Fig. 1. PIP₂/N-terminus interactions regulate DAT phosphorylation, a posttranslational modification that supports PIP₂ independent DA efflux.

a hDAT WT, hDAT K/N, or hDAT K/A cells were labeled with ³²P prior to vehicle or AMPH (10 μM, 30 min) incubation. Equal amounts of DAT determined by immunoblotting were immunoprecipitated and subjected to SDS-PAGE/auto-radiography. Left: representative autoradiographs showing hDAT phosphorylation after vehicle or AMPH exposure, along with respective immunoblots of total hDAT. Right: DAT phosphorylation is quantified as ³²P labeling normalized to basal hDAT ³²P labeling. hDAT WT phosphorylation increased post-AMPH treatment relative to vehicle (n = 4; F_{(2, 18)} = 5.05, p = 0.002). In hDAT K/N and hDAT K/A cells, displayed no changes in DAT phosphorylation in response to AMPH compared with vehicle (p > .05). b Left: representative traces of amperometric currents recorded from hDAT WT (black traces) and hDAT S/D (green traces) preincubated in either vehicle or PAO (20 μM, 10 min) and subsequently treated with AMPH (10 μM; indicated by arrow). Right: quantitation of mean peak current amplitudes in hDAT WT and hDAT S/D cells. Preincubation with PAO significantly decreased DA efflux in hDAT WT cells (0.33 ± 0.04 pA) with respect to vehicle (0.16 ± 0.02 pA; p = 0.03; n = 4), but not in hDAT S/D cells (vehicle: 0.38 ± 0.06 pA, PAO: 0.35 ± 0.05 pA; p > 0.05; n = 4). c Representative traces from hDAT S/D cells after whole-cell patch delivery of control peptide (3 μM, pal-HAQKHFEAAA) or PIP₂ sequestering peptide (3 μM, pal-HRQKHFEKRR) to the cytoplasm of the cell prior (10 min) to the application of AMPH (10 μM, indicated by arrow). Delivery of control or PIP₂ sequestering peptides resulted in comparable peak currents in response to AMPH (n = 4; p > 0.05). Data are presented as mean ± SEM. Two-way ANOVA with Bonferroni’s multiple comparison test: a, b; Student’s t test: c.