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Reaction of oxidized dopamine with endogenous cysteine residues in the human dopamine transporter
Author(s) -
Whitehead Rachel E.,
Ferrer Jasmine V.,
Javitch Jonathan A.,
Justice Joseph B.
Publication year - 2001
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 - 10.1046/j.1471-4159.2001.00125.x
Subject(s) - cysteine , dopamine transporter , dopamine , chemistry , tropane , dopamine plasma membrane transport proteins , biochemistry , transporter , stereochemistry , alanine , enzyme , amino acid , biology , gene , neuroscience
There is evidence to suggest that dopamine (DA) oxidizes to form dopamine ortho ‐quinone (DAQ), which binds covalently to nucleophilic sulfhydryl groups on protein cysteinyl residues. This reaction has been shown to inhibit dopamine uptake, as well as other biological processes. We have identified specific cysteine residues in the human dopamine transporter (hDAT) that are modified by this electron‐deficient substrate analog. DAQ reactivity was inferred from its effects on the binding of [ 3 H]2‐β‐carbomethoxy‐3‐β‐(4‐fluorophenyl)tropane (β‐CFT) to hDAT cysteine mutant constructs. One construct, X5C, had four cysteines mutated to alanine and one to phenylalanine (Cys 90 A, Cys 135 A, C306A, C319F and Cys 342 A). In membrane preparations 1 m m DAQ did not affect [ 3 H]β‐CFT binding to X5C hDAT, in contrast to its effect in wild‐type hDAT in which it reduced the B max value by more than half. Wild‐type cysteines were substituted back into X5C, one at a time, and the ability of DAQ to inhibit [ 3 H]β‐CFT binding was assessed. Reactivity of DAQ with Cys 90 increased the affinity of [ 3 H]β‐CFT for the transporter, whereas reactivity with Cys 135 decreased the affinity of [ 3 H]β‐CFT. DAQ did not change the K D for [ 3 H]β‐CFT binding to wild‐type. The reactivity of DAQ at Cys 342 decreased B max to the same degree as wild‐type. The latter result suggests that Cys 342 is the wild‐type residue most responsible for DAQ‐induced inhibition of [ 3 H]β‐CFT binding.