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.
Similar content being viewed by others
References
Zerangue N, Kavanaugh MP (1996) Flux coupling in a neuronal glutamate transporter. Nature 383:634–637
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
Pines G, Kanner BI (1990) Counterflow of L-glutamate in plasma membrane vesicles and reconstituted preparations from rat brain. Biochemistry 29:11209–11214
Wadiche JI, Amara SG, Kavanaugh MP (1995) Ion fluxes associated with excitatory amino acid transport. Neuron 15:721–728
Kanner BI, Sharon I (1978) Active transport of L-glutamate by membrane vesicles isolated from rat brain. Biochemistry 17:3949–3953
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
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
Zerangue N, Kavanaugh MP (1996) Interaction of L-cysteine with a human excitatory amino acid transporter. J Physiol 493(Pt 2):419–423
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
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
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
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
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
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
Yernool D, Boudker O, Jin Y, Gouaux E (2004) Structure of a glutamate transporter homologue from Pyrococcus horikoshii. Nature 431:811–818
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
Verdon G, Boudker O (2012) Crystal structure of an asymmetric trimer of a bacterial glutamate transporter homolog. Nat Struct Mol Biol 19:355–357
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
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
Reyes N, Ginter C, Boudker O (2009) Transport mechanism of a bacterial homologue of glutamate transporters. Nature 462:880–885
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
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
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
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
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
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
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
Wadiche JI, Kavanaugh MP (1998) Macroscopic and microscopic properties of a cloned glutamate transporter/chloride channel. J Neurosci 18:7650–7661
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
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
Otis TS, Jahr CE (1998) Anion currents and predicted glutamate flux through a neuronal glutamate transporter. J Neurosci 18:7099–7110
Wang J, Zhang K, Goyal P, Grewer C (2020) Mechanism and potential sites of potassium interaction with glutamate transporters. J Gen Physiol 152
Szatkowski M, Barbour B, Attwell D (1990) Non-vesicular release of glutamate from glial cells by reversed electrogenic glutamate uptake. Nature 348:443–446
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
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
Wadiche JI, Arriza JL, Amara SG, Kavanaugh MP (1995) Kinetics of a human glutamate transporter. Neuron 14:1019–1027
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Acknowledgements
This work was supported by a National Institutes of Health (NIH), Grant 1 R15 GM135843-01 awarded to CG.
Author information
Authors and Affiliations
Corresponding author
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Special Issue: In Honor of Prof. Baruch Kanner.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
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 47, 148–162 (2022). https://doi.org/10.1007/s11064-021-03247-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11064-021-03247-8