Cell surface trafficking of the antidepressant-sensitive norepinephrine transporter revealed with an ectodomain antibody.

Savchenko V, Sung U, Blakely RD
Mol Cell Neurosci. 2003 24 (4): 1131-50

PMID: 14697674 · DOI:10.1016/s1044-7431(03)00235-5

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.

MeSH Terms (20)

Amino Acid Sequence Animals Antidepressive Agents Cell Line Cell Membrane Cells, Cultured Female Humans Male Mice Mice, Inbred C57BL Mice, Knockout Molecular Sequence Data Norepinephrine Norepinephrine Plasma Membrane Transport Proteins Protein Transport Rats Rats, Sprague-Dawley Superior Cervical Ganglion Symporters

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