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

We have cloned the Na+-dependent neutral amino acid transporter B-0 (ATB(0)) from rabbit jejunum and from the human intestinal cell line Caco-2. Rabbit intestinal ATB(0) (riATB(0)) cDNA codes for a protein of 541 amino acids with 10 potential transmembrane domains. When expressed in HeLa cells, riATB(0) 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 riATB(0). When expressed in Xenopus laevis oocytes, riATB(0) 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, riATB(0) does interact with anionic amino acids at acidic pH. In oocytes expressing riATB(0), 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 ATB(0) from the human intestinal cell line Caco-2. The Caco-2 ATB(0) cDNA also codes for a protein of 541 amino acids that is essentially identical to the ATB(0) 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 ATB(0) identical to the ATB(0) expressed in Caco-2 cells.