title: Structural domains involved in substrate selectivity in two neutral amino acid transporters.
abstract: The ability of the two highly homologous Na(+)/Cl(-)-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 transport-associated 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.
authors.affiliation: Dept. of Structural and Functional Biology, University of Insubria, Via Dunant 3, 21100 Varese, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Soragna
authors.forename: Stefania Anna
authors.initials: SA
authors.lastname: Mari
authors.forename: Rossana
authors.initials: R
authors.lastname: Pisani
authors.forename: Antonio
authors.initials: A
authors.lastname: Peres
authors.forename: Michela
authors.initials: M
authors.lastname: Castagna
authors.forename: V Franca
authors.initials: VF
authors.lastname: Sacchi
authors.forename: Elena
authors.initials: E
authors.lastname: Bossi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000502:physiology*; D002678:Chimera; D003001:Cloning, Molecular; D004594:Electrophysiology; D018402:Manduca; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010802:Phylogeny; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins