Sodium-dependent norepinephrine-induced currents in norepinephrine-transporter-transfected HEK-293 cells blocked by cocaine and antidepressants.
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.
D000316:Adrenergic alpha-Agonists / Q000378:metabolism*; D000928:Antidepressive Agents / Q000494:pharmacology*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D003042:Cocaine / Q000494:pharmacology*; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology*; D006801:Humans; D017136:Ion Transport; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D009638:Norepinephrine / Q000378:metabolism*; D050484:Norepinephrine Plasma Membrane Transport Proteins; D018408:Patch-Clamp Techniques; D012964:Sodium / Q000378:metabolism; D027981:Symporters; D014162:Transfection
D000316:Adrenergic alpha-Agonists; D000928:Antidepressive Agents; D002352:Carrier Proteins; D018765:Dopamine Uptake Inhibitors; D050484:Norepinephrine Plasma Membrane Transport Proteins; C493227:SLC6A2 protein, human; D027981:Symporters; D012964:Sodium; D003042:Cocaine; D009638:Norepinephrine