Molecular and functional characterization of intestinal Na⁺-dependent neutral amino acid transporter B⁰

We have cloned the Na⁺-dependent neutral amino acid transporter B⁰ (ATB⁰) from rabbit jejunum and from the human intestinal cell line Caco-2. Rabbit intestinal ATB⁰ (riATB⁰) cDNA codes for a protein of 541 amino acids with 10 potential transmembrane domains. When expressed in HeLa cells, riATB⁰ mediates the transport of several neutral amino acids, including glutamine, in a Na⁺-dependent manner. Anionic amino acids, cationic amino acids, and N-methylated amino acids are excluded by riATB⁰. When expressed in Xenopus laevis oocytes, riATB⁰ increases the transport of neutral amino acids severalfold. The induced transport activity is specific for neutral amino acids, with no noticeable interaction with anionic, cationic, and N- methylated amino acids. However, riATB⁰ does interact with anionic amino acids at acidic pH. In oocytes expressing riATB⁰, the neutral amino acid threonine evokes inward currents at a holding potential of -50 mV. The amino acid-evoked current is sensitive to membrane potential. The inward current increases as the membrane potential is hyperpolarized, but the current reverses at about -30 to -40 mV. Threonine evokes outward currents if the membrane potential is depolarized beyond this value. We have also cloned the ATB⁰ from the human intestinal cell line Caco-2. The Caco-2 ATB⁰ cDNA also codes for a protein of 541 amino acids that is essentially identical to the ATB⁰ expressed in the human choriocarcinoma cell line JAR. Reverse transcription-polymerase chain reaction (RT-PCR) and restriction analysis of the RT-PCR products indicate that the human intestine and the human kidney proximal tubular cell line HKPT express an ATB⁰ identical to the ATB⁰ expressed in Caco-2 cells.