Investigating the functional interaction between the dopamine D2 receptor (D2R) and the dopamine transporter (DAT) using a novel plate-based assay

Disruption of synaptic dopamine (DA) levels is implicated in a number of neurological disorders such as Parkinson’s disease and schizophrenia (1). DA release and reuptake are regulated by the short isoform of the D2R (D2S) and DAT co-expressed in pre-synaptic dopaminergic nerve terminals. Co-expression of D2S with DAT has been reported to increase the uptake function of DAT (2). The aim of this study was to investigate whether co-expression of DAT and D2R affected the function of either protein.

CHO K1 cells stably expressed DAT tagged with green fluorescent protein (GFP) alone, or with either SNAP-tagged D2S (DAT-D2S) or long isoform D2R (D2L) (DAT-D2L). In assaying DAT function, cells on 96 well plates were incubated in buffer (HBSS, 50µM Brilliant Black BN) with ligand (20min, 37ºC), prior to addition of fluorescent DAT substrate 4-[4-(diethyl-amino)-styryl]-N-methylpyridinium iodide (ASP+, 10 µM) (3). Fluorescent substrate uptake after 30min at 37ºC was quantified using a FlexStation3 (ex:485 nm, em:590nm). D2R function was studied using CHO D2S, D2L, DAT-D2S and DAT-D2L membranes. Membranes were pre-incubated with ligand and 1µM GDP (1h, 21°C) in buffer (20mM HEPES, 10mM MgCl2, 100mM NaCl, pH 7.4) before incubation with 200pM [35S]GTPγS (20 min, 21ºC). Bound radioactivity was separated using GF/B filter plates, which were washed and dried before quantification of radioactivity (Packard TopCount). Concentration response curves were fitted in GraphPad Prism v6.

The rank order of potency of DAT inhibitors in the ASP+ assay was consistent across DAT, DAT-D2S and DAT-D2L cell lines: IN>GBR>JHW>BP (Table 1). D2R ligand effects were also consistent between cell lines: QP had no effect, AP and DA inhibited ASP+ accumulation (Table 1), and BC increased accumulation at >10µM. In the [35S]GTPγS assay, D2R agonists showed no difference in pEC50 in D2S, DAT-D2S, D2L and DAT-D2L cell lines; QP: 6.82±0.23, 6.60± 0.07, 6.94±0.21, 6.54±0.10; BC: 8.76±0.18, 8.77±0.18, 8.84±0.19, 8.40±0.10; AP: no effect; and DA: 7.11±0.21, 6.78 ±0.08, 6.66±0.08, 6.53±0.11 respectively (n=5). The maximum responses to AP and DA (represented as % 10µM QP response) were similar in all cell lines, while BC Rmax decreased with DAT co-expression; D2S 76±9, DAT-D2S 51±3 and D2L 70±11, DAT-D2L 34±9% (P<0.05; unpaired t-test, Welch’s correction). These data suggest that co-expression of either isoform of D2R with DAT does not affect DAT inhibitor potency; however BC efficacy at theD2S/L receptor is reduced with DAT co-expression, indicating an alteration in receptor function.

Table 1: ASP+ assay data. Summary of D2R agonist and DAT inhibitor responses in DAT, DAT-D2S and DAT-D2L expressing CHO cells.
Data are shown as mean ± SEM; n=5-19. NE=no effect.* = Increase in ASP+ accumulation at 10µM BC.

Supported by an MRC DTA studentship to PE.

(1) Beaulieu JM et al. (2015). IUPHAR 172: 1–23.

(2) Bolan E et al. (2007). Mol Pharm 71: 1222–1232.

(3) Mason JN. et al. (2005) J Neurosci Methods 143: 3–25.