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_oF₁-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 (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 Δψ-dependent L-glutamate uptake and a Na⁺-dependent P _i uptake.