Ion dependence of carrier-mediated release in dopamine or norepinephrine transporter-transfected cells questions the hypothesis of facilitated exchange diffusion

The mechanism of release mediated by the human dopamine and norepinephrine transporter (DAT and NET, respectively) was studied by a superfusion techni que in human embryonic kidney 293 cells stably transfected with the respect ive transporter cDNA and loaded with the metabolically inert substrate [H-3 ]1-methyl-4-phenylpyridinium. Release was induced by amphetamine, dopamine, and norepinephrine or by lowering the sodium or chloride concentration in the superfusion buffer (isoosmotic replacement by lithium and isethionate, respectively). Efflux of [H-3]1-methyl-4-phenylpyridinium was analyzed at 3 0-s time resolution. In both transporters, release induced by the substrate s amphetamine, dopamine, and norepinephrine followed the same time course a s release induced by the removal of chloride and was faster than that cause d by the removal of sodium. In the presence of low sodium (DAT: 10 mM; NET: 5 mM) none of the substrates was able to induce release from either type o f cell, but adding back sodium to control conditions promptly restored the releasing action. In the presence of low chloride (DAT: 3 mM; NET: 2 mM), h owever, amphetamine as well as the catecholamines stimulated release from b oth types of cell. In contrast with the ion dependence of release observed in superfusion experiments, uptake initial rates of substrates at concentra tions used in release experiments were the same or even higher at low sodiu m than at low chloride. The results indicate a decisive role of extracellul ar sodium for carrier-mediated release unrelated to the sodium-dependent up take of the releasing substrate, and suggest a release mechanism different from simple exchange diffusion considering only the amines as substrates.