Short communicationDopamine uptake and cocaine binding mechanisms: The involvement of charged amino acids from the transmembrane domains of the human dopamine transporter
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.
References (20)
- et al.
Structure and function of the dopamine transporter
Eur. J. Pharmacol.
(2000) - et al.
[3H]WIN 35,428 binding to the dopamine uptake carrier. I. Effect of tonicity and buffer composition
J. Neurosci. Methods
(1994) - et al.
The interaction of methylphenidate and benztropine with the dopamine transporter is different than other substrates and ligands
Biochem. Pharmacol.
(2005) - et al.
Probing conformational changes in neurotransmitter transporters: a structural context
Eur. J. Pharmacol.
(2003) - et al.
The dopamine hypothesis of reinforcing properties of cocaine
Trends Neurosci.
(1991) - et al.
Ligand autoradiographic receptor screening. II. Expression of receptor cDNA in transfected COS cells grown on polyester disks and its recovery
Brain Res. Mol. Brain Res.
(1991) - et al.
A human dopamine transporter cDNA predicts reduced glycosylation, displays a novel repetitive element and provides racially-dimorphic TaqI RFLPs
Brain Res. Mol. Brain Res.
(1992) - et al.
Cloning, pharmacological characterization, and chromosome assignment of the human dopamine transporter
Mol. Pharmacol.
(1992) - et al.
Dopamine transporter transmembrane domain polar mutants: DeltaG and DeltaDeltaG values implicate regions important for transporter function
Mol. Pharmacol.
(2000) - et al.
Dopamine transporter site-directed mutations differentially alter substrate transport and cocaine binding
Proc. Natl. Acad. Sci.
(1992)
Cited by (12)
Model systems for analysis of dopamine transporter function and regulation
2019, Neurochemistry InternationalCocaine-insensitive dopamine transporters with intact substrate transport produced by self-administration
2011, Biological PsychiatryHow dopamine transporter interacts with dopamine: Insights from molecular modeling and simulation
2007, Biophysical JournalCitation Excerpt :The overall stoichiometry is likely as dopamine/Na+/Cl− = 1:2:1 (16,17). Site-directed mutagenesis has been performed extensively on DAT to search for amino-acid residues responsible for the Na+ action, dopamine binding, and their coupling with the actions of cocaine (18–29). However, molecular mechanisms for these actions have appeared as complex and bewildering.
Alchemical Free Energy Calculations on Membrane-Associated Proteins
2023, Journal of Chemical Theory and ComputationQuantitative Assessment of the Energetics of Dopamine Translocation by Human Dopamine Transporter
2018, Journal of Physical Chemistry B
- 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.