Delineation of sodium-stimulated amino acid transport pathways in rabbit kidney brush border vesicles

Abstract

Summary We have confirmed previous demonstrations of sodium gradient-stimulated transport ofl-alanine, phenylalanine, proline, and β-alanine, and in addition demonstrated transport of N-methylamino-isobutyric acid (MeAIB) and lysine in isolated rabbit kidney brush border vesicles. In order to probe the multiplicity of transport pathways available to each of these¹⁴C-amino acids, we measured the ability of test amino acids to inhibit tracer uptake. To obtain a rough estimate of nonspecific effects, e.g., dissipation of the transmembrane sodium electrochemical potential gradient, we measured the ability ofd-glucose to inhibit tracer uptake.l-alanine and phenylalanine were completely mutually inhibitory. Roughly 75% of the¹⁴C-l-alanine uptake could be inhibited by proline and β-alanine, while lysine and MeAIB were no more effective thand-glucose. Roughly 50% of the¹⁴C-phenylalanine uptake could be inhibited by proline and β-alanine; lysine was as effective as proline and β-alanine, and the effects of pairs of these amino acids at 50mm each were not cumulative. MeAIB was no more effective thand-glucose. We conclude that three pathways mediate the uptake of neutral,l, α-amino acids. One system is inaccessible to lysine, proline, and β-alanine. The second system carries a major fraction of thel-alanine flux; it is sensitive to proline and β-alanine, but not to lysine. The third system carries half the¹⁴C-phenylalanine flux, and it is sensitive to proline, lysine, and β-alanine. Since the neutral,l, α-amino acid fluxes are insensitive to MeAIB, we conclude that they are not mediated by the classicalA system, and since all of thel-alanine flux is inhibited by phenylalanine, we conclude that it is not mediated by the classicalASC system.l-alanine and phenylalanine completely inhibit uptake of lysine. MeAIB is no more effective thand-glucose in inhibiting lysine uptake, while proline and β-alanine appear to inhibit a component of the lysine flux. We conclude that the¹⁴C-lysine fluxes are mediated by two systems, one, shared with phenylalanine, which is inhibited by proline, β-alanine, andl-alanine, and one which is inhibited byl-alanine and phenylalanine but inaccessible to proline, β-alanine, and MeAIB. Fluxes of¹⁴C-proline and¹⁴C-MeAIB are completely inhibited byl-alanine, phenylalanine, proline, and MeAIB, but they are insensitive to lysine. Proline and MeAIB, as well as alanine and phenylalanine, but not lysine, inhibit¹⁴C-β-alanine uptake. However, β-alanine inhibits only 38% of the¹⁴C-proline uptake and 57% of the MeAIB uptake. We conclude that two systems mediate uptake of proline and MeAIB, and that one of these systems also transports β-alanine.