Phosphorylation of dopamine transporter serine 7 modulates cocaine analog binding

J Biol Chem. 2013 Jan 4;288(1):20-32. doi: 10.1074/jbc.M112.407874. Epub 2012 Nov 16.


As an approach to elucidating dopamine transporter (DAT) phosphorylation characteristics, we examined in vitro phosphorylation of a recombinant rat DAT N-terminal peptide (NDAT) using purified protein kinases. We found that NDAT becomes phosphorylated at single distinct sites by protein kinase A (Ser-7) and calcium-calmodulin-dependent protein kinase II (Ser-13) and at multiple sites (Ser-4, Ser-7, and Ser-13) by protein kinase C (PKC), implicating these residues as potential sites of DAT phosphorylation by these kinases. Mapping of rat striatal DAT phosphopeptides by two-dimensional thin layer chromatography revealed basal and PKC-stimulated phosphorylation of the same peptide fragments and comigration of PKC-stimulated phosphopeptide fragments with NDAT Ser-7 phosphopeptide markers. We further confirmed by site-directed mutagenesis and mass spectrometry that Ser-7 is a site for PKC-stimulated phosphorylation in heterologously expressed rat and human DATs. Mutation of Ser-7 and nearby residues strongly reduced the affinity of rat DAT for the cocaine analog (-)-2β-carbomethoxy-3β-(4-fluorophenyl) tropane (CFT), whereas in rat striatal tissue, conditions that promote DAT phosphorylation caused increased CFT affinity. Ser-7 mutation also affected zinc modulation of CFT binding, with Ala and Asp substitutions inducing opposing effects. These results identify Ser-7 as a major site for basal and PKC-stimulated phosphorylation of native and expressed DAT and suggest that Ser-7 phosphorylation modulates transporter conformational equilibria, shifting the transporter between high and low affinity cocaine binding states.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Chromatography, Thin Layer / methods
  • Cocaine / analogs & derivatives
  • Cocaine / chemistry*
  • Corpus Striatum / drug effects
  • Corpus Striatum / metabolism
  • Dopamine Plasma Membrane Transport Proteins / chemistry*
  • Dopamine Plasma Membrane Transport Proteins / metabolism
  • Dopamine Uptake Inhibitors / chemistry
  • HEK293 Cells
  • Humans
  • Kinetics
  • Male
  • Mass Spectrometry / methods
  • Mutagenesis, Site-Directed
  • Mutation
  • Phosphorylation
  • Protein Binding
  • Protein Conformation
  • Rats
  • Rats, Sprague-Dawley
  • Serine / chemistry*


  • Dopamine Plasma Membrane Transport Proteins
  • Dopamine Uptake Inhibitors
  • Serine
  • Cocaine