Structural Rearrangements at the Translocation Pore of the Human Glutamate Transporter, EAAT1
2006; Elsevier BV; Volume: 281; Issue: 40 Linguagem: Inglês
10.1074/jbc.m604991200
ISSN1083-351X
AutoresBarbara H. Leighton, Rebecca P. Seal, Spencer D. Watts, Mary O. Skyba, Susan Amara,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoStructure-function studies of mammalian and bacterial excitatory amino acid transporters (EAATs), as well as the crystal structure of a related archaeal glutamate transporter, support a model in which TM7, TM8, and the re-entrant loops HP1 and HP2 participate in forming a substrate translocation pathway within each subunit of a trimer. However, the transport mechanism, including precise binding sites for substrates and co-transported ions and changes in the tertiary structure underlying transport, is still not known. In this study, we used chemical cross-linking of introduced cysteine pairs in a cysteine-less version of EAAT1 to examine the dynamics of key domains associated with the translocation pore. Here we show that cysteine substitution at Ala-395, Ala-367, and Ala-440 results in functional single and double cysteine transporters and that in the absence of glutamate or dl-threo-β-benzyloxyaspartate (dl-TBOA), A395C in the highly conserved TM7 can be cross-linked to A367C in HP1 and to A440C in HP2. The formation of these disulfide bonds is reversible and occurs intra-molecularly. Interestingly, cross-linking A395C to A367C appears to abolish transport, whereas cross-linking A395C to A440C lowers the affinities for glutamate and dl-TBOA but does not change the maximal transport rate. Additionally, glutamate and dl-TBOA binding prevent cross-linking in both double cysteine transporters, whereas sodium binding facilitates cross-linking in the A395C/A367C transporter. These data provide evidence that within each subunit of EAAT1, Ala-395 in TM7 resides close to a residue at the tip of each re-entrant loop (HP1 and HP2) and that these residues are repositioned relative to one another at different steps in the transport cycle. Such behavior likely reflects rearrangements in the tertiary structure of the translocation pore during transport and thus provides constraints for modeling the structural dynamics associated with transport. Structure-function studies of mammalian and bacterial excitatory amino acid transporters (EAATs), as well as the crystal structure of a related archaeal glutamate transporter, support a model in which TM7, TM8, and the re-entrant loops HP1 and HP2 participate in forming a substrate translocation pathway within each subunit of a trimer. However, the transport mechanism, including precise binding sites for substrates and co-transported ions and changes in the tertiary structure underlying transport, is still not known. In this study, we used chemical cross-linking of introduced cysteine pairs in a cysteine-less version of EAAT1 to examine the dynamics of key domains associated with the translocation pore. Here we show that cysteine substitution at Ala-395, Ala-367, and Ala-440 results in functional single and double cysteine transporters and that in the absence of glutamate or dl-threo-β-benzyloxyaspartate (dl-TBOA), A395C in the highly conserved TM7 can be cross-linked to A367C in HP1 and to A440C in HP2. The formation of these disulfide bonds is reversible and occurs intra-molecularly. Interestingly, cross-linking A395C to A367C appears to abolish transport, whereas cross-linking A395C to A440C lowers the affinities for glutamate and dl-TBOA but does not change the maximal transport rate. Additionally, glutamate and dl-TBOA binding prevent cross-linking in both double cysteine transporters, whereas sodium binding facilitates cross-linking in the A395C/A367C transporter. These data provide evidence that within each subunit of EAAT1, Ala-395 in TM7 resides close to a residue at the tip of each re-entrant loop (HP1 and HP2) and that these residues are repositioned relative to one another at different steps in the transport cycle. Such behavior likely reflects rearrangements in the tertiary structure of the translocation pore during transport and thus provides constraints for modeling the structural dynamics associated with transport. Mammalian excitatory amino acid transporters (EAATs) 2The abbreviations used are: EAATs, excitatory amino acid transporters; MTSET, methanethiosulfonate-ethyltrimethylammonium; dl-TBOA, dl-threo-β-benzyloxyaspartate; CuPh, copper phenanthroline; DTT, dl-dithiothreitol; PBS, phosphate-buffered saline; TM, transmembrane. reside on the plasma membrane of neurons and glia and are responsible for removing glutamate from the extracellular space, maintaining its concentration below neurotoxic levels (1Amara S.G. Fontana A.C. Neurochem. Int. 2002; 41: 313-318Crossref PubMed Scopus (203) Google Scholar, 2Kanai Y. Hediger M.A. Pfluegers Arch. 2004; 447: 469-479Crossref PubMed Scopus (345) Google Scholar). Disruption of this process is associated with several pathological conditions, including ischemia, stroke, and amyotrophic lateral sclerosis (3Danbolt N.C. Prog. Neurobiol. 2001; 65: 1-105Crossref PubMed Scopus (3775) Google Scholar, 4Hinoi E. Takarada T. Tsuchihashi Y. Yoneda Y. Curr. Drug Targets CNS Neurol. Disord. 2005; 4: 211-220Crossref PubMed Scopus (35) Google Scholar). This transporter family is comprised of five EAAT subtypes (EAATs 1–5) (5Arriza J.L. Eliasof S. Kavanaugh M.P. Amara S.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4155-4160Crossref PubMed Scopus (803) Google Scholar, 6Arriza J.L. Fairman W.A. Wadiche J.I. Murdoch G.H. Kavanaugh M.P. Amara S.G. J. Neurosci. 1994; 14: 5559-5569Crossref PubMed Google Scholar, 7Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. 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Konings W.N. Curr. Opin. Microbiol. 1998; 1: 248-253Crossref PubMed Scopus (29) Google Scholar). In eukaryotes, the transport of glutamate against its concentration gradient is driven by the co-transport of three sodium ions and a proton, and the counter transport of a potassium ion (14Zerangue N. Kavanaugh M.P. Nature. 1996; 383: 634-637Crossref PubMed Scopus (709) Google Scholar), resulting in a stoichiometric transport current. Glutamate binding to the transporter in the presence of sodium also activates an anion conductance that is not stoichiometrically coupled to transport (7Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. Nature. 1995; 375: 599-603Crossref PubMed Scopus (1014) Google Scholar, 15Wadiche J.I. Amara S.G. Kavanaugh M.P. Neuron. 1995; 15: 721-728Abstract Full Text PDF PubMed Scopus (454) Google Scholar). The prokaryotic carriers use sodium and/or protons to drive the concentrative uptake of glutamate across the cell membrane, and it is not yet known whether they also mediate an anion conductance (13Lolkema J.S. Poolman B. Konings W.N. Curr. Opin. Microbiol. 1998; 1: 248-253Crossref PubMed Scopus (29) Google Scholar). The current model of the transporter topology is based on cysteine-scanning accessibility studies of the mammalian and bacterial carriers (1Amara S.G. Fontana A.C. Neurochem. Int. 2002; 41: 313-318Crossref PubMed Scopus (203) Google Scholar, 16Bridges R.J. Esslinger C.S. Pharmacol. Ther. 2005; 107: 271-285Crossref PubMed Scopus (109) Google Scholar, 17Sobczak I. Lolkema J.S. Curr. Opin. Microbiol. 2005; 8: 161-167Crossref PubMed Scopus (32) Google Scholar), as well as a recently reported 3.5 Å crystal structure of a archaeal transporter, GltPh (18Yernool D. Boudker O. Jin Y. Gouaux E. Nature. 2004; 431: 811-818Crossref PubMed Scopus (668) Google Scholar). In this model, the first half of the protein appears to form six TM helices, and the second half is composed of two re-entrant loops in opposite orientations (HP1 and HP2), a seventh TM divided by a β-linker into two helices (TM7) and a final amphipathic transmembrane helix (TM8). Biochemical studies of the rat EAAT1–3 and a related bacterial transporter, GltTBsc, as well as the crystal structure indicate that the transporter exists as a trimer, although substrate binding and translocation are thought to occur within each subunit (19Gendreau S. Voswinkel S. Torres-Salazar D. Lang N. Heidtmann H. Detro-Dassen S. Schmalzing G. Hidalgo P. Fahlke C. J. Biol. Chem. 2004; 279: 39505-39512Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 20Haugeto O. Ullensvang K. Levy L.M. Chaudhry F.A. Honore T. Nielsen M. Lehre K.P. Danbolt N.C. J. Biol. Chem. 1996; 271: 27715-27722Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, 21Yernool D. Boudker O. Folta-Stogniew E. Gouaux E. Biochemistry. 2003; 42: 12981-12988Crossref PubMed Scopus (81) Google Scholar, 22Grewer C. Balani P. Weidenfeller C. Bartusel T. Tao Z. Rauen T. Biochemistry. 2005; 44: 11913-11923Crossref PubMed Scopus (122) Google Scholar). Indeed, within each monomer of the crystal the C-terminal domains form what appears to be a pore or binding pocket for a "trapped" glutamate molecule (18Yernool D. Boudker O. Jin Y. Gouaux E. Nature. 2004; 431: 811-818Crossref PubMed Scopus (668) Google Scholar). Our current understanding of the role of the C-terminal domains in transport is based on numerous structure-function and topology studies of the mammalian and bacterial transporters. In these studies, it was determined that consecutive, conserved serine residues in HP1 are in an aqueous environment and are accessible from both sides of the membrane (23Grunewald M. Kanner B.I. J. Biol. Chem. 2000; 275: 9684-9689Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar– 25Slotboom D.J. Sobczak I. Konings W.N. Lolkema J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14282-14287Crossref PubMed Scopus (112) Google Scholar). Moreover, the modification of cysteines substituted for each of these residues abolishes transport, supporting a model in which the residues are closely associated with the translocation pore (23Grunewald M. Kanner B.I. J. Biol. Chem. 2000; 275: 9684-9689Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 24Seal R.P. Leighton B.H. Amara S.G. Neuron. 2000; 25: 695-706Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 25Slotboom D.J. Sobczak I. Konings W.N. Lolkema J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14282-14287Crossref PubMed Scopus (112) Google Scholar). Similarly, most residues in the highly conserved TM7 are also in an aqueous environment and accessible from one or both sides of the membrane (26Pines G. Zhang Y. Kanner B.I. J. Biol. Chem. 1995; 270: 17093-17097Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 27Seal R.P. Amara S.G. Neuron. 1998; 21: 1487-1498Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 28Zhang Y. Bendahan A. Zarbiv R. Kavanaugh M.P. Kanner B.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 751-755Crossref PubMed Scopus (88) Google Scholar). Modification of a majority of these residues, after substitution with cysteine, also abolishes transport, and A395C is protected from modification by substrates and nontransported inhibitors (27Seal R.P. Amara S.G. Neuron. 1998; 21: 1487-1498Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Two of these residues influence the binding of co-transported ions, sodium and/or potassium (Tyr-403 and Glu-404 in Glt1), and when mutated shift the carrier from a unidirectional transport mode into an exchange mode (26Pines G. Zhang Y. Kanner B.I. J. Biol. Chem. 1995; 270: 17093-17097Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 28Zhang Y. Bendahan A. Zarbiv R. Kavanaugh M.P. Kanner B.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 751-755Crossref PubMed Scopus (88) Google Scholar, 29Kavanaugh M.P. Bendahan A. Zerangue N. Zhang Y. Kanner B.I. J. Biol. Chem. 1997; 272: 1703-1708Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). In HP2, two residues (Ser-440 and Ser-443 in Glt1) influence whether lithium can substitute for sodium to support transport (30Zhang Y. Kanner B.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1710-1715Crossref PubMed Scopus (76) Google Scholar), and one residue appears to be accessible from the cytoplasmic side of the membrane (31Grunewald M. Menaker D. Kanner B.I. J. Biol. Chem. 2002; 277: 26074-26080Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In addition, most all of the residues in the C-terminal portion of HP2 form a helix and reside in an aqueous environment, near what appears to be the extracellular mouth of the pore (32Leighton B.H. Seal R.P. Shimamoto K. Amara S.G. J. Biol. Chem. 2002; 277: 29847-29855Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Cross-linking of a cysteine introduced into HP1 to one introduced into HP2 suggested that these loops reside in close proximity to one another when the transporter is in the sodium-bound state (33Brocke L. Bendahan A. Grunewald M. Kanner B.I. J. Biol. Chem. 2002; 277: 3985-3992Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Based on this finding and on the crystal structure, the two re-entrant loops are hypothesized to act as internal (HP1) and external (HP2) gates in an alternating access model of transport (18Yernool D. Boudker O. Jin Y. Gouaux E. Nature. 2004; 431: 811-818Crossref PubMed Scopus (668) Google Scholar, 33Brocke L. Bendahan A. Grunewald M. Kanner B.I. J. Biol. Chem. 2002; 277: 3985-3992Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Finally, previous work showed that TM8 forms an amphipathic helix with one face lining an aqueous pathway (34Slotboom D.J. Konings W.N. Lolkema J.S. J. Biol. Chem. 2001; 276: 10775-10781Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). An arginine residue at the N-terminal side of this helix (Arg-77 in Glt1) is thought to interact with the γ-carboxylate group of glutamate and to influence potassium binding (35Bendahan A. Armon A. Madani N. Kavanaugh M.P. Kanner B.I. J. Biol. Chem. 2000; 275: 37436-37442Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Mutation of a second arginine (Arg-475 in Glt1) in this helix changes the substrate selectivity and transforms the carrier into a substrate-gated cation channel (36Borre L. Kanner B.I. J. Biol. Chem. 2004; 279: 2513-2519Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Thus, many of the previous studies, as well as the recent crystal structure, support a model in which these C-terminal domains are directly associated with the translocation pore. To better understand how these domains are spatially oriented relative to one another during the transport cycle, we carried out a cysteine cross-linking study. We created 22 double cysteine transporters by introducing pairs of cysteine residues into C-terminal domains of a cysteine-less version of EAAT1 (27Seal R.P. Amara S.G. Neuron. 1998; 21: 1487-1498Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), and we then measured their transport activity before and after application of copper phenanthroline (CuPh), a catalyst of disulfide bond formation. We also determined the effect of cross-linking on the kinetics of transport, as well as the effects of substrates, inhibitors, and sodium ions on the extent of crosslinking. Results from this work suggest that Ala-395 in TM7 resides at a central point in the translocation pathway near a residue at the tip of each re-entrant loop, Ala-367 in HP1 and Ala-440 in HP2, and that the spatial relationship between these domains is altered during the transport cycle. Constructs and Cell Transfections—To make the cysteine substitution mutants, cysteine residues were introduced into the Cys-less-EAAT1 transporter using PCR (QuikChange® site-directed mutagenesis kit; Stratagene). PCR products were subcloned into the pCMV5 vector and sequenced by dye terminator cycle sequencing (PerkinElmer Life Sciences). COS-7 cells were passaged and plated into 24-well plates and then transfected by the DEAE-dextran method. Experiments were performed 2 days post-transfection. Cys-less-EAAT1 mutant constructs were assessed for their ability to accumulate 10 μm l-[3H]glutamate (100 nm l-[3H]glutamate, 9.9 μm unlabeled l-glutamate (24 Ci/mmol)). Cells were lysed in 0.1% SDS and counted in a scintillation counter. Cells transfected with the pCMV5 vector served as a control for endogenous l-glutamate uptake under all conditions. Kinetics—Cells expressing cysteine substitution mutants or Cys-less-EAAT1 were assayed for the ability to accumulate l-glutamate (200 μm; 100 nm l-[3H]glutamate, 199.9 μm nonlabeled l-glutamate) as a function of time (6Arriza J.L. Fairman W.A. Wadiche J.I. Murdoch G.H. Kavanaugh M.P. Amara S.G. J. Neurosci. 1994; 14: 5559-5569Crossref PubMed Google Scholar). Uptake remained linear for 10 min. For Vmax and Km calculations, cells were incubated with 100 nml-[3H]glutamate in final unlabeled l-glutamate concentrations of 1, 3, 10, 50, 100, 300, 500, and 1000 μm for 10 min at room temperature. Endogenous l-glutamate uptake measured in cells transfected with the pCMV5 vector was subtracted from each concentration. The Cys-less-EAAT1 transport activity was typically at least 5-fold over the endogenous background activity. The Vmax is expressed as a percent of Cys-less-EAAT1. Km and Vmax values were derived by the Michaelis-Menten equation using KaleidaGraph (Synergy Software, Reading, PA). CuPh-catalyzed Disulfide Cross-linking—COS-7 cells expressing Cys-less-EAAT1 or Cys-less-EAAT1 with single or double cysteine mutations were washed with phosphate-buffered saline (PBS) and then incubated for 5 min at room temperature with various concentrations of copper phenanthroline (CuPh) (0.1–1.5 mm) in PBS. Prior to experimental use, CuSO4 and 1,10-phenanthroline were combined in a 1:2 ratio to produce the CuPh reagent. Cells were then washed two times with PBS plus 0.1 mm calcium chloride, 1 mm magnesium chloride (PBSCM), and transporter-mediated uptake was assayed with 10 μm l-[3H]glutamate (100 nm l-[3H]glutamate, 9.9 μm unlabeled l-glutamate (24 Ci/mmol)) in PBSCM for 10 min at room temperature. After washing twice with PBSCM, cells were dissolved in 0.1% SDS, and activity was measured by a scintillation counter. Data were plotted as a percentage of the carrier activity without CuPh-catalyzed cross-linking under the same conditions. Percent activity = 100 × uptake after/uptake before. DTT Application after CuPh Treatment—CuPh-catalyzed cross-linking was performed as described above. Cells were then washed twice with PBSCM and incubated with 20 mm dl-dithiothreitol (DTT) in PBSCM for 5 min. Cells were washed twice with PBSCM, and uptake was performed as described above. Data were plotted as a percent of transport activity measured in the absence of CuPh. CuPh Treatment without Sodium—COS-7 cells expressing mutant or Cys-less-EAAT1 transporters were washed once in choline buffer containing 138 mm choline-Cl, 8.1 mm Tris-H3PO4, 2.7 mm KCl, 1.5 mm KH2PO4 (pH 7.4). CuPh was applied at 8 or 300 μm for A395C/A440C or 300 or 700 μm for A395C/A367C for 5 min in either choline buffer or PBS. Incubation at these concentrations reduces transport activity maximally or half-maximally. Cells were then washed twice in PBSCM before radiolabeled glutamate uptake was performed in PBSCM as described above. Data are plotted as the percent of the carrier activity without CuPh treatment. Percent activity = 100 × uptake after/uptake before. CuPh Treatment in the Presence of Substrate—COS-7 cells expressing mutant or Cys-less-EAAT1 transporters were washed once with PBS and then incubated for 5 min in PBS or 10 mm l-glutamate with or without the addition of 300 μm CuPh. Cells were then washed twice with PBSCM, and 10 μm l-[3H]glutamate uptake was measured as described above. MTSET Modification after CuPh Treatment—Cells expressing mutant cysteine transporters were washed once with PBS, and then CuPh was applied at a concentration that results in maximal inhibition of transport. To remove the potential contribution of uncross-linked transporters, cells were then washed twice with PBS and incubated for 5 min with 1 mm MTSET, an impermeant sulfhydryl-modifying reagent established to completely inhibit A367C, A395C, and A440C (24Seal R.P. Leighton B.H. Amara S.G. Neuron. 2000; 25: 695-706Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Cells were again washed twice, and 10 μm l-[3H]glutamate uptake was measured as described above. dl-TBOA Inhibition of l-Glutamate Uptake—COS-7 cells were transfected with the cysteine substitution mutants and washed once with PBSCM and then preincubated with dl-TBOA, a nontransported substrate analog, at concentrations ranging from 64 nm to 5 mm in PBSCM for 5 min, and uptake of 10 μm l-glutamate (100 nml-[3H]glutamate) was measured, as described above. Cells were then washed and processed as described above for scintillation counting. Results are expressed as the inhibition of l-glutamate uptake as a function of the concentration of dl-TBOA. IC50 values were calculated using GraphPad Prism (GraphPad Software, Inc., San Diego). The IC50 values are the mean ± S.E. of 3–4 independent experiments done in triplicate. CuPh Treatment in the Presence of TBOA—COS-7 cells expressing cysteine substitution mutants or Cys-less-EAAT1 transporters were washed once with PBS or choline containing buffer and then incubated for 5 min in PBS or choline with and without 1 mm TBOA and in the presence or absence of CuPh (300 μm A395C/A440C or 700 μm A395C/A367C). Cells were then washed twice with PBSCM prior to 10 μm glutamate uptake as described above. Three-dimensional Modeling—The software Swiss-PDBViewer was used to generate a folding model of EAAT1 based on a manual sequence alignment with the glutamate transporter homolog from Pyrococcus horikoshii (GltPh) (18Yernool D. Boudker O. Jin Y. Gouaux E. Nature. 2004; 431: 811-818Crossref PubMed Scopus (668) Google Scholar). The model is depicted without refinement. Images were rendered using MacPymol (DeLano Scientific, San Carlos, CA). Effects of Cysteine Cross-linking on Transport—We constructed 22 double cysteine transporters for this cross-linking study using as the template a previously characterized and fully functional version of EAAT1 that lacks endogenous cysteines (Cys-less-EAAT1) (27Seal R.P. Amara S.G. Neuron. 1998; 21: 1487-1498Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). We focused on residues that we knew were similar in accessibility and in phenotype following modification (24Seal R.P. Leighton B.H. Amara S.G. Neuron. 2000; 25: 695-706Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and that could be protected from modification by substrates and inhibitors (data not shown). To determine whether the cysteine pair introduced into each transporter is capable of forming a disulfide bond, we expressed each transporter in COS-7 cells and then measured the accumulation of radiolabeled l-glutamate before and after exposure to the cross-linking reagent CuPh. From this assay, we identified two double cysteine transporters, A395C/A367C and A395C/A440C (Fig. 1), that exhibit a dramatic decrease in transport activity following exposure to CuPh. The other transporter mutants showed either impaired transport activity in the absence of 300 μm CuPh (A395C/T362C, A395C/S363C, A395C/S366C, A395C/I371C, A395C/A414C, A395C/T428C, A395C/I429C, A395C/A441C, A395C/G442C, A395C/T450C, A395C/M451C, and A395C/S457C) or no change in transport activity after exposure to CuPh (A367C/A435C, A367C/I438C, A367C/G439C, A367C/A440C, A367C/T462C, and A395C/G439C; see Fig. 2).FIGURE 2Transport activity of cysteine substitution mutants before and after CuPh treatment. Double and single cysteine transporters expressed in COS-7 cells were assayed for their ability to transport 10 μm l-glutamate before and after incubation with 300 μm CuPh. Results are expressed as a percent of the activity measured for Cys-less-EAAT1 and are the average ± S.E. of 4–8 experiments done in triplicate. The * denotes a significant difference from the untreated control, p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To better characterize the effect of CuPh on the A395C/A367C and A395C/A440C transporters, we measured glutamate transport activity as a function of CuPh concentration. For both transporters, we observed that increasing concentrations of the cross-linking agent (1–1500 μm) lead to a greater reduction in glutamate transport (data not shown). Because the transport activity of our control Cys-less-EAAT1 transporter declines slightly at 1 mm CuPh, in subsequent experiments, we used CuPh concentrations that maximize cross-linking but do not affect the Cys-less-EAAT1 transporter (300 μm for the A395C/A440C transporter and 700 μm for the A395C/A367C transporter). However, even at the highest concentrations of CuPh, we still observed some residual transport activity in the A395C/A367C and A395C/A440C transporters (data not shown). To determine whether this residual activity results from incomplete cross-linking, we modified uncross-linked sulfhydryl groups with MTSET, a compound shown to abolish transport in the A395C, A367C, and A440C transporters (24Seal R.P. Leighton B.H. Amara S.G. Neuron. 2000; 25: 695-706Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 27Seal R.P. Amara S.G. Neuron. 1998; 21: 1487-1498Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Indeed, application of MTSET after CuPh treatment abolishes the residual activity in both double cysteine transporters (Fig. 3, A and B) suggesting incomplete cross-linking of these residues. Our observations from cross-linking thus far suggest that Ala-395 in TM7 is indeed in close proximity to Ala-367 in HP1 and to Ala-440 in HP2, the two re-entrant loops postulated to gate the translocation pore. Disulfide Bond Formation Is Reversible and Intra-molecular— To determine whether the effect of CuPh treatment on the two double cysteine transporters is reversible, we incubated the carriers first with CuPh and then with the reducing agent, DTT, for 5 min. As expected, incubation with 20 mm DTT restored transport activity in both double cysteine transporters (Fig. 3, A and B). In rare instances CuPh can lead to the formation of covalent links between cysteine and other residues and thus the reversibility in the presence of DTT confirms the formation of a disulfide bond. To determine whether the cross-linking in these carriers occurs intra-molecularly or inter-molecularly, we examined the effect of CuPh on the transport activity of the single cysteine mutants, A367C, A395C, and A440C expressed either individually or in pairs (A395C with A367C and A395C with A440C) (Fig. 3, A and B). As shown in Table 1, the uptake activity of the individual transporters is similar to that observed for Cys-less-EAAT1 (A395C Km = 41 ± 10 μm, A440C Km = 30 ± 7 μm, and A367C Km = 42 ± 6 μm) and when expressed either alone or with the two single mutants together is unaffected by incubation with CuPh (Table 1). Thus, disulfide bonds formed in the double cysteine transporters are reversible and occur within single subunits rather than between subunits. These findings reinforce the idea that any functional effects of cross-linking observed in the A395C/A367C and A395C/A440C transporters likely arise from constraints within the translocation core of a single monomer.TABLE 1Cross-linking alters transport kineticsVmaxKm300 μm CuPhVmaxKmCys-less-EAAT110033 ± 697 ± 332 ± 4A395C/A440C19 ± 124 ± 320 ± 3167 ± 25**A395C65 ± 841 ± 1068 ± 438 ± 5A440C30 ± 530 ± 729 ± 222 ± 3VmaxKm700 μm CuPhVmaxKmCys-less-EAAT110041 ± 5102 ± 263 ± 12A395C/A367C37 ± 355 ± 914 ± 1**37 ± 7A395C67 ± 841 ± 1072 ± 164 ± 14A367C60 ± 442 ± 656 ± 350 ± 7 Open table in a new tab Cross-linking Alters Transport Kinetics—To examine the effect of intra-molecular cross-linking on the transport kinetics of these carriers, we measured the apparent transport affinity (Km) and maximum transport rate (Vmax) of glutamate before and after exposure to CuPh. In the absence of CuPh, the double cysteine transporters exhibit Km values that are comparable with Cys-less-EAAT1 (Cys-less-EAAT1, Km = 33 ± 6 μm; A395C/A440C, Km = 24 ± 3 μm; and A395C/A367C, Km = 55 ± 9 μm) (Table 1). After cross-linking, however, A395C/A367C exhibits the same apparent transport affinity (Km = 37 ± 7 μm) but a reduced Vmax (Table 1 and Fig. 4B). In contrast, A395C/A440C has a significantly lower apparent transport affinity (Km = 167 ± 25 μm) but the same Vmax (Table 1 and Fig. 4A). These data suggest that disulfide bond formation between A395C in TM7 and A367C in HP1 (the proposed intracellular gate) abolishes transport function, whereas a disulfide bond formed between the A395C and A440C in HP2 (the proposed extracellular gate) can surprisingly still permit maximal transport activity, albeit with a lower apparent transport affinity. Cross-linking
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