncated EAAT2 and COOH terminus of EAAT2 (CTE). In this study, we report that analysis of spinal cord homogenates prepared from mutant G93A-SOD1 mice reveals CTE to be of a higher molecular weight than expected because it is conjugated with SUMO-1. The sumoylated CTE fragment (CTE-SUMO-1) accumulates in the spinal cord of these mice as early as presymptomatic stage (70 days of age) and not in other central nervous system areas unaffected by the disease. The presence and accumulation of CTE-SUMO-1 is specific to ALS mice, since it does not occur in the R6/2 mouse model for Huntington disease. Furthermore, using an astroglial cell line, primary culture of astrocytes, and tissue samples from G93A-SOD1 mice, we show that CTE-SUMO-1 is targeted to promyelocytic leukemia nuclear bodies. Since one of the proposed functions of promyelocytic leukemia nuclear bodies is regulation of gene transcription, we suggest a possible novel mechanism by which the glial glutamate transporter EAAT2 could contribute to the pathology of ALS.

Conformationally sensitive reactivity to permeant sulfhydryl reagents of cysteine residues engineered into helical hairpin 1 of the glutamate transporter GLT-1.

In the central nervous system, glutamate transporters terminate the actions of this neurotransmitter by concentrating it into cells surrounding the synapse by a process involving sodium and proton cotransport followed by countertransport of potassium. These transporters contain two oppositely oriented helical hairpins 1 and 2. Hairpin 1 originates from the cytoplasm, but its tip is close to that of hairpin 2, which enters the transporter's lumen from the extracellular side. Here we address the question of whether hairpin 1 and/or domains surrounding it undergo conformational changes during the transport cycle. Therefore, we probed the reactivity of cysteines introduced into hairpin 1 and the cytoplasmic ends of transmembrane domains 6, 7, and 8 of the GLT-1 transporter to membrane-permeant N-ethylmaleimide. In each domain, except for transmembrane domain 6, cysteine mutants were found in which the inhibition of d-[(3)H]aspartate transport by the sulfhydryl reagent was increased when external sodium was replaced by potassium, a condition expected to increase the proportion of cytoplasmic-facing transporters. Conversely, the nontransportable blocker kainate protected against the inhibition in several of these mutants, presumably by locking the transporter in an outward-facing conformation. Moreover, external potassium decreased the oxidative cross-linking of two cysteines, each introduced at the tip of each hairpin. Our results are consistent with a model based on the crystal structure of an archeal homolog. According to this model, the inward movement of hairpin 1 results in the opening of a pathway between the binding pocket and the cytoplasm, lined by parts of transmembrane domains 7 and 8.

The importance of company: Na+ and Cl- influence substrate interaction with SLC6 transporters and other proteins.

SLC6 transporters, which include transporters for gamma-aminobutyric acid (GABA), norepinephrine, dopamine, serotonin, glycine, taurine, L-proline, creatine, betaine, and neutral cationic amino acids, require Na+ and Cl- for their function, and this review covers the interaction between transporters of this family with Na+ and Cl- from a structure-function standpoint. Because detailed structure-function information regarding ion interactions with SLC6 transporters is limited, we cover other proteins cotransporting Na+ or Cl- with substrate (SLClA2, PutP, SLC5A1, melB), or ion binding to proteins in general (rhodanese, ATPase, LacY, thermolysine, angiotensin-converting enzyme, halorhodopsin, CFTR). Residues can be involved in directly binding Na+ or Cl-, in coupling ion binding to conformational changes in transporter, in coupling Na+ or Cl- movement to transport, or in conferring ion selectivity. Coordination of ions can involve a number of residues, and portions of the substrate and coupling ion binding sites can be distal in space in the tertiary structure of the transporter, with other portions that are close in space thought to be crucial for the coupling process. The reactivity with methanethiosulfonate reagents of cysteines placed in strategic positions in the transporter provides a readout for conformational changes upon ion or substrate binding. More work is needed to establish the relationships between ion interactions and oligomerization of SLC6 transporters.

Identification of motifs involved in endoplasmic reticulum retention-forward trafficking of the GLT-1 subtype of glutamate transporter.

Glutamate transporters may exist as homomultimers, but little is known about the mechanisms that ensure proper assembly and surface expression. In the present study, we investigated the mechanisms that contribute to posttranslational processing of the GLT-1 subtype of glutamate transporter. An extracellular leucine-based motif was identified that after mutation to alanine (6L/6A GLT-1) prevented export of GLT-1 from the endoplasmic reticulum (ER) to the plasma membrane and displayed a glycosylation pattern characteristic of "immature" transporter. This 6L/6A variant had a selective dominant-negative effect on wild-type GLT-1 expression and formed coimmunoprecipitable complexes with GLT-1. Mutation of two downstream arginine residues to alanine partially restored maturation and functional activity of the 6L/6A variant. The fact that this additional mutation rescued maturation of GLT-1 essentially excludes the possibility that the 6L/6A mutant variant is not appropriately processed because of simple misfolding. When the domain containing these motifs was introduced into a topologically similar location in the interleukin 2alpha receptor subunit (Tac protein), the mutations had a similar effect on protein maturation. Topological models place at least the leucine-based motif in an extracellular domain, which would face the lumen of the ER during assembly. On the basis of these data, we suggest that an evolutionarily conserved arginine-based motif functions as an ER retention signal and a lumenal leucine motif is required for suppression of this signal. Interestingly, a high percentage of variably spliced GLT-1 mRNAs lacking parts of this domain are found in the CNS, suggesting that GLT-1 expression may be regulated during assembly.

Protein kinase C activation decreases cell surface expression of the GLT-1 subtype of glutamate transporter. Requirement of a carboxyl-terminal domain and partial dependence on serine 486.

Na(+)-dependent glutamate transporters are required for the clearance of extracellular glutamate and influence both physiological and pathological effects of this excitatory amino acid. In the present study, the effects of a protein kinase C (PKC) activator on the cell surface expression and activity of the GLT-1 subtype of glutamate transporter were examined in two model systems, primary co-cultures of neurons and astrocytes that endogenously express GLT-1 and C6 glioma cells transfected with GLT-1. In both systems, activation of PKC with phorbol ester caused a decrease in GLT-1 cell surface expression. This effect is opposite to the one observed for the EAAC1 subtype of glutamate transporter (Davis, K. E., Straff, D. J., Weinstein, E. A., Bannerman, P. G., Correale, D. M., Rothstein, J. D., and Robinson, M. B. (1998) J. Neurosci. 18, 2475-2485). Several recombinant chimeric proteins between GLT-1 and EAAC1 transporter subtypes were generated to identify domains required for the subtype-specific redistribution of GLT-1. We identified a carboxyl-terminal domain consisting of 43 amino acids (amino acids 475-517) that is required for PKC-induced GLT-1 redistribution. Mutation of a non-conserved serine residue at position 486 partially attenuated but did not completely abolish the PKC-dependent redistribution of GLT-1. Although we observed a phorbol ester-dependent incorporation of (32)P into immunoprecipitable GLT-1, mutation of serine 486 did not reduce this signal. We also found that chimeras containing the first 446 amino acids of GLT-1 were not functional unless amino acids 475-517 of GLT-1 were also present. These non-functional transporters were not as efficiently expressed on the cell surface and migrated to a smaller molecular weight, suggesting that a subtype-specific interaction is required for the formation of functional transporters. These studies demonstrate a novel effect of PKC on GLT-1 activity and define a unique carboxyl-terminal domain as an important determinant in cellular localization and regulation of GLT-1.

Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesis.

Sodium- and potassium-coupled transporters clear the excitatory neurotransmitter glutamate from the synaptic cleft. Their function is essential for effective glutamatergic neurotransmission. Glutamate transporters have an unusual topology, containing eight membrane-spanning domains and two reentrant loops of opposite orientation. We have introduced pairwise cysteine substitutions in several structural elements of the GLT-1 transporter. A complete inhibition of transport by Cu(II)(1,10-phenanthroline)(3) is observed in the double mutants A412C/V427C and A364C/S440C, but not in the corresponding single mutants. No inhibition is observed in more then 20 other double cysteine mutants. The Cu(II)(1,10-phenanthroline)(3) inhibition can be partly prevented by the nontransportable glutamate analogue dihydrokainate. Treatment with dithiothreitol restores much of the transport activity. Moreover, micromolar concentrations of cadmium ions reversibly inhibit transport catalyzed by A412C/V427C and A364C/S440C double mutants, but not by the corresponding single mutants. Inhibition by Cu(II)(1,10-phenanthroline)(3) and by cadmium is only observed when the cysteine pairs are introduced in the same polypeptide. Therefore, in both cases the proximity appears to be intra- rather than intermolecular. Positions 364 and 440 are located on reentrant loop I and II, respectively. Our results suggest that these two loops, previously shown to be essential for glutamate transport, come in close proximity.