Characterization of a recombinant human dopamine transporter in multiple cell lines

A. J. Eshleman, R. L. Neve, Aaron Janowsky, Kim Neve

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

A 3.5-kilobase cDNA encoding the dopamine transporter was isolated from a human substantia nigra cDNA library. Sequence analysis of the coding region of the transporter identified two nucleotide differences between the cDNA and published human dopamine transporter sequences. One of the substitutions changed an amino acid conserved among previously cloned dopamine (DA) and norepinephrine transporters, Arg-344, to a methionine. C6 glioma cells or COS-7 cells transfected with the cDNA (C6-hDAT and Cos7-hDAT cells) accumulated [3H]DA with high affinity (K(m) = 1.2 and 1.5 μM, respectively), and DA uptake inhibitors had similar potencies in both cell lines. [3H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane ([3H]CFT) bound to membranes prepared from both cell lines with high affinity (K(d) = 2-8 nM), although some experiments with C6-hDAT cell membranes indicated the presence of a second class of binding sites with lower affinity for the radioligand. Using the high-affinity K(d) value for [3H]CFT binding determined in Cos7- hDAT cells to calculate K(i) values, drug affinity for inhibition was highly correlated (r = .92) with affinity for inhibition of [3H]DA uptake, although transporter substrates were significantly more potent inhibitors of uptake than of [3H]CFT binding. The binding of [3H]l-[2-diphenylmethoxy]ethyl-4- (3-phenylpropyl)-piperazine ([3H]GBR-12935) to C6-hDAT cells could not be characterized due to high binding to untransfected C6 cells, but on Cos7- hDAT cells the radioligand labeled a single population of binding sites (K(d) = 1 nM). Inhibition of [3H]GBR-12935 binding by drugs correlated highly with inhibition of either [3H]CFT binding (r = .98) or of [3H]DA uptake (r = .95). These results demonstrate that expression of an hDAT cDNA provides a binding site for both [3H]CFT and [3H]GBR-12935, and that the structural features of the human transporter that determine drug affinity for binding are similar to the structural features responsible for inhibition of [3H]DA uptake.

Original languageEnglish (US)
Pages (from-to)276-283
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume274
Issue number1
StatePublished - 1995

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Dopamine Plasma Membrane Transport Proteins
Cell Line
Dopamine
Complementary DNA
Binding Sites
Dopamine Uptake Inhibitors
Pharmaceutical Preparations
Norepinephrine Plasma Membrane Transport Proteins
COS Cells
Substantia Nigra
Amino Acid Substitution
Gene Library
Glioma
Methionine
Sequence Analysis
Nucleotides
Cell Membrane
Membranes
Population
GBR 12935

ASJC Scopus subject areas

  • Pharmacology

Cite this

Characterization of a recombinant human dopamine transporter in multiple cell lines. / Eshleman, A. J.; Neve, R. L.; Janowsky, Aaron; Neve, Kim.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 274, No. 1, 1995, p. 276-283.

Research output: Contribution to journalArticle

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