FIGURE 2.

A and B, α-synuclein increases DAT-mediated inward currents and induces a rightward shift in the current-voltage I(V) relationship. A, the experiments were performed as in Fig. 1 except the dialysis electrode contained 5 μm α-synuclein. Briefly, after two tight seals were obtained, the recording electrode was transitioned into whole cell configuration. The whole cell currents were recorded as described in Fig. 1. The dialysis electrode containing 5 μm α-synuclein was then switched to whole cell configuration, dialyzing the recombinant α-synuclein into the cell for 10 min. Whole cell currents were recorded at this time (n = 4–5). B, shown are resting membrane potentials of cells expressing DAT only or DAT with and without α-synuclein (n = 6). C, active dopamine transporter is required for α-synuclein-stimulated increase in inward currents. A 5-min pre-application of a DAT blocker, GBR 12935 (5 μm), eliminates the effect of α-synuclein on inward currents and the rightward shift in the current voltage relationship. The black squares represent GBR12935 subtracted base-line currents. The circles represent the currents after the dialysis of α-synuclein into the cell when the transporter is blocked (n = 4) D, heat-inactivated α-synuclein does not increase the inward currents or affect the reversal potential. Averaged normalized whole cell currents before and after dialysis of heat-inactivated α-synuclein into the cell are shown; experiments were performed as in Figs. 2 and 3. Whole cell currents were recorded before and 10 min after dialysis of heat-inactivated α-synuclein into the cell. There is no effect on the reversal potential of the I(V) curve with intracellular heat-inactivated α-synuclein (n = 4).