Dynamic regulation of the dopamine transporter
Introduction
Dopamine, a major neurotransmitter in the mammalian central nervous system, is involved in the control of locomotor activity, and also in pathways regulating goal oriented behavior and reward (Schultz, 2002). The dopamine transporter clears neurotransmitter from the extracellular space and serves as an important regulator of signal amplitude and duration at dopaminergic synapses. The dopamine transporter also regulates the activation of extrasynaptic receptors and thus has a significant impact on volume neurotransmission, another established mode of dopamine signaling. The catalytic rate of the transporter itself is slow—less than one dopamine molecules/s in cultures of midbrain dopamine neurons—however, this low turnover rate may not be so surprising in that dopamine acts through G-protein coupled receptors that modulate intracellular events on a timescale of seconds (Prasad and Amara, 2001) The transporter is expressed selectively in dopaminergic neurons of the substantia nigra and the ventral tegmental area of the brain Ciliax et al., 1995, Freed et al., 1995. These dopaminergic neurons project to the striatum, nucleus accumbens and the prefrontal cortex and the transporters are expressed throughout the cell on axons, dendrites and the soma, but have not been found in the active zones of synapses (Nirenberg et al., 1997).
Psychostimulants, such as methylphenidate, cocaine, and amphetamines, exert many of their effects by acting on the dopamine transporter, and thus provide a compelling clinical rationale for understanding the function and regulation of the carrier. These drugs either block transport of substrates, as established for cocaine or methylphenidate, or are carrier substrates, such as amphetamines, which both inhibit uptake of extracellular dopamine and stimulate efflux of intracellular dopamine. The result is an increase in extracellular dopamine that can activate the well-known motor and reward pathways of the midbrain and trigger the increased locomotor activity and euphoria associated with drug use. Dopamine transporter gene disruption experiments in mice have demonstrated the importance of the carrier in psychostimulant action; mice that completely lack a functional dopamine transporter display intense hyperactivity and a profound persistence of extracellular dopamine, mimicking the prominent actions of the drugs. However, the striking behavioral effects and changes in the rate of dopamine clearance observed in homozygous knock out animals are not further enhanced by cocaine or amphetamine administration, providing additional support for the importance of the dopamine transporter as an obligate target for these stimulant drugs (Giros et al., 1996). It is important to note that all the behavioral effects of cocaine cannot be attributed solely to dopamine transporter, as these mice still show conditioned place preference for cocaine Rocha et al., 1998, Sora et al., 1998. Mice with a double disruption of both dopamine and serotonin transporter genes exhibit no conditioned place preference in response to cocaine, supporting the contribution of the serotonin transporter to cocaine reward and reinforcement (Sora et al., 2001). A mouse “knockdown” line which retains only 10% of wild type dopamine transport activity has provided a promising model for the counterintuitive calming effect of psychostimulants, such as methylphenidate, on patients with attention deficit hyperactivity disorder (ADHD) (Zhuang et al., 2001). These mice show the decreased dopamine clearance rates and hyperactivity characteristic of a hyperdopaminergic phenotype. Intriguingly, in this model, which retains some dopamine transporter function, psychostimulants also have a calming effect that resembles their therapeutic action in humans with ADHD.
The result of transporter knock outs have underscored how important short term and chronic regulation of the dopamine transporter are during normal dopaminergic neurotransmission, and in a variety of neurological and neuropsychiatric disorders, including ADHD, schizophrenia, psychostimulant addiction and Parkinson's disease. Both the chronic regulatory changes and the acute regulation of transport activity have been the subject of an excellent, comprehensive review on the biogenic amine transporters (Zahniser and Doolen, 2001). Activation of intracellular signaling systems has been proposed to acutely modulate transport activity directly by altering the phosphorylation state of carriers, or indirectly by changing the electrical and chemical gradients that drive transport or by regulating the trafficking of carriers to and from the cell surface. A summary of these processes is illustrated in Fig. 1. With an emphasis on the dopamine transporter, our review will consider on recent developments in our understanding of the dynamic cellular events that regulate short-term changes in transport activity.
Section snippets
Na+- and Cl−-dependent transporter family
The dopamine transporter belongs to the family of Na+/Cl− dependent neurotransmitter transporters. The cloning of two transporters by different groups initially facilitated the identification of this family. Amino acid sequence information from a γ-hydroxybutyric acid (GABA) transporter purified from rat brain was the basis for cloning the first GABA transporter, GAT1 (Guastella et al., 1990). A norepinephrine transporter was cloned using a functional expression assay based on its ability to
Voltage dependence of transport in cloned and endogenous carriers
As transport involves the electrogenic movement of charged substrates and ions, the amount of transport could vary with changes in membrane potential. Xenopus oocytes provided a unique system in which to address this issue because uptake can be measured while simultaneously maintaining a constant membrane potential. This approach was used to show that dopamine uptake by the human dopamine transporter is a modestly voltage-dependent process and that transport increases with hyperpolarization and
Impact of carrier phosporylation on activity
Because members of the Na+/Cl− dependent family of transporters possess in their amino acid sequences multiple consensus sites for phosphorylation by several different protein kinases, it has been speculated that a direct phosphorylation of the transporter could regulate the activity of the transporter. Possible consequences of such phosphorylation events include altering the catalytic rate of the carrier, shifting its apparent affinity for substrate and/or inhibitors, and marking the carrier
Arachidonic acid, ethanol, and nitric oxide
Several other molecules have been implicated in the regulation of dopamine transporter. Inhibitors of arachidonic acid metabolism are relatively potent inhibitors of dopamine uptake in rat striatal slices (Cass et al., 1991) an observation supported by a later study performed in striatal synaptosomes (L'hirondel et al., 1995). Others have shown in C6 rat glioma cells stably expressing the dopamine transporter that short incubations with arachidonic acid stimulate dopamine uptake while longer
Activation of protein kinase C alters activity
Several different intracellular signaling pathways have been studied and implicated in the regulation of dopamine transporters. By far the best characterized is the stimulation of protein kinase C (PKC) activation using phorbol esters. The most consistent result from these studies is a rapid down regulation of transport activity as a result of removal of transporters from the cell surface. This effect has been shown in striatal synaptosomes Copeland et al., 1996, Vaughan et al., 1997, in COS
Oligomerization
Recent studies have investigated whether the transporters work as single units or they form complexes with each other and whether this could have an effect on transport activity. Several different studies have addressed these issues, and it has in fact been possible to show that the transporters do exist as complexes or oligomers Hastrup et al., 2001, Sorkina et al., 2003, Torres et al., 2003.
It has been difficult to determine whether oligomerization is important for the activity of the
Conclusions
Both genetic and pharmacological studies have established that the dopamine transporter serves a pivotal role in limiting dopamine-mediated neurotransmission and ultimately in determining behavior. As the dopamine transporter is a major target for drugs used in treating depression, nicotine addiction, ADHD and for psychostimulant drugs of abuse it is important to understand the precise mechanisms that regulate its functions. It is now well recognized that the transporter can be regulated both
Acknowledgments
We thank Drs. Geoffrey Murdoch, Mark Sonders and Andreia Fontana for providing helpful comments and discussion on the manuscript. Work from our laboratory has been supported by NIH grants DA07595, DA12408, DA13838 and the Howard Hughes Medical Institute (SGA) and the Alfred Benzon Foundation (OVM).
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