Abstract
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 [3H]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 Km. 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.
Highlights
Tween members of this family, there are differences in ion coupling mechanisms
GAT, SERT, and NE transporter (NET) represent vesicles prepared from LLC-GAT, LLC-SERT, and LLC-PK1 cells expressing human NE transporter (LLC-NET) cells
Together with the previous kinetic data these results strongly suggest that NET, like SERT, cotransports one Naϩ ion with each molecule of substrate
Summary
Materials—The parental LLC-PK1 cells and cells expressing GABA transporter (LLC-GAT) were gifts from Dr Michael Caplan, Department of Cell and Molecular Physiology, Yale University. GAT-1 cDNA, encoding a rat brain GABA transporter, was donated generously by Dr Baruch Kanner, Hebrew University, Israel. The generation and characterization of LLC-PK1 cells expressing human NE transporter (LLC-NET) and the rat 5-HT transporter (LLC-SERT) were described previously [27]. Membrane Vesicle Preparation—Cells were grown to confluence as monolayers on 15-cm diameter tissue culture dishes. Following one wash with 10 ml of vesicle preparation buffer (VPB: 200 mM mannitol, 80 mM HEPES), adjusted to pH 7.4 with LiOH, 1 g/ml pepstatin, 17 g/ml phenylmethylsulfonyl fluoride, and 5 mM oxidized form of glutathione), cells were harvested in the same buffer (10 ml/dish) by scraping and collected at 2300 ϫ g for 10 min.
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