Fig. 3.

E313 is crucial for rVMAT2 activity in cells. (A) Predicted location of E313 in the cytoplasm-facing model of rVMAT2, viewed from the plane of the membrane with the cytoplasm at the bottom. Helices are shown as ribbons and using a surface representation, sliced through the center of the protein. The side chain of E313 is shown as sticks. (B) Schematic of the whole-cell transport system. HEK293 cells are permeabilized with digitonin and supplemented with ATP to generate the proton gradient by the V-type ATPase. (C) [³H]-serotonin transport into vesicles in cells transfected with rVMAT2 (■), E313Q (●), and E313D (▲). Digitonin-permeabilized cells were assayed after incubation with [³H]-serotonin. (D) Binding of the noncompetitive inhibitor of VMAT2, [³H]-TBZOH. (Inset) Western blot. Membranes were analyzed as described in Materials and Methods. Results presented are from duplicate experiments; error bars indicate SE. Experiments were repeated at least twice.