Rapid delivery of the dopamine transporter to the plasmalemmal membrane upon amphetamine stimulation

doi:10.1016/j.neuropharm.2005.08.018

Neuropharmacology

Volume 49, Issue 6, November 2005, Pages 750-758

https://doi.org/10.1016/j.neuropharm.2005.08.018 Get rights and content

Abstract

The dopamine transporter, DAT, is a primary regulator of dopamine (DA) signaling at the synapse. Persistent stimulation with the substrate amphetamine (AMPH) promotes DAT internalization. AMPH rapidly elicits DA efflux, yet its effect on DAT trafficking at short times is unknown. We examined the rapid effect of AMPH on DAT trafficking in rat striatal synaptosomes using biotinylation to label surface DAT. Within 30 s of treatment with 3 μM AMPH, synaptosomal DAT surface expression increased to 163% of control and remained elevated through at least 1 min before returning to control levels at 2.5 min. The increase in surface DAT was cocaine-sensitive but was not produced by DA itself. A 1-min preincubation with AMPH did not alter [³H]DA uptake, but did result in a higher basal DA efflux and efflux elicited in the presence of AMPH as compared to vehicle pretreatment. Reversible biotinylation experiments demonstrated that the AMPH-stimulated rise in surface DAT is due to an increase in the delivery of DAT to the plasmalemmal membrane rather than a reduction of the endocytic process. These studies suggest that AMPH has a biphasic effect on DAT trafficking and acts rapidly to regulate DAT in the plasmalemmal membrane.

Introduction

The plasmalemmal dopamine transporter (DAT) is a presynaptic protein that belongs to the SLC6A class of Na⁺/Cl⁻ dependent neurotransmitter transporters. DAT is characterized as having 12 trans-membrane segments and a large second extracellular loop with the amino and carboxyl terminals located intracellularly. There are also putative phosphorylation sites for protein kinase C (PKC), protein kinase A (PKA), and CAM kinase II (Giros and Caron, 1993). DAT is the primary regulator of the duration and strength of the dopamine (DA) signal in the synapse (Giros et al., 1996). DAT binds extracellular DA in a Na⁺/Cl⁻ dependent manner and transports it intracellularly, thus clearing it from the synaptic cleft. DA clearance can thus be modulated by turnover rate and density of the plasmalemmal transporter.

Whereas the plasmalemmal transporters were once considered to be stable in the membrane, it is now recognized that they actively traffic between intracellular pools and the cell surface (Melikian, 2004, Zahniser and Sorkin, 2004). DAT exhibits both constitutive and regulated traffickings. Constitutive internalization and recycling of DAT from distinct recycling endosomal pools have been demonstrated in hDAT–PC12 cells (Loder and Melikian, 2003). Under basal conditions DAT internalizes at a rate of 3–5% per minute (Li et al., 2004, Loder and Melikian, 2003).

In addition to the constitutive recycling, DAT exhibits regulated trafficking. Evidence from heterologous expression cell systems and synaptosomes demonstrates that DAT expression at the cell surface can be rapidly regulated by second messenger systems, glycosylation, and DAT substrates and inhibitors (Mortensen and Amara, 2003). Persistent activation of PKC reduces DAT uptake capacity and surface expression (Melikian, 2004, Reith et al., 1997, Zahniser and Sorkin, 2004). N-linked glycosylation of DAT is important for DAT stability and activity of surface DAT (Li et al., 2004). Importantly, DAT ligands themselves regulate DAT surface trafficking. Notable DAT ligands are the psychostimulant drugs, amphetamine (AMPH) and cocaine, which elevate synaptic DA concentrations. Both regulate DAT surface trafficking but apparently do so in different ways. Cocaine, a dopamine transporter blocker, prevents DA uptake into the synaptic terminal, increasing synaptic DA. Acute treatment with cocaine elicited an increase in DAT surface expression and DA uptake activity in striatum from rats given cocaine in vivo or in hDAT-expressing heterologous cells (Daws et al., 2002, Little et al., 2002). Although AMPH also increases synaptic DA, it is a substrate of DAT and competitively inhibits the binding of DA. Following its transport into the synaptic terminal, AMPH elicits a reversal of the transporter which results in DA efflux. AMPH, however, appears to regulate DAT trafficking differently from cocaine and very similar to that seen upon PKC activation. Persistent exposure (>20 min) to amphetamine in rats in vivo and in heterologous expression systems in vitro results in a decrease in DAT transport activity and ionic currents that are ascribed to a decrease in surface expression (Gulley et al., 2002, Melikian, 2004, Saunders et al., 2000, Zahniser and Sorkin, 2004).

However, AMPH elicits pharmacological responses, such as DA efflux, within seconds (Chen and Justice, 2000, Jones et al., 1999). The very rapid effects of AMPH on DAT trafficking are poorly understood, therefore we investigated the effect of AMPH on DAT surface expression and trafficking at times and concentrations closer to its pharmacological effects. These studies were performed in rat synaptosomes to demonstrate the rapid effects of AMPH on trafficking in a physiologically relevant system.

Section snippets

Synaptosome preparation

Rat striata were homogenized in 10 vol of homogenization buffer containing 0.32 M sucrose, 1 mM EDTA and a cocktail of protease inhibitors (Complete Mini, Roche), pH 7.4. Homogenates were centrifuged at 1000 × g for 10 min to remove cell debris. The supernatant was saved and the pellet was resuspended in homogenization buffer and centrifuged again. The combined supernatant fractions were centrifuged for 30 min at 15,000 × g at 4 °C. The supernatant was removed and the pellet (P2) was resuspended in

Rapid effect of AMPH on DAT surface expression

As shown in Fig. 1, incubation of rat striatal synaptosomes with 3 μM AMPH increased surface DAT (biotinylated DAT) within 30 s. The measurement was specific for surface DAT because very little biotinylated actin was detected. We detected only a 0.14% surface-labeling of total actin as compared to an approximately 7% surface-labeling of total DAT. (Note that all of the biotinylated sample is used in the immunoblot, while only 10% of the lysate and non-biotinylated fractions are used.) The

Discussion

A number of studies have demonstrated that persistent treatment of DAT with AMPH will down-regulate the surface expression of DAT through internalization of the transporter (Gulley et al., 2002, Kahlig et al., 2004, Saunders et al., 2000). In this study, we have made the novel observation that AMPH modulates and increases DAT surface expression rapidly within 1 min, a time commensurate with its pharmacological effect. The AMPH-stimulated increase in surface DAT is substrate-specific since 30 μM

Acknowledgements

Funded by NIH grant DA11697 and Pharmacological Sciences Training Grant GM07767. This work was supported in part by the Michigan Diabetes Research and Training Center Grant 5P60DK-20572 from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health. We would like to thank Dr. Steven Lentz for his help in obtaining and processing the confocal images and Dr. Roxanne Vaughan for the MAB16 anti-DAT monoclonal antibody. We would also like to thank Dr.

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Rapid delivery of the dopamine transporter to the plasmalemmal membrane upon amphetamine stimulation

Abstract

Introduction

Section snippets

Synaptosome preparation

Rapid effect of AMPH on DAT surface expression

Discussion

Acknowledgements

Mol. Brain Res.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Trends Pharmacol. Sci.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Pharmacol. Ther.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

J. Biol. Chem.

Neuropharmacology

Substrate-induced trafficking of the dopamine transporter in heterologously expressing cells and in rat striatal synaptosomal preparations

J. Pharmacol. Exp. Ther.