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Substrates and inhibitors display different sensitivity to expression level of the dopamine transporter in heterologously expressing cells
Author(s) -
Chen Nianhang,
Reith Maarten E. A.
Publication year - 2007
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2006.04384.x
Subject(s) - mazindol , dopamine transporter , hek 293 cells , dopamine plasma membrane transport proteins , dopamine , chemistry , dopamine uptake inhibitors , heterologous expression , tropane , potency , transporter , norepinephrine transporter , microbiology and biotechnology , biochemistry , pharmacology , biology , dopaminergic , in vitro , endocrinology , stereochemistry , recombinant dna , gene
The use of heterologous expression systems for studying dopamine (DA) transporter (DAT) function has provided important information corroborating and complementing in situ obtained knowledge. Preliminary experiments with human embryonic kidney cells (HEK293) heterologously expressing varying amounts of DAT suggested fluctuations in the potency of cocaine in inhibiting DA uptake and led to the present systematic assessment of the impact of the density of DAT on its function. Transiently expressing intact HEK293 cells, transfected with increasing amounts of DAT cDNA, displayed increasing levels of surface DAT, binding of the cocaine analog [ 3 H]2β‐carbomethoxy‐3β‐(4‐fluorophenyl)tropane ([ 3 H]CFT), and uptake of [ 3 H]DA, [ 3 H] N ‐methyl‐4‐phenylpyridinium ([ 3 H]MPP + ), [ 3 H]norepinephrine, and [ 3 H]serotonin. However, the amount of DAT cDNA and the DAT expression level required to produce 50% of maximal activity was threefold higher for CFT binding than for DA uptake. Increased DAT expression was accompanied by weakened potency in inhibiting [ 3 H]DA uptake for cocaine, CFT, benztropine, and its analog JHW025, GBR 12909 and mazindol; their potency in inhibiting [ 3 H]CFT binding was unaffected. Inhibition of uptake by the substrates DA, m‐tyramine, d‐amphetamine, or MPP + was also unaffected. Increasing DAT in stably expressing HEK293 cells by stimulation of gene expression with sodium butyrate also decreased the uptake inhibitory potency of a number of the above blockers without affecting the interaction between substrates and DAT. The present results prompt discussion of models explaining how factors regulating DAT expression at the plasma membrane can regulate DAT function and pharmacology.