Dopamine transporter (DAT) is a highly specialized membrane-spanning protein that aids in terminating dopaminergic neurotransmission by sodium-dependent reuptake of released dopamine into presynaptic neurons.
In present study, the cells which stably expressed DAT were prepared by transfection of CV-1 cells (monkey kidney cell line) with plasmid pRc/CMV-DAT which is the expression vector pRc/CMV-based plasmid containing bovine DAT cDNA. CV-1 cells, transfected with pRc/CMV-DAT, stably expresses a high affinity DAT in dopamine uptake assay, the cpm values uptaken by transfected cells was 30-50 folds higher than those of nontransfectants.
The transfected cells accumulated 〔^(3)H〕dopamine in a dose-dependent manner with a Km of 991. 6nM. Even though high doses of norepinephrine, epinephrine, serotonin, and choline neurotransmitters inhibited the uptake of 〔^(3)H〕dopamine, DAT expressed in transfected cells was proven to be much more specific to dopamine. The psychotropic drugs such as GBR12909. CFT(cocaine congener), normifensine, clomipramine, desipramine, and imipramine inhibited significantly the dopamin uptake in tissue culture cells stably transfected with DAT cDNA.
It has been known that rat and human DAT generally have the activities of Parkinsonism-inducing neurotoxin, MPP^(+)-uptake and cocaine-binding. However, MPP^(+) uptake activity of expressed bovine DAT was significantly lesser than that of expressed rat DAT, and no CFT binding activity was seen. Radioactive in situ hybridization was done to map the cellular localization of DAT mRNA containing cells in the adult rat central nervous system.
The strong hybridization signals were detected only in the substantia nigra pars compacta and ventral tegmental area. The restricted anatomical localization of DAT mRNA-containing cells confirms the DAT as a presynaptic marker of dopamine containing cells confirms the DAT as a presynaptic marker of dopamine containing cells in the rat brain.
All of ?? species homologues of the DAT(rat, bovine, and human) possess consensus sites ?? phosphorylation by cAMP-dependent protein kinase A(PKA), Ca^(2+)-calmodulin-dependent kinase Ⅱ(CaM kinase Ⅱ), and protein kinase C(PKC). The existence of putative phosphorylation sites indicates that the funcitions of DAT may be regulated by protein kinases or phosphatases. Concentration- and time- course preincubations of DAT expressing cells with protein kinase activators or inhibitors such as phorbol 12-myristrate 13-acetate(PMA, a protein kinase C(PKC) activator), staurosphorin(a nonspecific protein kinase inhibitor), calphostin C(a specific PKC inhibitor), forskolin (a cAMP-dependent protein kinase A activator), and EGTA (Ca^(2+)-chelating agent, a effector of Ca^(2+)-calmodulin-dependent kinase Ⅱ) at 37℃ were followed by DAT functional assays.
Dopamine uptake into DAT expressing cells was severely decreased by PMA pretreatment, and increased by staurosphorin or calphostin C. PKC activator and inhibitors showed similar effects on MPP+ uptake of DAT. However, DAT functions were not altered by forskolin or EGTA. These results implies that the functions of DAT are modulated by protein kinase C.
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