Sodium-Dependent Norepinephrine-Induced Currents in Norepinephrine-Transporter-Transfected Hek-293 Cells Blocked by Cocaine and Antidepressants

Abstract

Transport of norepinephrine (NE+) by cocaine- and antidepressant-sensitive transporters in presynaptic terminals is predicted to involve the cotransport of Na+ and Cl-, resulting in a net movement of charge per transport cycle. To explore the relationship between catecholamine transport and ion permeation through the NE transporter, we established a human norepinephrine transporter (hNET) cell line suitable for biochemical analysis and patch-clamp recording. Stable transfection of hNET cDNA into HEK-293 (human embryonic kidney) cells results in lines exhibiting (1) a high number of transporter copies per cell (10(6)), as detected by radioligand binding and hNET-specific antibodies, (2) high-affinity, Na(+)-dependent transport of NE, and (3) inhibitor sensitivities similar to those of native membranes. Whole-cell voltage-clamp of hNET-293 cells reveals NE-induced, Na(+)-dependent currents blocked by antidepressants and cocaine that are absent in parental cells. In addition to NE-dependent currents, transfected cells posses an NE-independent mode of charge movement mediated by hNET. hNET antagonists without effect in non-transfected cells abolish both NE-dependent and NE-independent modes of charge movement in transfected cells. The magnitude of NE-dependent currents in these cells exceeds the expectations of simple carrier models using previous estimates of transport rates. To explain our observations, we propose that hNETs function as ion-gated ligand channels with an indefinite stoichiometry relating ion flux to NE transport. In this view, external Na+ and NE bind to the transporter with finite affinities in a cooperative fashion. However, coupled transport may not predict the magnitude or the kinetics of the total current through the transporter. We propose instead that Na+ gates NE transport and also the parallel inward flux of an indeterminate number of ions through a channel-like pore.

Probing the functional limits of the norepinephrine transporter with self-reporting, fluorescent stilbazolium dimers

“…This behavior is ideal for imaging applications and monitoring binding or transport as the probe "turns-on" when bound or accumulated intracellularly. 11 We screened 1-4 against HEK293 cells stably expressing hNET 12 and compared the emission intensities against untransfected HEK293 cells, which served as controls. Cells were treated with 500 nM solutions of the dyes for 30 min then imaged by confocal microscopy with excitation via 405 nm or 488 nm laser lines; details of imaging experiments are provided in the ESI.…”

Antidepressant Principles of the Roots of Polygala tenuifolia

“…Compounds 1 − 5 were tested in vitro for their ability to inhibit isotope-labeled RTI-55 binding to norepinephrine transporter protein . In this membrane-binding assay, polygalatenosides A ( 1 ) and B ( 2 ) showed significant inhibitory activities, with IC 50 values of 30.0 and 6.04 μM, respectively.…”

Investigation of the Optical Isomers of Methcathinone, and Two Achiral Analogs, at Monoamine Transporters and in Intracranial Self-Stimulation Studies in Rats

“…The number of total cells analyzed per dose–response curve (including all concentrations) ranges from 227 to 832 per compound. Since many cells are analyzed per concentration, the SEM of the mean response for each concentration is small and sometimes is not visible in the graph. , The EC 50 values reported in this assay are a functional estimation of the potency of the tested compounds to produce structural transition at transporters that promote inward current associated with the uptake of substrates; association between depolarizing currents and substrate uptake has been described previously. − …”