Abstract
The plasma membrane transporter ASCT2 is a well-known Na+-dependent obligatory antiporter of neutral amino acids. The crucial role of the residue C467 in the recognition and binding of the ASCT2 substrate glutamine, has been highlighted by structure/function relationship studies. The reconstitution in proteoliposomes of the human ASCT2 produced in P. pastoris is here employed to unveil another role of the C467 residue in the transport reaction. Indeed, the site-directed mutant C467A displayed a novel property of the transporter, i.e., the ability of mediating a low but measurable unidirectional transport of [3H]-glutamine. This reaction conforms to the main features of the ASCT2-mediated transport, namely the Na+-dependence, the pH dependence, the stimulation by cholesterol included in the proteoliposome membrane, and the specific inhibition by other common substrates of the reconstituted human ASCT2. Interestingly, the WT protein cannot catalyze the unidirectional transport of [3H]-glutamine, demonstrating an unspecific phenomenon. This difference is in favor of a structural conformational change between a WT and C467A mutant that triggers the appearance of the unidirectional flux; this feature has been investigated by comparing the available 3D structures in two different conformations, and two homology models built on the basis of hEAAT1 and GLTPh.
Highlights
Published: 20 January 2022ASCT2 (SLC1A5) is a membrane transporter of paramount importance in both physiological and pathological conditions due to its involvement in maintaining the homeostasis of some neutral amino acids
We provide evidence that a unidirectional transport appears in the C467A mutant, which is absent in the WT isoform, proposing a role for this residue in the antiport mechanism of the hASCT2 transport cycle
To investigate the role of the C467 residue in the antiport reaction mediated by ASCT2, a reliable comparison between the antiport and the unidirectional transport activities was mandatory
Summary
ASCT2 (SLC1A5) is a membrane transporter of paramount importance in both physiological and pathological conditions due to its involvement in maintaining the homeostasis of some neutral amino acids. ASCT2 belongs to the SLC1 family that is constituted by two subgroups of proteins: the first one includes five high affinity glutamate transporters (EAATs); the second one includes two neutral amino acid transporters, ASCT1 and ASCT2. The two subgroups have been described and studied separately because they are distinguished in terms of transport modes, substrate specificities, and tissue distributions [4,5]. Each subgroup is further characterized by different ion coupling, in terms of both ion type and flux direction: EAATs (SLC1A1, A2, A3, A6, A7) are involved in glutamate and aspartate uptake, coupled to exchange of 3Na+ ex :1H+ ex , with K+ in [6]. The involvement of extracellular calcium in the transport process has been proposed [8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
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.
