Fig. 2.

Characterization of vesicular glutamate transport by VGLUT2 in transiently transfected PC12 cells. A, Transfection efficiency close to 100% and high expression levels are obtained as illustrated by the strong labeling visible after 30 min incubation with X-gal at 37°C in β-galactosidase-expressing PC12 cells.B, VGLUT2 is detected in transfected but not in mock-transfected PC12 cell homogenates. C, Time course of glutamate uptake in VGLUT2-transfected (●) and mock-transfected (○) PC12 cells. D, Saturation analysis of glutamate uptake by VGLUT2. VGLUT2-specific (mock-subtracted) uptake velocity reaches a plateau at ∼2 mm substrate.Inset, Lineweaver-Burk analysis of VGLUT2 initial velocity determines a K_m of 0.8 mm and a V_max of 190 pmol · min⁻¹ · mg⁻¹protein. E, VGLUT2 is highly specific for glutamate. Transport of glutamate (50 μm) was measured in 4 mm Cl⁻-containing medium in the absence (100%) or presence of various amino acids (10 mm), K₂PO₄ (20 mm), or trypan blue (TB) (1 μm). F, VGLUT2 activity is H⁺ dependent. H⁺-ATPase inhibitors and dissipaters of the H⁺ electrochemical gradient abolish transport. Vesicle preparations were preincubated for 2 min with 1 μm bafilomycin A1 (bafilo), 200 μmN-ethyl maleimide (NEM), 50 μm carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP), or 20 mm KSCN (SCN). G, Anion specificity of VGLUT2 stimulation. Vesicle preparations were incubated in the absence or presence of the potassium salts of various inorganic anions (4 mm), chloride (Cl⁻), bromide (Br⁻), iodide (I⁻), phosphate (PO₄⁻), thiocyanate (SCN⁻), and in the presence of Cl⁻ plus 1 μmDIDS or 4 mm SCN⁻.H, Transport is dependent on Δψ but can be driven by ΔpH. Vesicle preparations were incubated in the absence (control) or presence of 1 μmnigericin (+Nig), 1 μm valinomycin (+Val), or both (+Nig,+Val), in a medium containing 4 or 40 mm Cl⁻ and 5 mm Mg-ATP. Nonspecific transport (corresponding mock values) was subtracted.