Accepted Article
Article Type: Original Article
Title: Molecular determinants of transport stimulation of EAAT2 are located at interface between the trimerization and substrate transport domains
Author list: Ole V. Mortensen,† José L. Liberato,‡ Joaquim Coutinho-Netto,§ Wagner F.
dos Santos‡ and Andréia C. K. Fontana† †
Department of Pharmacology and Physiology, Drexel University College of Medicine,
Philadelphia, PA, 19102, USA ‡
Neurobiology and Venoms Laboratory, Department of Biology, College of Philosophy,
Sciences and Literature of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil §
Department of Biochemistry and Immunology, Ribeirão Preto School of Medicine,
University of São Paulo, SP, Brazil Address correspondence and reprint requests to Dr Andréia C. K. Fontana, Drexel University College of Medicine, Department of Pharmacology and Physiology, NCB, MS 488, 245 N. 15th Street, Philadelphia, PA 19012, USA. Phone: (215) 762-4399, Fax:
(215) 762- 2999. E-mail address: [email protected]
Running title: Region of EAAT2 important for transport stimulation
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jnc.13047 This article is protected by copyright. All rights reserved.
Accepted Article
Keywords: Excitatory Amino Acid Transporter 2 (EAAT2), glutamate transporter, glutamate uptake, Parawixia bistriata, transport enhancement
Footnote: § Joaquim Coutinho-Netto, in memoriam, September 10, 2012.
Abbreviations used: D-PBS, Dulbecco’s phosphate-buffered saline; EAAC1, excitatory amino acid carrier 1; EAATs, excitatory amino acid transporters; EAAT1-3, human excitatory amino acid transporter subtypes 1-3; EAAT2, human glutamate transporter 2; GLAST, glutamate and aspartate transporter; GLT-1, rat glutamate transporter 1; HP, hairpin loop; NaOH, sodium hydroxide; PBS-CM, D-PBS with 0.1 mM CaCl2 and 1 mM MgCl2 added; SDS, sodium dodecyl sulfate; TM, transmembrane; WT, wild type.
ABSTRACT Excitatory amino acid transporters (EAATs) regulate glutamatergic signal transmission by clearing extracellular glutamate. Dysfunction of these transporters has been implicated in the pathogenesis of various neurological disorders. Previous studies have shown that venom from the spider Parawixia bistriata and a purified compound (Parawixin1) stimulate EAAT2 activity and protect retinal tissue from ischemic damage. In the present study the EAAT2 subtype specificity of this compound was explored, employing chimeric proteins between EAAT2 and EAAT3 transporter subtypes and mutants to characterize the structural region targeted by the compound. This identified a critical residue (Histidine-71 in EAAT2 and Serine-45 in EAAT3) in transmembrane domain 2 (TM2) to be important for the selectivity between EAAT2 and EAAT3 and for
This article is protected by copyright. All rights reserved.
Accepted Article
the activity of the venom. Using the identified residue in TM2 as a structural anchor, several neighboring amino acids within TM5 and TM8 were identified to also be important for the activity of the venom. This structural domain of the transporter lies at the interface of the rigid trimerization domain and the central substrate binding transport domain. Our studies suggest that the mechanism of glutamate transport enhancement involves an interaction with the transporter that facilitates the movement of the transport domain.
INTRODUCTION Glutamate is the predominant excitatory amino acid neurotransmitter in the
mammalian central nervous system (CNS) and is essential for normal brain function including cognition, memory, learning, developmental plasticity, and long-term potentiation (McEntee & Crook 1993, Weiler et al. 1995, Peng et al. 2011). The termination of glutamate neurotransmission is achieved by rapid uptake of the released glutamate by presynaptic and astrocytic sodium-dependent transporters (Beart & O'Shea 2007, Danbolt 2001, Vandenberg & Ryan 2013). The transport cycle consists of several steps, including co-transport of glutamate with three sodium ions, followed by counter transport of potassium (Amato et al. 1994, Zerangue & Kavanaugh 1996). The translocation of glutamate over the cell membrane is an energetically unfavorable process, taking place against a glutamate concentration gradient. To overcome this, transporters harness the energy of preexisting electrochemical gradients of ions and the excitatory amino acid transporters (EAATs) are referred to as symporters, since transport of substrate co-occur with the co-transport of sodium. The transport cycle is
This article is protected by copyright. All rights reserved.
Accepted Article
initiated by concomitant binding of a substrate molecule, three sodium ions and a proton to an outward-facing conformation of the EAAT (Zerangue & Kavanaugh 1996). This triggers a conformational cascade resulting in the EAAT adopting an inward-facing conformation from which the sodium ions, protons and the substrates are released into the cytoplasm of the cell. Subsequently, the transporter returns to its outward facing conformation via the counter-transport of a potassium ion and becomes accessible for a new substrate molecule (Danbolt et al. 1992, Levy et al. 1998, Robinson 1998).
Five structurally distinct subtypes of Na+-dependent glutamate transporters have
been cloned to date, including GLAST or EAAT1 (Storck et al. 1992) and GLT-1 or EAAT2 (Pines et al. 1992), both present in astroglial cells (Rothstein et al. 1994);
EAAC1 or EAAT3, present in neurons (Kanai & Hediger 1992), EAAT4, found in the cerebellum (Fairman et al. 1995, Gegelashvili & Schousboe 1998) and EAAT5, expressed in vertebrate retina (Arriza et al. 1997).
Glutamate transporter EAAT2 /GLT-1 (human/rat homolog) is expressed throughout
the brain, in the spinal cord, primarily in astrocytes, and also in neurons and oligodendrocytes, and accounts for approximately 95% of glutamate transport in the CNS (Suchak et al. 2003), demonstrating its central role in maintenance of extracellular glutamate homeostasis (Maragakis et al. 2004, Sheldon & Robinson 2007, Lauriat & McInnes 2007, Kim et al. 2011).
This article is protected by copyright. All rights reserved.
Accepted Article
on glutamate transport mediated by EAAT2 WT and EAAT2 mutants H71S, D83E, and M86V, and by EAAT3 WT and EAAT3 mutants S45H and K49V. (d) Effects of P. bistriata spider venom on glutamate transport mediated by L290A, L295A, G298A, K299A, and P443A mutated EAAT2. Assays were performed in COS-7 cells transiently transfected with appropriate cDNA (chimeras or mutants) or empty vector. Cells were incubated with varied concentrations (a) or 2 μg/mL (b-d) of venom for 10 min at 37oC
and 5 min with 50 nM of 3H-L-glutamate. Results are normalized to percentage of control (vehicle) and expressed as mean ± SEM of three independent experiments (One Way Anova followed by Bonferroni’s Multiple Comparison post-hoc test using vehicle as control, **p
Article Type: Original Article
Title: Molecular determinants of transport stimulation of EAAT2 are located at interface between the trimerization and substrate transport domains
Author list: Ole V. Mortensen,† José L. Liberato,‡ Joaquim Coutinho-Netto,§ Wagner F.
dos Santos‡ and Andréia C. K. Fontana† †
Department of Pharmacology and Physiology, Drexel University College of Medicine,
Philadelphia, PA, 19102, USA ‡
Neurobiology and Venoms Laboratory, Department of Biology, College of Philosophy,
Sciences and Literature of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil §
Department of Biochemistry and Immunology, Ribeirão Preto School of Medicine,
University of São Paulo, SP, Brazil Address correspondence and reprint requests to Dr Andréia C. K. Fontana, Drexel University College of Medicine, Department of Pharmacology and Physiology, NCB, MS 488, 245 N. 15th Street, Philadelphia, PA 19012, USA. Phone: (215) 762-4399, Fax:
(215) 762- 2999. E-mail address: [email protected]
Running title: Region of EAAT2 important for transport stimulation
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jnc.13047 This article is protected by copyright. All rights reserved.
Accepted Article
Keywords: Excitatory Amino Acid Transporter 2 (EAAT2), glutamate transporter, glutamate uptake, Parawixia bistriata, transport enhancement
Footnote: § Joaquim Coutinho-Netto, in memoriam, September 10, 2012.
Abbreviations used: D-PBS, Dulbecco’s phosphate-buffered saline; EAAC1, excitatory amino acid carrier 1; EAATs, excitatory amino acid transporters; EAAT1-3, human excitatory amino acid transporter subtypes 1-3; EAAT2, human glutamate transporter 2; GLAST, glutamate and aspartate transporter; GLT-1, rat glutamate transporter 1; HP, hairpin loop; NaOH, sodium hydroxide; PBS-CM, D-PBS with 0.1 mM CaCl2 and 1 mM MgCl2 added; SDS, sodium dodecyl sulfate; TM, transmembrane; WT, wild type.
ABSTRACT Excitatory amino acid transporters (EAATs) regulate glutamatergic signal transmission by clearing extracellular glutamate. Dysfunction of these transporters has been implicated in the pathogenesis of various neurological disorders. Previous studies have shown that venom from the spider Parawixia bistriata and a purified compound (Parawixin1) stimulate EAAT2 activity and protect retinal tissue from ischemic damage. In the present study the EAAT2 subtype specificity of this compound was explored, employing chimeric proteins between EAAT2 and EAAT3 transporter subtypes and mutants to characterize the structural region targeted by the compound. This identified a critical residue (Histidine-71 in EAAT2 and Serine-45 in EAAT3) in transmembrane domain 2 (TM2) to be important for the selectivity between EAAT2 and EAAT3 and for
This article is protected by copyright. All rights reserved.
Accepted Article
the activity of the venom. Using the identified residue in TM2 as a structural anchor, several neighboring amino acids within TM5 and TM8 were identified to also be important for the activity of the venom. This structural domain of the transporter lies at the interface of the rigid trimerization domain and the central substrate binding transport domain. Our studies suggest that the mechanism of glutamate transport enhancement involves an interaction with the transporter that facilitates the movement of the transport domain.
INTRODUCTION Glutamate is the predominant excitatory amino acid neurotransmitter in the
mammalian central nervous system (CNS) and is essential for normal brain function including cognition, memory, learning, developmental plasticity, and long-term potentiation (McEntee & Crook 1993, Weiler et al. 1995, Peng et al. 2011). The termination of glutamate neurotransmission is achieved by rapid uptake of the released glutamate by presynaptic and astrocytic sodium-dependent transporters (Beart & O'Shea 2007, Danbolt 2001, Vandenberg & Ryan 2013). The transport cycle consists of several steps, including co-transport of glutamate with three sodium ions, followed by counter transport of potassium (Amato et al. 1994, Zerangue & Kavanaugh 1996). The translocation of glutamate over the cell membrane is an energetically unfavorable process, taking place against a glutamate concentration gradient. To overcome this, transporters harness the energy of preexisting electrochemical gradients of ions and the excitatory amino acid transporters (EAATs) are referred to as symporters, since transport of substrate co-occur with the co-transport of sodium. The transport cycle is
This article is protected by copyright. All rights reserved.
Accepted Article
initiated by concomitant binding of a substrate molecule, three sodium ions and a proton to an outward-facing conformation of the EAAT (Zerangue & Kavanaugh 1996). This triggers a conformational cascade resulting in the EAAT adopting an inward-facing conformation from which the sodium ions, protons and the substrates are released into the cytoplasm of the cell. Subsequently, the transporter returns to its outward facing conformation via the counter-transport of a potassium ion and becomes accessible for a new substrate molecule (Danbolt et al. 1992, Levy et al. 1998, Robinson 1998).
Five structurally distinct subtypes of Na+-dependent glutamate transporters have
been cloned to date, including GLAST or EAAT1 (Storck et al. 1992) and GLT-1 or EAAT2 (Pines et al. 1992), both present in astroglial cells (Rothstein et al. 1994);
EAAC1 or EAAT3, present in neurons (Kanai & Hediger 1992), EAAT4, found in the cerebellum (Fairman et al. 1995, Gegelashvili & Schousboe 1998) and EAAT5, expressed in vertebrate retina (Arriza et al. 1997).
Glutamate transporter EAAT2 /GLT-1 (human/rat homolog) is expressed throughout
the brain, in the spinal cord, primarily in astrocytes, and also in neurons and oligodendrocytes, and accounts for approximately 95% of glutamate transport in the CNS (Suchak et al. 2003), demonstrating its central role in maintenance of extracellular glutamate homeostasis (Maragakis et al. 2004, Sheldon & Robinson 2007, Lauriat & McInnes 2007, Kim et al. 2011).
This article is protected by copyright. All rights reserved.
Accepted Article
on glutamate transport mediated by EAAT2 WT and EAAT2 mutants H71S, D83E, and M86V, and by EAAT3 WT and EAAT3 mutants S45H and K49V. (d) Effects of P. bistriata spider venom on glutamate transport mediated by L290A, L295A, G298A, K299A, and P443A mutated EAAT2. Assays were performed in COS-7 cells transiently transfected with appropriate cDNA (chimeras or mutants) or empty vector. Cells were incubated with varied concentrations (a) or 2 μg/mL (b-d) of venom for 10 min at 37oC
and 5 min with 50 nM of 3H-L-glutamate. Results are normalized to percentage of control (vehicle) and expressed as mean ± SEM of three independent experiments (One Way Anova followed by Bonferroni’s Multiple Comparison post-hoc test using vehicle as control, **p
