N‐terminus Regulation of Vesicular Monoamine Transporter 2 activity

Abstract

The 20 amino acid N‐terminus of the vesicular monoamine transporter 2 (VMAT2) was examined as an enabler and regulator of transport activity. Relative to accumulation of 3H 5HT substrate by WT VMAT2 in transfected SH‐SY5Y cells, accumulation by truncants, lacking the first 16 or 19 N‐terminus amino acids, was reduced by over 50%. The N‐terminus contains 2 putative protein kinase C (PKC) phosphorylation sites at serines 15 and 18. A construct of the N‐terminus N‐terminally linked to GST underwent specific incorporation of 32P in the presence of 32P‐γ‐ATP and PKC at serines 15 and 18. A peptide of VMAT2 N‐terminus residues 1–20 inhibited transport by WT VMAT2 in digitonin permeabilized COS cells with a Ki of 100μM. However, peptides comprised of residues 1–11 or 12–20, a randomized peptide as well as a peptide in which serines 15 and 18 were completely phosphorylated, all failed to inhibit transport. Phospho‐mimetic mutations of serines 15 and 18 to aspartate resulted in a 50% loss in transport activity by the full length VMAT2 molecule in transfected permeabilized COS cells, whereas mutation to alanine retained full transport activity. Antisera raised against the phosphorylated N‐terminus showed specific immunoreactivity toward denucleated membranes obtained from WT VMAT2 transfected COS cells. These findings suggest the N‐terminal domain enables and regulates VMAT2 transport. supported by NIH R01NS033650‐08