Structural domains involved in substrate selectivity in two neutral amino acid transporters. Am J Physiol Cell Physiol 287: C754 -C761, 2004. First published May 12, 2004 10.1152/ajpcell.00016.2004The ability of the two highly homologous Na ϩ /Cl Ϫ -dependent neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the larva Manduca sexta, to transport different amino acids depends on the cotransported ion, on pH, and on the membrane voltage. Different organic substrates give rise to transportassociated currents with their own characteristics, which are notably distinct between the two proteins. Differences in amplitude, kinetics, and voltage dependence of the transport-associated currents have been observed, as well as different substrate selectivity patterns measured by radioactive amino acid uptake assays. These diversities represent useful tools to investigate the structural determinants involved in the substrate selectivity. To identify these regions, we built four chimeric proteins between the two transporters. These proteins, heterologously expressed in Xenopus laevis oocytes, were analyzed by two-electrode voltage clamp and uptake measurements. Initially, we exchanged the first three domains, obtaining the chimeras C3K9 and K3C9 (where numbers indicate the transmembrane domains and letters represent the original proteins), which showed electrophysiological and [ 3 H]amino acid uptake characteristics resembling those of KAAT1 and CAATCH1, respectively. Subsequent substitution of the last four domains in C3K9 and K3C9 gave the proteins C3K5C4 and K3C5K4, which showed the same behavior as KAAT1 and CAATCH1 in electrophysiological and transport determinations. These results suggest that in KAAT1 and CAATCH1, only the central transmembrane domains (from 4 to 8) of the protein are responsible for substrate selectivity. structure and function; Manduca sexta COMPARING SEQUENCES AND FUNCTIONS of related proteins can give some insights on the physiological role of particular domains. In the present study, we analyzed two amino acid transporters cloned from the Manduca sexta larva, the tobacco hornworm, which is a lepidopteran of great agroeconomical importance and is one of the best-studied insects as a result of the ease in rearing it on an artificial diet. The intestine of this larva has been the source for the cloning of the two transporters used in the present work: K ϩ -coupled amino acid transporter 1 (KAAT1) (6) and cation-amino acid transporter/channel 1 (CAATCH1) (9). The two proteins, of 634 and 633 amino acids, respectively, are very similar to each other (90% amino acid identity), and their 35-45% identity with the members of the Na ϩ /Cl Ϫ -dependent neurotransmitter transporters allows them to be included in this important superfamily (6, 9). Accordingly, their hydropathicity profiles suggest the presence of 12 transmembrane domains with cytosolic carboxy and amino termini. The two transporters share the peculiar property of being able to utilize the K ϩ gradient to en...