N-terminus regulation of VMAT2 mediates methamphetamine-stimulated efflux

• The VMAT2 N-terminus enables sequestration.
• A GST-construct of the N-terminus undergoes phosphorylation by PKC at serines 15 and/or 18.
• The charged state of N-terminus serines 15 and 18 regulates sequestration by the VMAT2.
• The charged state of N-terminus serines 15 and 18 regulates the VMAT2 efflux response to methamphetamine.

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 [3H]-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 [3H]-5HT sequestration and reduced methamphetamine (METH)-stimulated efflux of preloaded [3H]-5HT. In contrast, mutation of serines 15 and 18 to alanines maintained intact net substrate sequestration but eliminated METH-stimulated efflux of pre-accumulated [3H]-5HT. In summary, these data suggest a model in which the VMAT2 N-terminus regulates monoamine sequestration.