Reconstitution into liposomes of the B°-like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na+ dependent [H-3],glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K+. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization. the protein concentration, the cletergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [H-3]glutamine accumulation in proteoliposomes occurred only in the presence of external Na+ and internal K+. The transporter showed low if there is any tolerance towards the substitution of Na+ of K+ for other cations. Valinomycin strongly Stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na+ concentration cooperativity index close to 1 was derived, indicating that 1 Na+ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na+ transport. Optimal rate of 0.1 MM [H-3]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport Fate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5-7.0. [H-3]glutamine (and [H-3]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl2, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1] heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue alpha-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was Protected by the presence of the substrate. On the basis of the Na+ dependence. the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B degrees W-like. (c) 2008 Elsevier B.V. All rights reserved.