Vesicular glutamate transporter contains two independent transport machineries

Narinobu Juge, Yumi Yoshida, Shouki Yatsushiro, Hiroshi Omote, Yoshinori Moriyama

Research output: Contribution to journalArticlepeer-review

85 Citations (Scopus)

Abstract

Vesicular glutamate transporters (VGLUTs) are responsible for the vesicular storage of L-glutamate and play an essential role in glutamatergic signal transmission in the central nervous system. The molecular mechanism of the transport remains unknown. Here, we established a novel in vitro assay procedure, which includes purification of wild and mutant VGLUT2 and their reconstitution with purified bacterial FoF1-ATPase (F-ATPase) into liposomes. Upon the addition of ATP, the proteoliposomes facilitated L-glutamate uptake in a membrane potential (Δψ)-dependent fashion. The ATP-dependent L-glutamate uptake exhibited an absolute requirement for ∼4 mM Cl-, was sensitive to Evans blue, but was insensitive to D,L-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg184, His128, and Glu191 showed a dramatic loss in L-glutamate transport activity, whereas Na+-dependent inorganic phosphate (Pi) uptake remained comparable to that of the wild type. Furthermore, Pi transport did not require Cl- and was not inhibited by Evans blue. Thus, VGLUT2 appears to possess two intrinsic transport machineries that are independent of each other: a Δψ-dependent L-glutamate uptake and a Na+-dependent P i uptake.

Original languageEnglish
Pages (from-to)39499-39506
Number of pages8
JournalJournal of Biological Chemistry
Volume281
Issue number51
DOIs
Publication statusPublished - Dec 22 2006

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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