Model systems for analysis of dopamine transporter function and regulation

Abstract

The dopamine transporter (DAT) plays a critical role in dopamine (DA) homeostasis by clearing transmitter from the extraneuronal space after vesicular release. DAT serves as a site of action for a variety of addictive and therapeutic reuptake inhibitors, and transport dysfunction is associated with transmitter imbalances in disorders such as schizophrenia, attention deficit hyperactive disorder, bipolar disorder, and Parkinson disease. In this review, we describe some of the model systems that have been used for in vitro analyses of DAT structure, function and regulation, and discuss a potential relationship between transporter kinetic values and membrane cholesterol.

Keywords: CFT; Cholesterol; Kinetic parameters; Neuronal cultures; Nucleus accumbens; Striatum.

Fig. 1.. Morphology of cell systems used to study DAT function and regulation.

Panels show phase contrast microscopic images of several cell systems studied in our labs. Images were acquired using a 20X objective on an inverted Olympus microscope equipped with a digital camera. The scale bar indicates 50 μm and is the same for all panels. The dopaminergic neuron image showing tyrosine hydroxylase positive midbrain neurons (arrow) is modified from (Branton and Clarke, 1999).

Fig. 2.. Distribution of K_m(DA) values across model systems.

Scatterplot representation of reported K_m(DA) values for rat, mouse, human, and nonhuman primate DATs expressed in cell or brain tissue systems.

Fig 3.. Membrane cholesterol content from DAT model systems.

Membranes were prepared as previously described from cells (Moritz et al., 2013) or rat striatal synaptosomes (Foster and Vaughan, 2011) and assayed for total cholesterol using a colorimetric total cholesterol kit from Fujifilm Wako Diagnostics (Richmond, VA) and total protein using the BCA method as previously described (Foster et al., 2008). Values shown are means ± SEM (n=3-6). Statistical analysis was performed by ANOVA with a Dunnett’s multiple comparison post-hoc test. ***, p<0.001 indicated cells vs. synaptosomes; ^†, p<0.05 PC-12 cells vs. LLC-PK₁ cells; n.s. no statistical difference for indicated groupings.

Fig. 4.. Inverse correlation between K_m and membrane cholesterol content.

(A) Plot of pooled K_m(DA) values (means ± SEM) vs. membrane cholesterol for the indicated cell lines and tissues. The line indicates the linear regression analysis (r²=0.78; Pearson correlation coefficient of r = −0.88 ;*** p= 0.0008).(B) Plot of K_d(CFT) values (means ± SEM) vs. membrane cholesterol for the indicated cell lines and tissues. The line indicates linear regression analysis (r²=0.16, Pearson correlation coefficient of r = ‒0.4; p = 0.43). Asterisk indicates significant difference (*, p<0.05) between K_d(CFT) LLC-PK₁ vs synaptosomes. Numbers in parentheses indicate the number of reports for each system and where absent error bars lie within the symbol.