Sodium-Dependent Norepinephrine-Induced Currents in Norepinephrine-Transporter-Transfected Hek-293 Cells Blocked by Cocaine and Antidepressants
Open Access
- 1 October 1995
- journal article
- Published by The Company of Biologists in Journal of Experimental Biology
- Vol. 198 (10), 2197-2212
- https://doi.org/10.1242/jeb.198.10.2197
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 (106), 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 possess 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.This publication has 50 references indexed in Scilit:
- A GABA transporter operates asymmetrically and with variable stoichiometryNeuron, 1994
- Permeation Properties of Neurotransmitter TransportersAnnual Review of Pharmacology and Toxicology, 1994
- Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytesNeuron, 1993
- Advances in molecular biology of neurotransmitter transportersCurrent Opinion in Psychiatry, 1992
- Steady-state current-voltage relationship of the Na/K pump in guinea pig ventricular myocytes.The Journal of general physiology, 1989
- Depolarization Without Calcium Can Release γ-Aminobutyric Acid from a Retinal NeuronScience, 1987
- Effects of Monovalent Ions on the Transport of Noradrenaline Across the Plasma Membrane of Neuronal Cells (PC‐12 Cells)Journal of Neurochemistry, 1985
- A major role for chloride in (3H)- noradrenaline transport by rat heart adrenergic nervesLife Sciences, 1977
- Role of sodium and potassium ions in storage of norepinephrine by sympathetic nerve endingsLife Sciences, 1966
- Lack of Uptake of Catecholamines after Chronic Denervation of Sympathetic NervesNature, 1961