Fig.7. Flot1 is required for PKC-triggered internalization and AMPH-induced reverse transport of DA in primary DAergic neurons.

a. Representative DAT-mediated transient currents recorded from DA neurons transduced with lentiviri carrying shCtrl or shFlot1 following a voltage jump to −140 mV from a holding potential of −20 mV. The integral of the DAT-mediated transient current is proportional to the number of transporters on the cell surface². Transient currents were obtained first with vehicle, then in the presence of PMA. Bars represent the mean ratios of PMA transient currents to pre-PMA responses in the same cell. PMA did not reduce DAT-mediated transient currents in shFlot1 neurons, suggesting that Flot1 is necessary for PMA-triggered DAT trafficking. b. Loss of Flot1 leads to an inhibition of AMPH-mediated DA efflux. Neurons were loaded via the patch pipette with 2 mM DA and 30 mM Na⁺. Amperometric current-voltage relationship was obtained by stepping the voltage in 20 mV intervals from 0 mV to +100 mV from a holding potential of −60 mV. DAT-mediated DA efflux is defined as the current recorded in the presence of AMPH, minus the current recorded after the addition of cocaine to the bath with AMPH still present from shFlot1 (red, n = 4) and shCtrl (black, n = 4). Values are reported as mean ± SEM (* indicates p < 0.05). shFlot1 significantly reduced DA efflux as compared to shCtrl at voltages greater than +40 mV (* p < 0.05, ** p < 0.01, One-way ANOVA followed by Bonferroni posthoc tests).