Figure 5

Synthetic isoketals form adducts with human dopamine transporter (hDAT) and decrease its function. (a) Immunoblots with anti-γ-KA protein adduct and anti-DAT antibodies using green fluorescent protein (GFP)-DAT immunoprecipitates from hDAT cells treated with synthetic isoketals (1 μm) for 1 h at 37 °C. (b) Quantification of immunoprecipitates as a ratio of γ-KA:DAT. Each value represents mean±s.e.m. of ⩾ four independent experiments (t(6)=3.94; **P<0.01 by Student’s t-test; n=4). (c) Uptake of [³H]DA in hDAT cells treated with vehicle (open circles) or with 1 μm synthetic isoketals (gray triangles) for 1 h at 37 °C. The kinetic parameters (d) Vmax and (e) Km were obtained from the Michaelis–Menten fit to [³H]DA uptake. Each value represents mean±s.e.m. of three independent experiments (Vmax: t(4)=4.21; *P<0.05 by Student’s t-test; n=3; Km: t(4)=0.88; *P=0.43 by Student’s t-test). (f) Influx of [³H]DA in hDAT cells pretreated with salicylamine (0.5 mm) and then treated with vehicle (open circles, 0.1% ethanol in Krebs–Henseleit buffer), Fe⁺³/Asc (open triangles) or with 40 μm AA for 1 h at 37 °C in the absence (gray squares) or presence of Fe⁺³/Asc (gray triangles). The kinetic parameters (g) Vmax and (h) Km were obtained from the Michaelis–Menten fit to the influx of [³H]DA. Each value represents mean±s.e.m. of ⩾ three independent experiments. No significant differences were observed between the groups. The statistical analysis was performed with analysis of variance. AA, arachidonic acid; Veh, vehicle.