Efeitos da fosforilação do transportador vesicular de acetilcolina em seu tráfego e localização em células PC12

The vesicular acetylcholine transporter (VAChT) participates effectively the cholinergic transmission. Studies in PC12 cells have shown that serine 480 is the s te of phosphorylation of VAChT. Experiments have shown that protein kinase C (PKC) is invol ved in the phosphorylation of VAChT, and that substitution of serine 480 to glutamate mimicking the phosphorylation event increases the expression of VAChT in LCDVs ( larger vesicles which are usually the monoamine transporters or VMATs). To verify that phosphorylation of VAChT alters their traffic in PC12 cells, we made two mutants of VAChT. At first, it was replaced the serine 480 (phosphorylation site for PKC), by alanine (a n amino acid no load, and therefore cannot be phosphorylated), this mutant was designated GFP -S480A VAChT. Then, the serine has been replaced by glutamate (an amino acid t hat mimics the phosphorylated state of VAChT), this mutant was designated GFP-S480E V AChT. The VAChT was tagged with GFP, a fluorescent protein that allows vis ualization of VAChT in PC12 cells by confocal microscopy. The aim of this study was to determine whether there are differences in traffic and location the GFP-S480E VAChT and GFPS480A VAChT in PC12 cells. The VAChT phosphorylated (GFP-S480E VAChT) located in the highe st concentration in cells of varicosities (36% more fluorescence in the cell body) , where ACh is released. Already VAChT in non-phosphorylated state (GFP-S480A VAChT) located i n highest concentration in the cell body (20% more fluorescence in the body in re lation to varicosities). These results indicate that phosphorylation of VAChT affects its raffic and, consequently, its location in PC12 cells.