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Dopamine uptake and cocaine binding mechanisms: The involvement of charged amino acids from the transmembrane domains of the human dopamine transporter

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Abstract

The wild type human dopamine transporter (DAT) and five DAT mutants were transfected into COS-7 cells and their ability to uptake dopamine or to bind cocaine was examine three days later. In each mutant, a single charged amino acid, located in areas that initial hydrophobic analysis had indicated were DAT transmembrane domains was substituted by alanine. Mutants used in this study were lysines 257 and 525 (termed K257A and K525A), arginines 283 and 521 (termed R283A and R521A), and glutamate 491 (termed E491A). Dopamine affinity was significantly enhanced in the K257A and R283A mutants, and the IC50 for displacement of the radioactive cocaine analog 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (CFT) by cocaine was significantly elevated in the E491A mutant. All mutants displayed a reduction or complete loss of the maximal velocity (Vm) of dopamine transport.

Introduction

The dopamine transporter (DAT) is the dopamine uptake site, and the binding site of cocaine (Ritz et al., 1987). Cocaine causes its euphoric and reinforcing effects by inhibiting the DAT-mediated reuptake of dopamine (Kuhar et al., 1991).

The DAT is a member of a family of Na+ and Cl dependent biogenic amine transporters that also includes the norepinephrine transporter (NET) and the serotonin transporter (SERT) (Norregaard and Gether, 2001, Chen and Reith, 2000). Initial hydrophobicity analysis predicts these transporters to have 12 transmembrane domains with the N- and C-termini having an intracellular orientation (Pacholczyk et al., 1991). Recently, the bacterial leucine (Yamashita et al., 2005) transporter, LeuTAa, a homologue of the eukaryotic Na+/Cl transporters, was crystallized and found to be composed of 12 transmembrane domains with intracellular N- and C-termini, as predicted. The LeuTAa transmembrane domains discovered, however, only partially overlap with those suggested for the eukaryotic transporters.

Cloning of DAT cDNA (Shimada et al., 1991, Vandenbergh et al., 1992) has allowed more direct investigation of its structure, function and drug interactions. Since the crystal structure of the human DAT (hDAT) is not yet available, studies that mutate specific DAT amino acids are often used to elucidate DAT structure and function (Kitayama et al., 1992, Lin et al., 1999, Lin et al., 2000a, Lin et al., 2000b). In previous mutagenesis studies, the roles of the DAT's aromatic (Lin et al., 1999, Lin et al., 2000b) cyclic (Lin et al., 2000a) and polar (Itokawa et al., 2000) residues were extensively investigated, but little information was gathered about the role of acidic (aspartate and glutamate) or basic (lysine, arginine and histidine) DAT amino acids. The exception was an early study examining DATs mutated at aspartic acid 79, which found this residue to be significant for both dopamine uptake and cocaine binding (Kitayama et al., 1992).

In this study, the role of charged DAT amino acids was investigated in relation to dopamine transport and cocaine binding mechanisms. According to all analyses, most DAT transmembrane domains consist primarily of aliphatic amino acids, and only a few charged amino acids. Charged DAT amino acids that are conserved in 2 or all of the biogenic amine transporters and that are located in the transmembrane domains, according to the initial hydrophobic analysis (Pacholczyk et al., 1991), were mutated to alanine, transfected to COS-7 cells and tested for their ability to bind cocaine or accumulate dopamine.

Section snippets

Materials

[3H]CFT (84.5 ci/mmol) was from Perkin-Elmer Life Science (Boston, MA, USA). [3H]dopamine (46 ci/mmol) was from Pharmacia Amersham (Amersham, England). Dulbecco's modified eagle's medium (DMEM), fetal calf serum (FCS) and trypsin/EDTA were from Life Technologies (Gaithesburg, MD, USA). Fish sperm DNA was from Hoffmann-La Roche Inc (Nutley, NJ, USA). Cocaine hydrochloride was supplied by the National Institute on Drug Abuse.

Preparation of DAT mutants

The cDNA that encodes wild type hDAT (Vandenbergh et al., 1992) in

Results

Lysine 257, arginine 283, glutamate 491, arginine 521 and lysine 525, which were presumed to be located within transmembrane (TM) domains 4, 5, 10 and 11, according to the initial hydrophobic analysis (Pacholczyk et al., 1991), were mutated to alanine. DAT mutants were expressed in COS-7 cells and tested for their ability to bind cocaine by displacing radiolabeled CFT, and for the ability to accumulate radiolabeled dopamine. Except from the E491A mutant, all DAT mutants displayed some degree of

Discussion

Hydrophobic analysis shows 2.7–5.4% of DAT transmembrane sequence to be comprised of charged amino acids (Pacholczyk et al., 1991, Goldberg et al., 2003), while LeuTAa superposition puts the figure at 8.7% (Yamashita et al., 2005). According to the hydrophobic analysis conducted by Goldberg et al. (2003), all the described amino acids, except Lysine 257, are located within their related transmembrane domains. Lysine 257 is the first amino acid found outside the membrane, towards its inner part.

Conclusions

Mutation of the DAT transmembrane domain charged amino acids targeted in this study resulted in a reduction or complete loss of the Vm of dopamine uptake. The affinity of dopamine for the K257A and R283A mutants was significantly enhanced, and the affinity of cocaine for the E491A mutant was significantly reduced, compared to their affinities for wild type DAT. The major findings of this study are that glutamate 491 is significant in the general functioning of the dopamine transporter and that

Acknowledgments

The authors acknowledge the financial support of the National Institute on Drug Abuse–Intramural Research Program.

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1

Current address: University of Southern Nevada, College of Pharmacy, II Sunset Way, Henderson, NV 89014, USA.

2

Current address: Center for Developmental and Health Genetics and Department of Biobehavioral Health, The Pennsylvania State University, 101 Amy Gardener House, University Park, PA 16802, USA.

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