Involvement of threonine 258 and serine 259 motif in amphetamine-induced norepinephrine transporter endocytosis

J Neurochem. 2010 Oct;115(1):23-35. doi: 10.1111/j.1471-4159.2010.06898.x. Epub 2010 Jul 30.


D-amphetamine (AMPH) down-regulates the norepinephrine transporter (NET), although the exact trafficking pathways altered and motifs involved are not known. Therefore, we examined the cellular and molecular mechanisms involved in AMPH-induced NET regulation in human placental trophoblast cells expressing the wild-type (WT)-hNET and the hNET double mutant (DM)-bearing protein kinase C (PKC)-resistant T258A + S259A motif. NET function and surface expression were significantly reduced in cells expressing WT-hNET but not in cells expressing hNET-DM following AMPH treatment. AMPH inhibited plasma membrane recycling of both WT-hNET and hNET-DM. In contrast, AMPH stimulated endocytosis of WT-hNET, and did not affect hNET-DM endocytosis. Although PKC or calcium/calmodulin- dependent kinase-II (CaMKII) inhibition or depletion of calcium failed to block AMPH-mediated down-regulation of WT-hNET, NET-specific blocker desipramine completely prevented AMPH-induced down-regulation. Furthermore, AMPH treatment had no effect on phospho-CaMKII immunoreactivity. The inhibitory potency of AMPH was highest on hNET-DM, intermediary on T258A and S259A single mutants and lowest on WT-hNET. Single mutants exhibited partial resistance to AMPH-mediated down-regulation. AMPH accumulation was similar in cells expressing WT-hNET or hNET-DM. The results demonstrate that reduced plasma membrane insertion and enhanced endocytosis account for AMPH-mediated NET down-regulation, and provide the first evidence that T258/S259 motif is involved only in AMPH-induced NET endocytosis that is desipramine-sensitive, but PKC and CaMKII independent.

Publication types

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

MeSH terms

  • Adrenergic Uptake Inhibitors / pharmacology
  • Amphetamine / pharmacology*
  • Biotinylation
  • Calcium / physiology
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / antagonists & inhibitors
  • Cell Membrane / drug effects
  • Central Nervous System Stimulants / pharmacology*
  • Desipramine / pharmacology
  • Down-Regulation / drug effects
  • Endocytosis / drug effects*
  • Endocytosis / genetics*
  • Female
  • Humans
  • Mutation / physiology
  • Norepinephrine Plasma Membrane Transport Proteins / antagonists & inhibitors
  • Norepinephrine Plasma Membrane Transport Proteins / genetics*
  • Norepinephrine Plasma Membrane Transport Proteins / physiology*
  • Placenta / cytology
  • Pregnancy
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism
  • Protein Kinase Inhibitors / pharmacology
  • Serine / genetics*
  • Signal Transduction / drug effects
  • Threonine / genetics*
  • Trophoblasts / drug effects


  • Adrenergic Uptake Inhibitors
  • Central Nervous System Stimulants
  • Norepinephrine Plasma Membrane Transport Proteins
  • Protein Kinase Inhibitors
  • Threonine
  • Serine
  • Amphetamine
  • Protein Kinase C
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Calcium
  • Desipramine