title: N-terminus regulation of VMAT2 mediates methamphetamine-stimulated efflux.
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.
authors: University of Wisconsin, Madison, Department of Neuroscience, 1300 University Avenue, Madison, WI 53706, USA.
authors: B
authors: B
authors: Torres
authors: University of Wisconsin, Madison, Department of Neuroscience, 1300 University Avenue, Madison, WI 53706, USA. Electronic address: aeruoho@wisc.edu.
authors: A E
authors: AE
authors: Ruoho
mesh_terms: D018759:Adrenergic Uptake Inhibitors / Q000493:pharmacokinetics; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D000697:Central Nervous System Stimulants / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D004797:Enzyme-Linked Immunosorbent Assay; D008694:Methamphetamine / Q000494:pharmacology*; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D009447:Neuroblastoma; D010766:Phosphorylation / Q000187:drug effects; D012701:Serotonin / Q000493:pharmacokinetics; D013747:Tetrabenazine / Q000493:pharmacokinetics; D014162:Transfection; D014316:Tritium / Q000493:pharmacokinetics; D050493:Vesicular Monoamine Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D018759:Adrenergic Uptake Inhibitors; D000697:Central Nervous System Stimulants; D050493:Vesicular Monoamine Transport Proteins; D014316:Tritium; D012701:Serotonin; D008694:Methamphetamine; D013747:Tetrabenazine
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