Abstract
To determine the role of a neutral and basic amino acid transporter (NBAT) in amino acid transport, we microinjected several COOH-terminal deletion mutants of NBAT cRNA into Xenopus oocytes and measured transport activity for arginine, leucine, and cystine in the presence and absence of sodium. Wild-type NBAT significantly stimulated the uptake of all three amino acids 10-20-fold compared with controls. On the other hand, no mutant, except a Delta511-685 mutant, stimulated the uptake of these amino acids. The Delta511-685 mutant significantly increased the uptake of arginine. In the presence of sodium, the Delta511-685 mutant also increased the uptake of leucine. The Delta511-685 mutant did not stimulate cystine uptake in the presence or absence of sodium. The stimulation of arginine uptake by the Delta511-685 mutant was inhibited by a 100-fold excess of unlabeled leucine in the presence of sodium. Inhibition of L-arginine uptake by L-homoserine was seen only in the presence of sodium, and an increase in the inhibition of L-arginine uptake by L-histidine was seen when the extracellular pH was decreased. Furthermore, an inward current in oocytes injected with the Delta511-685 mutant was recorded electrophysiologically when basic amino acids were applied. Homoserine was also taken up, but sodium was necessary for their transport. These properties of the Delta511-685 mutant correspond to those of the y+ amino acid transporter. If NBAT is a component of the b0,+-like amino acid transport system, it is unlikely that a mutant protein (Delta511-685) is able to stimulate an endogenous y+-like transport system. These results suggest that NBAT functions as a activator of the amino acid transport system in Xenopus oocytes.
Highlights
Virus receptor contains several membrane-spanning domains and has no homology with 4F2 antigen [15, 16]
Mutant NBAT (⌬511– 685) cRNA-stimulated, sodium-independent uptake of L-arginine was inhibited completely by L-leucine in the presence of sodium. The stimulation both of sodium-dependent uptake of L-arginine and L-leucine induced by mutant NBAT (⌬511– 685) cRNA was inhibited completely by basic L-amino acids
Dibasic amino acids induced inward currents in Xenopus oocytes injected with the ⌬511– 685 mutant, but neutral amino acids did not induce outward currents
Summary
To measure amino acid uptake, six or seven oocytes from each experimental group were incubated in medium containing L-[3H]arginine, L-[3H]leucine, and L-[35S]cystine (Amersham). Oocytes first were washed for 30 s in solution A (100 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM Hepes/Tris, pH 7.5). The oocytes were washed three times with cold Naϩfree uptake solution containing 5 mM amino acids. Oocytes were superfused with a medium containing either 100 mM NaCl or tetramethylammonium chloride and including 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM Hepes (pH 7.5) with Tris base (superfusion medium). Samples were mixed with 10 g of affinity-purified antibody (anti-human NBAT rabbit IgG) and 8 l of protein A-Sepharose. The antiserum after the fourth boost was affinity purified using a gel column (Cellulofine; Seikagaku-Kogyo, Tokyo, Japan) [27]
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