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.
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