Structure and function of the dopamine transporter

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Abstract

The dopamine transporter mediates uptake of dopamine into neurons and is a major target for various pharmacologically active drugs and environmental toxins. Since its cloning, much information has been obtained regarding its structure and function. Binding domains for dopamine and various blocking drugs including cocaine are likely formed by interactions with multiple amino acid residues, some of which are separate in the primary structure but lie close together in the still unknown tertiary structure. Chimera and site-directed mutagenesis studies suggest the involvement of both overlapping and separate domains in the interaction with substrates and blockers, whereas recent findings with sulfhydryl reagents selectively targeting cysteine residues support a role for conformational changes in the binding of blockers such as cocaine. The dopamine transporter can also operate in reverse, i.e. in an efflux mode, and recent mutagenesis experiments show different structural requirements for inward and outward transport. Strong evidence for dopamine transporter domains selectively influencing binding of dopamine or cocaine analogs has not yet emerged, although the development of a cocaine antagonist at the level of the transporter remains a possibility.

Introduction

The present article is linked to Professor De Wied and the Rudolf Magnus Institute in two ways. First, one of the present authors (M.E.A.R) is fortunate to have experienced the inspiring leadership of Professor De Wied while studying for the Masters degree in the Rudolf Magnus Institute, and he is indebted to De Wied's mentorship while preparing for the PhD degree. De Wied's role model of scholarship, discipline, and creativity has been crucial in developing the experiments in this laboratory that are part of the present review of the dopamine transporter area. Second, De Wied's pioneering work on the role of neuropeptides in learning and memory has important ramifications for our understanding of mechanisms involved in the action of drugs of abuse, which clearly involve components of learning and memory. One class of drugs of abuse, the psychostimulants, has the dopamine transporter as a major target, and it is this protein that is the subject of the present mini-review.

The dopamine transporter or carrier, located on the plasma membrane of nerve terminals, transports dopamine across the membrane. By taking up synaptic dopamine into neurons, it plays a critical role in terminating dopamine neurotransmission and in maintaining dopamine homeostasis in the central nervous system Giros et al., 1996, Jones et al., 1998a, Jones et al., 1998b. Many substances, such as the psychostimulant amphetamine, the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and various sympathomimetic amines, structurally resemble dopamine. They are thus substrates for the dopamine carrier and can be transported Jones et al., 1998a, Jones et al., 1998b, Miller et al., 1999a, Miller et al., 1999b. The dopamine transporter is also a major molecular target for the addictive drug cocaine and, to a lesser extent, antidepressants Tatsumi et al., 1997, Amara and Sonders, 1998. These drugs cannot be transported but can bind to the dopamine carrier to block dopamine transport. Therefore, interactions with the dopamine transporter protein can have profound neurobiological, pathophysiological, and pharmacological consequences. In the last decade, since the cloning of the dopamine transporter Giros et al., 1991, Kilty et al., 1991, Shimada et al., 1991, Usdin et al., 1991, major advances have been made in the characterization of dopamine carriers. The focus of this review is on the recent progress in elucidating structure–function relationships for the dopamine transporter.

Section snippets

Molecular and pharmacological characteristics of the dopamine transporter

The dopamine transporter has been identified from brains of various species and from Caenorhabditis elegans (for references see Table 1). A transporter with neuronal dopamine transporter properties and a partial cDNA clone has also been characterized from the African green monkey kidney (COS) cell line (Sugamori et al., 1999). The mammalian dopamine transporters exhibit high sequence identity (Table 1). The reported longer C-terminus of the bovine dopamine transporter was due to an error of the

Structure of dopamine transporter

Up to date, no X-ray crystallographic or high-resolution structural information is available for the topological assignments of the transporters. Hydropathy analysis of the primary sequences of mammalian monoamine transporters predicts a topology with 12 transmembrane segments connected by alternating extracellular and intracellular loops with the N- and C-termini located in the cytosol. A study using cysteine/lysine-modifying reagents and biotinylated probe scanning has agreed with the

Substrate structure/active form and substrate recognition

The structural requirements for the interaction of substrates with the dopamine transporter have been examined by comparing the transport of phenethylamine derivatives in striatal slice preparations (Meiergerd and Schenk, 1994). These studies indicate that the dopamine transporter requires molecules that possess a phenyl ring with a primary ethylamine side chain for optimal activity, and the β rotamer of the extended conformation of catecholamines is transported preferentially (Meiergerd and

Substrate transport: inward and outward

Among 62 single or double/triple dopamine transporter mutants published so far, 14 of the 54 showing near normal membrane expression or CFT binding display reductions in turnover rates for dopamine to less than one-third of wild-type values (Table 2). Except for Phe155 in the rat dopamine transporter, all the other 13 residues are conserved throughout mammal dopamine and norepinephrine carriers. Except for Glu218 in the human dopamine transporter, which is located on the second extracellular

Inhibitor structure and recognition

Among inhibitors, most information is available for cocaine and the related analog CFT, as described in the next section. General indications as to which dopamine transporter domains interact with various inhibitors have been obtained in chimera studies. Thus, chimeras between the transporters for norepinephrine and dopamine constructed in two different laboratories Giros et al., 1994, Buck and Amara, 1995 point to transmembrane domains 5–8 for conferring inhibitor sensitivity to a variety of

Relationship between substrate permeation way and cocaine binding sites

This issue has been explored extensively by chimeric, photoaffinity labeling, and site-directed mutagenesis strategies. In identification of regions essential for function and transport inhibition, of most interest are structural components important for cocaine recognition but not for dopamine transport. This could provide significant insights into a transporter site that might recognize a dopamine-sparing cocaine antagonist and could even provide information about the structure of the cocaine

Acknowledgements

We would like to thank the National Institute on Drug Abuse (DA 08379 and DA 11978 to M.E.A.R.).

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