Figure 3. hDAT-mediated depolarization activates L-type Ca²⁺ channels in Flp-hDAT cells.

Intracellular Ca²⁺ concentration was determined by fluorescence microscopy in Flp-hDAT or the parental Flp-In^TM T-REx^TM 293 (Flp-In) cells (no hDAT expression) cells co-transfected with Ca_V1.2 (A, C, E) or Ca_V1.3 (B, D,F) plus β₃, α₂δ and EGFP plasmids, using the Ca²⁺ sensitive dye Fura-2AM. The experiments were carried out under constant perfusion at 35°C. The transfected cells were identified by their EGFP signal. A, B. Cells were briefly exposed to DA 10 μM, S(+)AMPH 5 μM or high potassium external solution 130 mM (K⁺) as indicated in each panel. C, D. The blockade of hDAT with methylenedioxypyrovalerone (MDPV, 1 μM) prevented Ca²⁺ signals induced by hDAT substrates. E, F. Cells were exposed to external solution containing Li⁺ (equimolar substitution of Na⁺) or external solution with high potassium as indicated in the timeline of each panel. Li⁺-induced Ca²⁺ signals only take place in cells expressing hDAT. Traces represent the mean ± s.e.m. of n ≥ 30 cells per condition.