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-sen sitive transporters in presynaptic terminals is predicted to involve t he cotransport of Na+ and Cl-, resulting in a net movement of charge p er transport cycle. To explore the relationship between catecholamine transport and ion permeation through the NE transporter, we establishe d a human norepinephrine transporter (hNET) cell line suitable for bio chemical analysis and patch-clamp recording. Stable transfection of hN ET cDNA into HEK-293 (human embryonic kidney) cells results in lines e xhibiting (1) a high number of transporter copies per cell (10(6)), as detected by radioligand binding and hNET-specific antibodies, (2) hig h-affinity, Na+-dependent transport of NE, and (3) inhibitor sensitivi ties 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 addi tion to NE-dependent currents, transfected cells possess an NE-indepen dent mode of charge movement mediated by hNET, hNET antagonists withou t effect in non-transfected cells abolish both NE-dependent and NE-ind ependent modes of charge movement in transfected cells, The magnitude of NE-dependent currents in these cells exceeds the expectations of si mple carrier models using previous estimates of transport rates. To ex plain our observations, we propose that hNETs function as ion-gated li gand 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 tran sport may not predict the magnitude or the kinetics of the total curre nt through the transporter. We propose instead that Na+ gates NE trans port and also the parallel inward flux of an indeterminate number of i ons through a channel-like pore.