Phorbol ester induced trafficking-independent regulation and enhanced phosphorylation of the dopamine transporter associated with membrane rafts and cholesterol

J Neurochem. 2008 Jun;105(5):1683-99. doi: 10.1111/j.1471-4159.2008.05262.x. Epub 2008 Feb 1.


We examined the mechanisms involved in protein kinase C (PKC)-dependent down-regulation of dopamine transporter (DAT) activity and cell surface expression by treating heterologously expressing cells with the clathrin-mediated endocytosis inhibitor concanavalin A (Con A) or the cholesterol depleter/membrane raft disrupter methyl-beta-cyclodextrin (MbetaC) prior to treatment with the PKC activator phorbol 12-myristate, 13-acetate (PMA). Con A blocked PMA-induced surface reductions of DAT but only partially inhibited down-regulation, while MbetaC partially blocked down-regulation but did not inhibit loss of cell surface DAT, demonstrating that PKC-induced DAT down-regulation occurs by a combination of trafficking and non-trafficking processes. Using density-gradient centrifugation, we found that DATs are distributed approximately equally between Triton-insoluble, cholesterol-rich membrane rafts and Triton-soluble non-raft membranes. DATs in both populations are present at the cell surface and are active for dopamine and cocaine binding. PMA-induced loss of cell surface DAT occurred only from non-raft populations, demonstrating that non-raft DATs are regulated by trafficking events and indicating the likelihood that the cholesterol-dependent non-trafficking regulatory mechanism occurs in rafts. PMA did not affect the DAT raft-non-raft distribution but stimulated the phosphorylation of DAT to a substantially greater level in rafts than non-rafts. These findings reveal a previously unknown role for cholesterol in DAT function and demonstrate the presence of distinct subcellular DAT populations that possess multiple regulatory differences that may impact dopaminergic neurotransmission.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cholesterol / genetics
  • Cholesterol / metabolism*
  • Dopamine Plasma Membrane Transport Proteins / genetics
  • Dopamine Plasma Membrane Transport Proteins / metabolism*
  • Humans
  • LLC-PK1 Cells
  • Membrane Microdomains / drug effects
  • Membrane Microdomains / genetics
  • Membrane Microdomains / metabolism*
  • Phorbol Esters / pharmacology*
  • Phosphorylation / drug effects
  • Protein Transport / drug effects
  • Protein Transport / physiology
  • Rats
  • Swine


  • Dopamine Plasma Membrane Transport Proteins
  • Phorbol Esters
  • phorbol-12-myristate
  • Cholesterol