Ion coupling stoichiometry for the norepinephrine transporter in membrane vesicles from stably transfected cells

We prepared membrane vesicles from stable LLC-PK1 cells expressing serotonin (5-HT) gamma-aminobutyric acid (GABA) and norepinephrine (NE) transporters (SERT, GAT-1, and NET), These vesicles accumulate transport substrates when the appropriate transmembrane ion gradients are imposed, For NET, accumulation of [H-3]dopamine (DA) was stimulated by imposition of Na+ and Cl- gradients (out > in) and of a K+ gradient (in > out), The presence of Na+ or Cl-, even in the absence of a gradient, stimulated DA accumulation by NET, but K+ had little or no effect in the absence of a K+ gradient, Stimulation by a K+ gradient was markedly enhanced by increasing the K+ permeability with valinomycin, suggesting that net positive charge is transported together with DA. Cationic DA is likely to be the major substrate for NET, since varying pH did not affect K-m. We estimated the Na+:DA stoichiometry by measuring the effect of the transmembrane Na+ gradient on peak DA accumulation, The results suggest a 1:1 cotransport of Na+ with DA. Taken together, the results suggest that NET catalyzes cotransport of one cationic substrate molecule with one Na+ ion, and one Cl- ion, and that K+ does not participate directly in the transport process.