title: Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini.
abstract: The norepinephrine transporter (NET) mediates reuptake of synaptically released norepinephrine 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. Phorbol 12-myristate 13-acetate increased intracellular accumulation of JHC 1-64-labeled NET and caused a parallel reduction in uptake capacity. Internalized NET strongly colocalized 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 differentiated CATH.a cells. In contrast to NET, the dopamine transporter displayed markedly less constitutive internalization and limited sorting to the Rab11 recycling compartment in the differentiated CATH.a 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.
authors.affiliation: From the Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute 18.6, University of Copenhagen, DK-2200 Copenhagen, Denmark,; the Department of Pharmacology, Drug Development, and Therapeutics, University of Turku, Turku FI-20014, Finland,; the Unit of Clinical Pharmacology, Turku University Hospital, Turku FI-20520, Finland, and.
authors.forename: Anne
authors.initials: A
authors.lastname: Vuorenpää
authors.affiliation: From the Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute 18.6, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Trine N
authors.initials: TN
authors.lastname: Jørgensen
authors.affiliation: the Medicinal Chemistry Section, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224.
authors.forename: Amy H
authors.initials: AH
authors.lastname: Newman
authors.affiliation: From the Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute 18.6, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Madsen
authors.affiliation: the Department of Pharmacology, Drug Development, and Therapeutics, University of Turku, Turku FI-20014, Finland,; the Unit of Clinical Pharmacology, Turku University Hospital, Turku FI-20520, Finland, and.
authors.forename: Mika
authors.initials: M
authors.lastname: Scheinin
authors.affiliation: From the Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Panum Institute 18.6, University of Copenhagen, DK-2200 Copenhagen, Denmark,. Electronic address: gether@sund.ku.dk.
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D050483:Dopamine Plasma Membrane Transport Proteins / Q000032:analysis / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009638:Norepinephrine / Q000378:metabolism; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000032:analysis / Q000378:metabolism*; D010703:Phorbol Esters / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D013194:Staining and Labeling; D020691:rab GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D010703:Phorbol Esters; C493227:SLC6A2 protein, human; D020691:rab GTP-Binding Proteins; D009638:Norepinephrine
keywords: dopamine transporter; internalization; monoamine transporter; neurotransmitter transport; norepinephrine transporter; protein chimera; recycling; trafficking; transporter regulation