Transport mechanism and regulatory properties of the human amino acid transporter ASCT2 (SLC1A5)

• Published in: Amino acids Vol. 46; no. 11; pp. 2463 - 2475
• Main Authors: Scalise, Mariafrancesca, Pochini, Lorena, Panni, Simona, Pingitore, Piero, Hedfalk, Kristina, Indiveri, Cesare
• Format: Journal Article
• Language: English
• Published: Vienna Springer Vienna 01.11.2014; Springer Nature B.V
• Subjects: Amino Acid Transport System ASC - metabolism; Amino acids; Amino Acids - chemistry; Analytical Chemistry; Animals; Biochemical Engineering; Biochemistry; Biologi; Biological Sciences; Biological Transport; Biomedical and Life Sciences; Biomedical materials; Carrier Proteins - metabolism; Constants; Cross-Linking Reagents - chemistry; crosslinking; Electrochemistry; Gene Expression Regulation; Glutamine; Glutamine - chemistry; HeLa Cells; Human; Humans; Kinetics; Life Sciences; Liposomes - chemistry; Membrane potential; Minor Histocompatibility Antigens; Neurobiology; Original Article; Pichia - metabolism; potassium; Potassium - chemistry; Proteomics; Rats; Reaction kinetics; Recombinant Proteins - metabolism; scaffolding proteins; sodium; Sodium - chemistry; Surgical implants; Transport; Transporter; Valinomycin; Valinomycin - chemistry; Amino acid; Liposomes; Transport; Scaffold protein; Plasma membrane; Glutamine
• ISSN: 0939-4451; 1438-2199; 1438-2199
• DOI: 10.1007/s00726-014-1808-x

The kinetic mechanism of the transport catalyzed by the human glutamine/neutral amino acid transporter hASCT2 over-expressed in P. pastoris was determined in proteoliposomes by pseudo-bi-substrate kinetic analysis of the Na + -glutamine ex /glutamine in transport reaction. A random simultaneous mechanism resulted from the experimental analysis. Purified functional hASCT2 was chemically cross-linked to a stable dimeric form. The oligomeric structure correlated well with the kinetic mechanism of transport. Half-saturation constants (Km) of the transporter for the other substrates Ala, Ser, Asn and Thr were measured both on the external and internal side. External Km were much lower than the internal ones confirming the asymmetry of the transporter. The electric nature of the transport reaction was determined imposing a negative inside membrane potential generated by K + gradients in the presence of valinomycin. The transport reaction resulted to be electrogenic and the electrogenicity originated from external Na + . Internal Na + exerted a stimulatory effect on the transport activity which could be explained by a regulatory, not a counter-transport, effect. Native and deglycosylated hASCT2 extracted from HeLa showed the same transport features demonstrating that the glycosyl moiety has no role in transport function. Both in vitro and in vivo interactions of hASCT2 with the scaffold protein PDZK1 were revealed.