title: Cell surface trafficking of the antidepressant-sensitive norepinephrine transporter revealed with an ectodomain antibody.
abstract: The antidepressant-sensitive L-norepinephrine (NE) transporter (NET;SLC6A2) is a critical determinant of neurotransmitter inactivation following NE release at synapses. Although regulated trafficking of NET has been documented in transfected cells, a lack of reagents suitable for reporting native NET surface exposition has limited validation of this concept in neurons. In the current report, we document the utility of a novel antibody (43408) directed at conserved sequences in the NET second extracellular loop. Using human NET (hNET) stably transfected cells, we document loss of NET surface expression following acute (30 min) phorbol ester treatments. In superior cervical ganglion (SCG) cultures, NET surface expression is prominent on varicosities defined by FM1-43 labeling of living neurons or synaptophysin labeling of fixed preparations. Moreover, NET surface density can be rapidly augmented by brief depolarization (5 min, 40 mM K(+)). Similarly, in brainstem cultures, we demonstrate an increase in NET surface labeling following either depolarization or angiotensin II stimulation. These findings provide the first evidence for regulated trafficking of NET in neurons and support the suggestion that activity-dependent NET trafficking may provide additional modulatory capacity for noradrenergic signaling.
authors.affiliation: Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, TN 37232-8548, USA.
authors.forename: Valentina
authors.initials: V
authors.lastname: Savchenko
authors.forename: Uhna
authors.initials: U
authors.lastname: Sung
authors.forename: Randy D
authors.initials: RD
authors.lastname: Blakely
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000928:Antidepressive Agents / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009638:Norepinephrine / Q000378:metabolism* / Q000494:pharmacology; D050484:Norepinephrine Plasma Membrane Transport Proteins; D021381:Protein Transport / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017783:Superior Cervical Ganglion / Q000378:metabolism; D027981:Symporters / Q000235:genetics / Q000378:metabolism*
chemical_list: D000928:Antidepressive Agents; D050484:Norepinephrine Plasma Membrane Transport Proteins; C493227:SLC6A2 protein, human; C493228:Slc6a2 protein, mouse; C493226:Slc6a2 protein, rat; D027981:Symporters; D009638:Norepinephrine