Vesicular glutamate transporter contains two independent transport machineries.
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 F(o)F(1)-ATPase (F-ATPase) into liposomes. Upon the addition of ATP, the proteoliposomes facilitated l-glutamate uptake in a membrane potential (DeltaPsi)-dependent fashion. The ATP-dependent l-glutamate uptake exhibited an absolute requirement for approximately 4 mm Cl(-), was sensitive to Evans blue, but was insensitive to d,l-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg(184), His(128), and Glu(191) showed a dramatic loss in l-glutamate transport activity, whereas Na(+)-dependent inorganic phosphate (P(i)) uptake remained comparable to that of the wild type. Furthermore, P(i) 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 DeltaPsi-dependent l-glutamate uptake and a Na(+)-dependent P(i) uptake.
Juge
Narinobu
N
Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8530, Japan.
Yoshida
Yumi
Y
Yatsushiro
Shouki
S
Omote
Hiroshi
H
Moriyama
Yoshinori
Y
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001692:Biological Transport; D002712:Chlorides / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D007313:Insecta; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D009154:Mutation; D010710:Phosphates / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D050599:Vesicular Glutamate Transport Protein 2 / Q000737:chemistry / Q000502:physiology*
D002712:Chlorides; D010710:Phosphates; C493475:Slc17a6 protein, rat; D050599:Vesicular Glutamate Transport Protein 2; D018698:Glutamic Acid; D006639:Histidine; D000255:Adenosine Triphosphate; D001120:Arginine; D012964:Sodium