Regulation of the high affinity choline transporter (CHT1) by ubiquitin ligase Nedd4‐2

The high affinity choline transporter (CHT1) is specifically expressed in cholinergic neurons, and plays a critical role in the regulation of acetylcholine synthesis. Little is known about the mechanisms that regulate the cell surface expression and trafficking of CHT1. Recently membrane proteins such as receptors, channels and transporters have been shown to be ubiquitinated resulting in the regulation of their endocytosis and trafficking. We have examined the role of ubiquitination in the function and trafficking of CHT1, focusing on E3 ubiquitin ligase Nedd4‐2 which is reported to ubiquitinate dopamine transporter and some neuronal membrane proteins. Coexpression of Nedd4‐2 with CHT1 in HEK293 cells was found to decrease the amount of cell surface CHT1 by 40%, which was assessed as the binding activity with hemicholinium‐3 (a membrane‐impermeable, specific inhibitor of CHT1). Choline uptake activity was also decreased by 40% in the same cells, indicating that coexpression of Nedd4‐2 did not affect the specific activity of the cell surface CHT1. Coexpression of catalytically inactive Nedd4‐2 mutant did not affect either hemicholinium‐3 binding or choline uptake activity. These results indicate that the trafficking, but not the specific activity, of CHT1 is regulated by Nedd4‐2.