The norepinephrine (NE) transporter (NET) mediates high-affinity uptake of the neurotransmitter NE in central and peripheral noradrenergic neurons. NET has a critical function in controlling the synaptic levels of NE and modulating several fundamental brain and body functions, including arousal, vigilance, attention, learning and autonomic functions. In this study, the cellular regulation of NET distribution, trafficking and function were investigated in sympathetic neurons, and compared to the related transporter of dopamine (DA), i.e. DAT, in neuronal cell models. In addition, a kinome-wide siRNA screen was conducted in heterologous cell models to investigate the possible roles of the regulatory kinases in the functions of NET, DAT and the serotonin transporter SERT. 

NET was found to undergo marked constitutive internalization and sorting mainly to Rab 11 -positive recycling endosomes in sympathetic neurons. Direct comparison in neuronal cell models demonstrated that NET was sequestered from the plasma membrane to a significantly greater extent than DAT, and was subsequently largely recycled back to the plasma membrane, whereas internalized DAT was mainly targeted to degradation. The different trafficking itineraries of NET and DAT were found to be determined by non-conserved structural elements in the intracellular N-termini of the transporter proteins. The PDZ domain protein PICK1 was shown to associate with NET and to be important for stabilizing NET in the plasma membrane of noradrenergic neurons. RNA interference -mediated depletion of PICK1 reduced both total and surface-expressed NET and decreased NET uptake capacity, but had no effect on DAT that also binds PICK1. Furthermore, the results of the kinome-wide siRNA screen indicated that the functions of monoamine transporters are likely to be regulated by several kinases, such as the cyclic adenosine monophosphate (cAMP)- dependent protein kinases, the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) and the mitogen-activated protein kinase (MAPK). In addition, protein kinase, X-Linked (PrKX) was identified as a novel kinase possible involved in the regulation of monoamine transporter function.