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. 2010 Apr 28;30(17):6048–6057. doi: 10.1523/JNEUROSCI.5094-09.2010

Figure 1.

Figure 1.

D2R signaling sustains hDAT A559V-mediated ADE through a Gi/Go-dependent pathway. A, Top, Representative oxidative currents, filtered at 1 Hz, in hDAT A559V cells following pretreatment with vehicle (left) or 1 μm raclopride (right). Arrows indicate application of cocaine. Bottom, Data reported as mean amperometric current ± SEM in hDAT A559V cells expressed as a percentage of current recorded in hDAT cells, both for vehicle control conditions (n = 7) and with raclopride (n = 6) (t = 5.6, df = 11, p = 0.0002, Student's t test). B, Top, qPCR demonstrating the presence of endogenous D2R in HEK 293T cells. Traces from real-time PCR (top, left) and representative PCR gel (top, right) demonstrating that when RNA isolated from HEK 293T cells is reverse transcribed (+RT), PCR product crosses threshold near cycle 30 (arrow) (cT = 31.2 ± 0.1, n = 8), whereas if no reverse transcription is performed (−RT), no product reaches threshold (arrow). Middle, Representative immunoblot for D2R (top) and actin (bottom) in HEK 293T cells transfected with the indicated cDNA, confirming D2R expression. Mouse striatum, heavily enriched with endogenous D2R, was used as a positive control (n = 3). Bottom, Representative confocal images of single sections demonstrating that endogenous D2R and transfected hDAT are expressed in HEK 293T cells (upper row) and that cotransfection of hDAT and GFP leads to their coexpression (lower row). C, Top, Representative oxidative currents recorded from hDAT A559V cells pretreated for 4 h with vehicle control (left) or with 200 ng/ml PTX (right). Bottom, Data reported as mean amperometric current ± SEM in hDAT A559V cells expressed as a percentage of current in hDAT cells, both in vehicle control conditions (n = 3) and with PTX (n = 5) (t = 5.2, df = 6, p = 0.0019, Student's t test). D, Top, Representative oxidative currents in hDAT S/D cells [5 most distal hDAT N-terminal serines (Ser-2, Ser-4, Ser-7, Ser-12, and Ser-13) substituted by aspartates to mimic phosphorylated state] following pretreatment with vehicle (left) or 1 μm raclopride (right). Bottom, Data reported as mean amperometric current ± SEM in hDAT S/D cells, expressed as a percentage of current in hDAT cells both in control conditions (hDAT, n = 6; hDAT S/D, n = 6) and with raclopride (hDAT, n = 6; hDAT S/D n = 4) (t = 0.8, df = 8, p = 0.44, Student's t test). **p < 0.05.