N-terminus regulation of VMAT2 mediates methamphetamine-stimulated efflux

Neuroscience. 2014 Feb 14;259:194-202. doi: 10.1016/j.neuroscience.2013.11.059. Epub 2013 Dec 7.

Abstract

The 20 amino acid (AA) N-terminus of the vesicular monoamine transporter 2 (VMAT2) was examined as a regulator of VMAT2 function. Removal of the first 16 or 19 AAs of the N-terminus resulted in a molecule with reduced ability to sequester [(3)H]-5HT. A glutathione-S-transferase-construct of the N-terminus underwent phosphorylation in the presence of PKC at serines 15 and 18. These putative phosphorylation sites were examined for effects on function. Phospho-mimetic substitution of serines 15 and 18 with aspartate in the full-length VMAT2 resulted in reduced [(3)H]-5HT sequestration and reduced methamphetamine (METH)-stimulated efflux of preloaded [(3)H]-5HT. In contrast, mutation of serines 15 and 18 to alanines maintained intact net substrate sequestration but eliminated METH-stimulated efflux of pre-accumulated [(3)H]-5HT. In summary, these data suggest a model in which the VMAT2 N-terminus regulates monoamine sequestration.

Keywords: AAs; AMPH; BSA; CPM; CaCl(2); DAT; DPM; GST; HA; KLH; KSR; M-PMTs; METH; MgCl(2); PBS; PD; PIP(2); PKC; PS; PVDF; Parkinson’s disease; RSP; SDS–PAGE; SH; TBZ; TBZ-OH; VMAT2; [(3)H]-tetrabenazine-OH; amino acids; amphetamine; bovine serum albumin; calcium chloride; counts per minute; decays per minute; diolyl phosphatidyl serine; dopamine transporter; efflux; glutathione-S-transferase; hemagglutinin; kDa; ketansarin; keyhole limpet hemocyanin; kilodalton; kinase; magnesium chloride; methamphetamine; monoamine; monoamine plasma membrane transporters; pN-term Ab; phosphate-buffered saline; phosphatidylinositol-4,5-bisphosphate; phospho-specific N-term antibody; polyvinylidene fluoride; protein kinase C; reserpine; sodium dodecyl sulfate–polyacrylamide gel; sucrose HEPES; tetrabenazine; transporter; vesicular monoamine transporter 2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adrenergic Uptake Inhibitors / pharmacokinetics
  • Animals
  • COS Cells
  • Cell Line
  • Central Nervous System Stimulants / pharmacology*
  • Chlorocebus aethiops
  • Enzyme-Linked Immunosorbent Assay
  • Methamphetamine / pharmacology*
  • Models, Biological
  • Mutation / genetics
  • Neuroblastoma
  • Phosphorylation / drug effects
  • Serotonin / pharmacokinetics
  • Tetrabenazine / pharmacokinetics
  • Transfection
  • Tritium / pharmacokinetics
  • Vesicular Monoamine Transport Proteins / chemistry
  • Vesicular Monoamine Transport Proteins / genetics
  • Vesicular Monoamine Transport Proteins / metabolism*

Substances

  • Adrenergic Uptake Inhibitors
  • Central Nervous System Stimulants
  • Vesicular Monoamine Transport Proteins
  • Tritium
  • Serotonin
  • Methamphetamine
  • Tetrabenazine