Abstract
The human plasma membrane transporter ASCT2 is responsible for mediating Na- dependent antiport of neutral amino acids. New insights into structure/function relationships were unveiled by a combined approach of recombinant over-expression, site-directed mutagenesis, transport assays in proteoliposomes and bioinformatics. WT and Cys mutants of hASCT2 were produced in P. pastoris and purified for functional assay. The reactivity towards SH reducing and oxidizing agents of WT protein was investigated and opposite effects were revealed; transport activity increased upon treatment with the Cys reducing agent DTE, i.e., when Cys residues were in thiol (reduced) state. Methyl-Hg, which binds to SH groups, was able to inhibit WT and seven out of eight Cys to Ala mutants. On the contrary, C467A loses the sensitivity to both DTE activation and Methyl-Hg inhibition. The C467A mutant showed a Km for Gln one order of magnitude higher than that of WT. Moreover, the C467 residue is localized in the substrate binding region of the protein, as suggested by bioinformatics on the basis of the EAAT1 structure comparison. Taken together, the experimental data allowed identifying C467 residue as crucial for substrate binding and for transport activity modulation of hASCT2.
Highlights
ASCT2 (SLC1A5) is a neutral amino acid transporter of plasma membrane that belongs to the SLC1 family together with ASCT1 (SLC1A4) and five glutamate transporters (SLC1A1–3 and SLC1A5–6).Tissue distribution of ASCT2 is quite broad, being expressed in kidney, intestine, brain, lung, skeletal muscle, placenta and pancreas where ASCT2 is mainly involved in traffic of neutral amino acids [1].Murine ASCT2 members have been firstly studied in different experimental models, i.e., cells and proteoliposomes [2,3]
To investigate the influence of the redox state of Cys on transport function, the thiol residues of the protein were reduced by DTE and the Na+ -dependent [3 H]Glnex /Glnin antiport was measured in proteoliposomes in which the recombinant ASCT2 is inserted with the same orientation as in the native membrane (Figure 1) [4]
Initial transport rates were calculated as k limit from the first order rate equation used to plot the experimental data
Summary
ASCT2 (SLC1A5) is a neutral amino acid transporter of plasma membrane that belongs to the SLC1 family together with ASCT1 (SLC1A4) and five glutamate transporters (SLC1A1–3 and SLC1A5–6).Tissue distribution of ASCT2 is quite broad, being expressed in kidney, intestine, brain, lung, skeletal muscle, placenta and pancreas where ASCT2 is mainly involved in traffic of neutral amino acids [1].Murine ASCT2 members have been firstly studied in different experimental models, i.e., cells and proteoliposomes [2,3]. ASCT2 (SLC1A5) is a neutral amino acid transporter of plasma membrane that belongs to the SLC1 family together with ASCT1 (SLC1A4) and five glutamate transporters (SLC1A1–3 and SLC1A5–6). Tissue distribution of ASCT2 is quite broad, being expressed in kidney, intestine, brain, lung, skeletal muscle, placenta and pancreas where ASCT2 is mainly involved in traffic of neutral amino acids [1]. Murine ASCT2 members have been firstly studied in different experimental models, i.e., cells and proteoliposomes [2,3]. In these studies, some important features of ASCT2 have been described such as the specificity towards amino acids and the mode of transport; in particular, Gln was revealed to be one.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
