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

Neurochemical Research (2021)Cite this article

Abstract

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.

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References

  1. 1.

    Zerangue N, Kavanaugh MP (1996) Flux coupling in a neuronal glutamate transporter. Nature 383:634–637

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  2. 2.

    Tanaka K, Watase K, Manabe T, Yamada K, Watanabe M, Takahashi K, Iwama H, Nishikawa T, Ichihara N, Kikuchi T, Okuyama S, Kawashima N, Hori S, Takimoto M, Wada K (1997) Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276:1699–1702

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  3. 3.

    Pines G, Kanner BI (1990) Counterflow of L-glutamate in plasma membrane vesicles and reconstituted preparations from rat brain. Biochemistry 29:11209–11214

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  4. 4.

    Wadiche JI, Amara SG, Kavanaugh MP (1995) Ion fluxes associated with excitatory amino acid transport. Neuron 15:721–728

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  5. 5.

    Kanner BI, Sharon I (1978) Active transport of L-glutamate by membrane vesicles isolated from rat brain. Biochemistry 17:3949–3953

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 6.

    Kavanaugh MP, Bendahan A, Zerangue N, Zhang Y, Kanner BI (1997) Mutation of an amino acid residue influencing potassium coupling in the glutamate transporter GLT-1 induces obligate exchange. J Biol Chem 272:1703–1708

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  7. 7.

    Zhang Y, Bendahan A, Zarbiv R, Kavanaugh MP, Kanner BI (1998) Molecular determinant of ion selectivity of a (Na+ + K+)-coupled rat brain glutamate transporter. Proc Natl Acad Sci U S A 95:751–755

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. 8.

    Zerangue N, Kavanaugh MP (1996) Interaction of L-cysteine with a human excitatory amino acid transporter. J Physiol 493(Pt 2):419–423

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Levy LM, Warr O, Attwell D (1998) Stoichiometry of the glial glutamate transporter GLT-1 expressed inducibly in a Chinese hamster ovary cell line selected for low endogenous Na+-dependent glutamate uptake. J Neurosci 18:9620–9628

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Bergles DE, Tzingounis AV, Jahr CE (2002) Comparison of coupled and uncoupled currents during glutamate uptake by GLT-1 transporters. J Neurosci 22:10153–10162

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Machtens JP, Kortzak D, Lansche C, Leinenweber A, Kilian P, Begemann B, Zachariae U, Ewers D, de Groot BL, Briones R, Fahlke C (2015) Mechanisms of anion conduction by coupled glutamate transporters. Cell 160:542–553

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Fairman WA, Vandenberg RJ, Arriza JL, Kavanaugh MP, Amara SG (1995) An excitatory amino-acid transporter with properties of a ligand-gated chloride channel. Nature 375:599–603

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    Arriza JL, Eliasof S, Kavanaugh MP, Amara SG (1997) Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. Proc Natl Acad Sci U S A 94:4155–4160

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Leary GP, Stone EF, Holley DC, Kavanaugh MP (2007) The glutamate and chloride permeation pathways are colocalized in individual neuronal glutamate transporter subunits. J Neurosci 27:2938–2942

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Yernool D, Boudker O, Jin Y, Gouaux E (2004) Structure of a glutamate transporter homologue from Pyrococcus horikoshii. Nature 431:811–818

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Grewer C, Balani P, Weidenfeller C, Bartusel T, Tao Z, Rauen T (2005) Individual subunits of the glutamate transporter EAAC1 homotrimer function independently of each other. Biochemistry 44:11913–11923

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17.

    Verdon G, Boudker O (2012) Crystal structure of an asymmetric trimer of a bacterial glutamate transporter homolog. Nat Struct Mol Biol 19:355–357

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    Koch HP, Brown RL, Larsson HP (2007) The glutamate-activated anion conductance in excitatory amino acid transporters is gated independently by the individual subunits. J Neurosci 27:2943–2947

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Boudker O, Ryan RM, Yernool D, Shimamoto K, Gouaux E (2007) Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature 445:387–393

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Reyes N, Ginter C, Boudker O (2009) Transport mechanism of a bacterial homologue of glutamate transporters. Nature 462:880–885

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 21.

    Crisman TJ, Qu S, Kanner BI, Forrest LR (2009) Inward-facing conformation of glutamate transporters as revealed by their inverted-topology structural repeats. Proc Natl Acad Sci U S A 106:20752–20757

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Bendahan A, Armon A, Madani N, Kavanaugh MP, Kanner BI (2000) Arginine 447 plays a pivotal role in substrate interactions in a neuronal glutamate transporter. J Biol Chem 275:37436–37442

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Kanner BI, Bendahan A (1982) Binding order of substrates to the sodium and potassium ion coupled L-glutamic acid transporter from rat brain. Biochemistry 21:6327–6330

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Wang J, Albers T, Grewer C (2018) Energy Landscape of the Substrate Translocation Equilibrium of Plasma-Membrane Glutamate Transporters. J Phys Chem B 122:28–39

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    Shabaneh M, Rosental N, Kanner BI (2014) Disulfide cross-linking of transport and trimerization domains of a neuronal glutamate transporter restricts the role of the substrate to the gating of the anion conductance. J Biol Chem 289:11175–11182

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Grewer C, Watzke N, Wiessner M, Rauen T (2000) Glutamate translocation of the neuronal glutamate transporter EAAC1 occurs within milliseconds. Proc Natl Acad Sci U S A 97:9706–9711

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Zhang Z, Tao Z, Gameiro A, Barcelona S, Braams S, Rauen T, Grewer C (2007) Transport direction determines the kinetics of substrate transport by the glutamate transporter EAAC1. Proc Natl Acad Sci U S A 104:18025–18030

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Wadiche JI, Kavanaugh MP (1998) Macroscopic and microscopic properties of a cloned glutamate transporter/chloride channel. J Neurosci 18:7650–7661

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. 29.

    Watzke N, Bamberg E, Grewer C (2001) Early intermediates in the transport cycle of the neuronal excitatory amino acid carrier EAAC1. J Gen Physiol 117:547–562

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Wang J, Zielewicz L, Grewer C (2019) A K(+)/Na(+) co-binding state: Simultaneous versus competitive binding of K(+) and Na(+) to glutamate transporters. J Biol Chem

  31. 31.

    Otis TS, Jahr CE (1998) Anion currents and predicted glutamate flux through a neuronal glutamate transporter. J Neurosci 18:7099–7110

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Wang J, Zhang K, Goyal P, Grewer C (2020) Mechanism and potential sites of potassium interaction with glutamate transporters. J Gen Physiol 152

  33. 33.

    Szatkowski M, Barbour B, Attwell D (1990) Non-vesicular release of glutamate from glial cells by reversed electrogenic glutamate uptake. Nature 348:443–446

    CAS  PubMed  Article  Google Scholar 

  34. 34.

    Marvin JS, Borghuis BG, Tian L, Cichon J, Harnett MT, Akerboom J, Gordus A, Renninger SL, Chen TW, Bargmann CI, Orger MB, Schreiter ER, Demb JB, Gan WB, Hires SA, Looger LL (2013) An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat Methods 10:162–170

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. 35.

    Grewer C, Zhang Z, Mwaura J, Albers T, Schwartz A, Gameiro A (2012) Charge compensation mechanism of a Na+-coupled, secondary active glutamate transporter. J Biol Chem 287:26921–26931

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Wadiche JI, Arriza JL, Amara SG, Kavanaugh MP (1995) Kinetics of a human glutamate transporter. Neuron 14:1019–1027

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Ryan RM, Mitrovic AD, Vandenberg RJ (2004) The chloride permeation pathway of a glutamate transporter and its proximity to the glutamate translocation pathway. J Biol Chem 279:20742–20751

    CAS  PubMed  Article  Google Scholar 

  38. 38.

    Zhang W, Zhang X, Qu S (2019) Substrate-Induced Motion between TM4 and TM7 of the Glutamate Transporter EAAT1 Revealed by Paired Cysteine Mutagenesis. Mol Pharmacol 95:33–42

    CAS  PubMed  Article  Google Scholar 

  39. 39.

    Zhang Y, Zhang X, Qu S (2014) Cysteine mutagenesis reveals alternate proximity between transmembrane domain 2 and hairpin loop 1 of the glutamate transporter EAAT1. Amino Acids 46:1697–1705

    CAS  PubMed  Article  Google Scholar 

  40. 40.

    Qu S, Zhang W, He S, Zhang X (2019) Paired-Cysteine Scanning Reveals Conformationally Sensitive Proximity between the TM4b-4c Loop and TM8 of the Glutamate Transporter EAAT1. ACS Chem Neurosci 10:2541–2550

    CAS  PubMed  Article  Google Scholar 

  41. 41.

    Rong X, Tan F, Wu X, Zhang X, Lu L, Zou X, Qu S (2016) TM4 of the glutamate transporter GLT-1 experiences substrate-induced motion during the transport cycle. Sci Rep 6:34522

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Zhang X, Qu S (2011) Proximity of transmembrane segments 5 and 8 of the glutamate transporter GLT-1 inferred from paired cysteine mutagenesis. PLoS One 6:e21288

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. 43.

    Aprico K, Beart PM, Crawford D, O’Shea RD (2004) Binding and transport of [3H](2S,4R)- 4-methylglutamate, a new ligand for glutamate transporters, demonstrate labeling of EAAT1 in cultured murine astrocytes. J Neurosci Res 75:751–759

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Zielewicz L, Wang J, Ndaru E, Grewer CT (2019) Transient Kinetics Reveal Mechanism and Voltage Dependence of Inhibitor and Substrate Binding to Glutamate Transporters. ACS Chem Biol

  45. 45.

    Mennerick S, Shen W, Xu W, Benz A, Tanaka K, Shimamoto K, Isenberg KE, Krause JE, Zorumski CF (1999) Substrate turnover by transporters curtails synaptic glutamate transients. J Neurosci 19:9242–9251

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Guskov A, Jensen S, Faustino I, Marrink SJ, Slotboom DJ (2016) Coupled binding mechanism of three sodium ions and aspartate in the glutamate transporter homologue GltTk. Nat Commun 7:13420

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Niu L, Wieboldt R, Ramesh D, Carpenter BK, Hess GP (1996) Synthesis and characterization of a caged receptor ligand suitable for chemical kinetic investigations of the glycine receptor in the 3-microseconds time domain. Biochemistry 35:8136–8142

    CAS  PubMed  Article  Google Scholar 

  48. 48.

    Canepari M, Nelson L, Papageorgiou G, Corrie JE, Ogden D (2001) Photochemical and pharmacological evaluation of 7-nitroindolinyl-and 4-methoxy-7-nitroindolinyl-amino acids as novel, fast caged neurotransmitters. J Neurosci Methods 112:29–42

    CAS  PubMed  Article  Google Scholar 

  49. 49.

    Wieboldt R, Gee KR, Niu L, Ramesh D, Carpenter BK, Hess GP (1994) Photolabile precursors of glutamate: synthesis, photochemical properties, and activation of glutamate receptors on a microsecond time scale. Proc Natl Acad Sci U S A 91:8752–8756

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Zielewicz L, Grewer C (2019) Genetically Encoded Halide Sensor-Based Fluorescent Assay for Rapid Screening of Glutamate Transport and Inhibition. ACS Sens 4:2358–2366

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  51. 51.

    Arena ET, Rueden CT, Hiner MC, Wang S, Yuan M, Eliceiri KW (2017) Quantitating the cell: turning images into numbers with ImageJ. Wiley Interdiscip Rev Dev Biol 6

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Acknowledgements

This work was supported by a National Institutes of Health (NIH), Grant 1 R15 GM135843-01 awarded to CG.

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  1. Department of Chemistry, Binghamton University, 4400 Vestal Parkway East, Binghamton, NY, 13902, USA

    Jiali Wang, Laura Zielewicz, Yang Dong & Christof Grewer

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  1. Jiali Wang
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  2. Laura Zielewicz
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  4. Christof Grewer
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Correspondence to Christof Grewer.

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Wang, J., Zielewicz, L., Dong, Y. et al. Pre-Steady-State Kinetics and Reverse Transport in Rat Glutamate Transporter EAAC1 with an Immobilized Transport Domain. Neurochem Res (2021). https://doi.org/10.1007/s11064-021-03247-8

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Keywords

  • Glutamate transporter
  • Crosslink
  • Electrophysiology
  • Kinetics
  • Laser-photolysis