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.

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  • Author(s): Shabaneh M;Shabaneh M; Rosental N; Rosental N; Kanner BI; Kanner BI
  • Source:
    The Journal of biological chemistry [J Biol Chem] 2014 Apr 18; Vol. 289 (16), pp. 11175-11182. Date of Electronic Publication: 2014 Feb 28.
  • Publication Type:
    Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology Country of Publication: United States NLM ID: 2985121R Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1083-351X (Electronic) Linking ISSN: 00219258 NLM ISO Abbreviation: J Biol Chem Subsets: MEDLINE
    • Publication Information:
      Publication: 2021- : [New York, NY] : Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology
      Original Publication: Baltimore, MD : American Society for Biochemistry and Molecular Biology
    • Subject Terms:
      Excitatory Amino Acid Transporter 3/*metabolism ; Ion Channel Gating/*physiology ; Protein Multimerization/*physiology; Amino Acid Substitution ; Animals ; Dithiothreitol/pharmacology ; Excitatory Amino Acid Transporter 3/genetics ; HeLa Cells ; Humans ; Ion Channel Gating/drug effects ; Ion Transport/drug effects ; Ion Transport/physiology ; Mutation, Missense ; Organometallic Compounds/pharmacology ; Protein Multimerization/drug effects ; Protein Structure, Tertiary ; Rabbits ; Xenopus laevis
    • Abstract:
      Excitatory amino acid transporters remove synaptically released glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. A structure of an archeal homologue, which reflects an early intermediate on the proposed substrate translocation path, has been suggested to be similar to an anion conducting conformation. To probe this idea by functional studies, we have introduced two cysteine residues in the neuronal glutamate transporter EAAC1 at positions predicted to be close enough to form a disulfide bond only in outward-facing and early intermediate conformations of the homologue. Upon treatment of Xenopus laevis oocytes expressing the W441C/K269C double mutant with dithiothreitol, radioactive transport was stimulated >2-fold but potently inhibited by low micromolar concentrations of the oxidizing reagent copper(II)(1,10-phenanthroline)3. The substrate-induced currents by the untreated double mutant, reversed at approximately -20 mV, close to the reversal potential of chloride, but treatment with dithiothreitol resulted in transport currents with the same voltage dependence as the wild type. It appears therefore that in the oocyte expression system the introduced cysteine residues in many of the mutant transporters are already cross-linked and are only capable of mediating the substrate-gated anion conductance. Reduction of the disulfide bond now allows these transporters to execute the full transport cycle. Our functional data support the idea that the anion conducting conformation of the neuronal glutamate transporter is associated with an early step of the transport cycle.
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    • Grant Information:
      R01 NS016708 United States NS NINDS NIH HHS; R56 NS016708 United States NS NINDS NIH HHS; NS 16708 United States NS NINDS NIH HHS
    • Contributed Indexing:
      Keywords: Gating; Glutamate; Membrane Biophysics; Membrane Energetics; Membrane Transport; Neurotransmitter Transport
    • Accession Number:
      0 (Excitatory Amino Acid Transporter 3)
      0 (Organometallic Compounds)
      0 (copper(2)-(10-phenanthroline)2 tetrachloride)
      T8ID5YZU6Y (Dithiothreitol)
    • Publication Date:
      Date Created: 20140304 Date Completed: 20140604 Latest Revision: 20211021
    • Publication Date:
      20240829
    • Accession Number:
      PMC4036256
    • Accession Number:
      10.1074/jbc.M114.550277
    • Accession Number:
      24584931