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.

ASCT2 is a neutral amino acid transporter, which catalyzes a sodium-dependent obligatory antiport among glutamine and other neutral amino acids. The human ASCT2 over-expressed in Pichia pastoris and reconstituted in proteoliposomes has been employed for identifying alternative substrates of the transporter. The experimental data highlighted that hASCT2 also catalyzes a sodium-dependent antiport of glutamate with glutamine. This unconventional antiport shows a preferred sidedness: glutamate is inwardly transported in exchange for glutamine transported in the counter direction. The orientation of the transport protein in proteoliposomes is the same as in the cell membrane; then, the observed sidedness corresponds to the transport of glutamate from the extracellular to the intracellular compartment. The competitive inhibition exerted by glutamate on the glutamine transport together with the docking analysis indicates that the glutamate binding site is the same as that of glutamine. The affinity for glutamate is lower than that for neutral amino acids, while the transport rate is comparable to that measured for the asparagine/glutamine antiport. Differently from the neutral amino acid antiport that is insensitive to pH, the glutamate/glutamine antiport is pH-dependent with optimal activity at acidic pH on the external (extracellular) side. The stimulation of glutamate transport by a pH gradient suggests the occurrence of a proton flux coupled to the glutamate transport. The proton transport has been detected by a spectrofluorometric method. The rate of proton transport correlates well with the rate of glutamate transport indicating a 1:1 stoichiometry H + : glutamate. The glutamate/glutamine antiport is also active in intact HeLa cells. On a physiological point of view, the described antiport could have relevance in some districts in which a glutamate/glutamine cycling is necessary, such as in placenta.

The plasma membrane transporter xCT belongs to the SLC7 family and has the physiological role of mediating the exchange of glutamate and cystine across the cell plasma membrane, being crucial for redox control. The xCT protein forms a heterodimer with the ancillary protein CD98. Over the years, xCT became a hot pharmacological target due to the documented over-expression in virtually all human cancers, which rely on cystine availability for their progression. Notwithstanding, several unknown aspects of xCT biology still exist that require a suitable single protein experimental model, to be addressed. To this aim, the recombinant host Escherichia coli has been exploited to over-express the human isoform of xCT. In this widely used and low-cost system, the optimization for growth and protein production has been achieved by acting on the metabolic needs of the bacterial strains. Then, the His-tagged protein has been purified by Ni2+-chelating chromatography and reconstituted in proteoliposomes for transport activity assays. The expressed protein was in a folded/active state allowing functional and kinetic characterization. Interestingly, the features of the recombinant protein meet those of the native one extracted from intact cells, further confirming the suitability of E. coli as a host for the expression of human proteins. This study opens perspectives for elucidating other molecular aspects of xCT, as well as for studying the interaction with endogenous and exogenous compounds, relevant to human health.

Alanine, serine, cysteine transporter 2 (ASCT2) is a membrane amino acid transporter with relevance to human physiology and pathology, such as cancer. Notwithstanding, the study on the ASCT2 transport cycle still has unknown aspects, such as the role of Na + in this process. We investigate this issue using recombinant hASCT2 reconstituted in proteoliposomes. Changes in the composition of purification buffers show the crucial role of Na + in ASCT2 functionality. The transport activity is abolished when Na + is absent or substituted by Li + or K + in purification buffers. By employing a Na + fluorometric probe, we measured an inwardly directed flux of Na + and, by combining fluorometric and radiometric assays, determined a 2Na + : 1Gln stoichiometry. Kinetics of Na + transport suggest that pH-sensitive residues are involved in Na + binding/transport. Our results clarify the role of Na + on human ASCT2 transporter activity.

The Novel Organic Cation Transporter, OCTN1, is the first member of the OCTN subfamily; it belongs to the wider Solute Carrier family SLC22, which counts many members including cation and anion organic transporters. The tertiary structure has not been resolved for any cation organic transporter. The functional role of OCNT1 is still not well assessed despite the many functional studies so far conducted. The lack of a definitive identification of OCTN1 function can be attributed to the different experimental systems and methodologies adopted for studying each of the proposed ligands. Apart from the contradictory data, the international scientific community agrees on a role of OCTN1 in protecting cells and tissues from oxidative and/or inflammatory damage. Moreover, the involvement of this transporter in drug interactions and delivery has been well clarified, even though the exact profile of the transported/interacting molecules is still somehow confusing. Therefore, OCTN1 continues to be a hot topic in terms of its functional role and structure. This review focuses on the most recent advances on OCTN1 in terms of functional aspects, physiological roles, substrate specificity, drug interactions, tissue expression, and relationships with pathology.