Identification of the differentiation-associated Na+/PI transporter as a novel vesicular glutamate transporter expressed in a distinct set of glutamatergic synapses - PubMed (original) (raw)

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 m

m

substrate.Inset, Lineweaver-Burk analysis of VGLUT2 initial velocity determines a _K_m of 0.8 m

m

and a _V_max of 190 pmol · min−1 · mg−1protein. E, VGLUT2 is highly specific for glutamate. Transport of glutamate (50 μ

m

) was measured in 4 m

m

Cl−-containing medium in the absence (100%) or presence of various amino acids (10 m

m

), K2PO4 (20 m

m

), or trypan blue (TB) (1 μ

m

). 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 μ

m

bafilomycin A1 (bafilo), 200 μ

m

_N-_ethyl maleimide (NEM), 50 μ

m

carbonyl cyanide_p-_trifluoromethoxyphenylhydrazone (FCCP), or 20 m

m

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 m

m

), chloride (Cl−), bromide (Br−), iodide (I−), phosphate (PO4−), thiocyanate (SCN−), and in the presence of Cl− plus 1 μ

m

DIDS or 4 m

m

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 μ

m

nigericin (+Nig), 1 μ

m

valinomycin (+Val), or both (+Nig,+Val), in a medium containing 4 or 40 m

m

Cl− and 5 m

m

Mg-ATP. Nonspecific transport (corresponding mock values) was subtracted.