Differential internalization rates and postendocytic sorting of the norepinephrine and dopamine transporters are controlled by structural elements in the N-termini
: Vuorenpää A, Jørgensen TN, Newman AH, Madsen KL, Scheinin M, Gether U
Publisher: American Society for Biochemistry and Molecular Biology, Inc.
: 2016
: Journal of Biological Chemistry
: 291
: 11
: 5634
: 5651
: 18
: 0021-9258
: 1083-351X
DOI: https://doi.org/10.1074/jbc.M115.702050
Abstract
The norepinephrine transporter (NET) mediates reuptake of synaptically released norepinephrine (NE) in central and peripheral noradrenergic neurons. The molecular processes governing availability of NET in the plasma membrane are poorly understood. Here we use the fluorescent cocaine analogue JHC 1-64, as well as several other approaches, to investigate the trafficking itinerary of NET in live noradrenergic neurons. Confocal imaging revealed extensive constitutive internalization of JHC 1-64-labeled NET in the neuronal somata, proximal extensions and presynaptic boutons. The phorbol ester PMA increased intracellular accumulation of JHC 1-64-labeled NET and caused a parallel reduction in NE uptake capacity. Internalized NET strongly co-localized with the "long loop" recycling marker Rab11, whereas less overlap was seen with the "short loop" recycling marker Rab4 and the late endosomal marker Rab7. Moreover, mitigating Rab11 function by overexpression of dominant negative Rab11 impaired NET function. Sorting of NET to the Rab11 recycling compartment was further supported by confocal imaging and reversible biotinylation experiments in transfected CAD cells. In contrast to NET, the dopamine transporter (DAT) displayed markedly less constitutive internalization and limited sorting to the Rab11 recycling compartment in the CAD cells. Exchange of domains between the two homologous transporters revealed that this difference was determined by non-conserved structural elements in the intracellular N-terminus. We conclude that NET displays a distinct trafficking itinerary characterized by continuous shuffling between the plasma membrane and the Rab11 recycling compartment, and that the functional integrity of the Rab11 compartment is critical for maintaining proper presynaptic NET function.