Figure 4
PKCβ inhibition restores AMPH-induced hDAT A559V internalization. (a) Representative immunoblots of phospho-PKCβII in hDAT and hDAT A559V cells. The top bands represent PKCβ in the cytosolic fraction. The middle bands represent PKCβ in the membrane fraction. PMA (100 nM for 20 min) was used as a positive control to demonstrate phosphorylation and translocation of PKCβ to the membrane in hDAT cells. The membrane protein Na/K ATPase (bottom bands) is used as a loading control for the membrane fraction. The bar graph shows that basal levels of phospho-PKCβII in the membrane fraction are higher in hDAT A559V cells than hDAT cells (*P<0.05, Student’s t-test, n=6–8). (b) Cells were treated for 20 min with the PKCβ inhibitor 3-(1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione (300 nM) before treatment with vehicle or AMPH (10 μM, 60 min). Top, representative immunoblot of DAT biotinylated proteins (‘surface DAT’) and total lysate (‘total DAT’). Bottom, quantification of surface to total DAT ratio. PKCβ inhibition restores AMPH-induced trafficking of hDAT A559V (AMPH vs vehicle, *P<0.05 Student’s t-test, n=4). (c) Transient currents were recorded as an index of surface DAT in hDAT A559V cells. Cells were dialyzed under whole-cell patch clamp for 10 min with internal solution containing either the PKCβII inhibitor peptide (N-Myr-SLNPEWNET, 10 μM) or a scramble peptide (10 μM). Top, subsequent DAT-mediated currents following a voltage step from −20 mV to −140 mV are plotted for control (dotted lines) compared with currents after extracellular AMPH treatment (10 μM AMPH for 10 min, solid lines). PKCβII inhibition restores the AMPH-induced decrease in transient current in hDAT A559V cells. Bottom, AMPH decreases transient charge movement Q in hDAT A559V cells treated with PKCβII inhibitor peptide (*P<0.05, QAMPH vs Qcontrol Student’s t-test, n=4) but not scamble peptide (P>0.05, QAMPH vs Qcontrol Student’s t-test, n=4). (d) hDAT A559V S/A cells were treated with vehicle or AMPH (10 μM, 60 min). Top, representative immunoblot of DAT biotinylated proteins (‘surface DAT’) and total cellular lysate (‘total DAT’). Bottom, quantification of surface to total DAT ratio. Mutation of N-terminal Ser to Ala in hDAT A559V restores AMPH-induced trafficking (AMPH vs vehicle, *P<0.05 Student’s t-test, n=4). AMPH, amphetamine; DAT, dopamine transporter; hDAT, human dopamine transporter.
