The involvement of sodium in the function of the human amino acid transporter ASCT2

Mazza, Tiziano; Scalise, Mariafrancesca; Pappacoda, Gilda; Pochini, Lorena; Indiveri, Cesare

doi:10.1002/1873-3468.14224

Cited by 13 publications

(14 citation statements)

References 44 publications

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“…This finding is in agreement with the hypothesis that Na + plays a modulatory role in ASCTs, as it is not providing a driving force for amino acid exchange. Overall, these data are in excellent agreement with a recent report on ASCT2 showing a Na + /glutamine stoichiometry of 2:1 and, for the first time, directly demonstrating sodium flux across the membrane during amino acid transport [ 46 ].…”

Section: Discussionsupporting

confidence: 91%

Functional and Kinetic Comparison of Alanine Cysteine Serine Transporters ASCT1 and ASCT2

2022

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Neutral amino acid transporters ASCT1 and ASCT2 are two SLC1 (solute carrier 1) family subtypes, which are specific for neutral amino acids. The other members of the SLC1 family are acidic amino acid transporters (EAATs 1–5). While the functional similarities and differences between the EAATs have been well studied, less is known about how the subtypes ASCT1 and 2 differ in kinetics and function. Here, by performing comprehensive electrophysiological analysis, we identified similarities and differences between these subtypes, as well as novel functional properties, such as apparent substrate affinities of the inward-facing conformation (in the range of 70 μM for L-serine as the substrate). Key findings were: ASCT1 has a higher apparent affinity for Na+, as well as a larger [Na+] dependence of substrate affinity compared to ASCT2. However, the general sequential Na+/substrate binding mechanism with at least one Na+ binding first, followed by amino acid substrate, followed by at least one more Na+ ion, appears to be conserved between the two subtypes. In addition, the first Na+ binding step, presumably to the Na3 site, occurs with high apparent affinity (<1 mM) in both transporters. In addition, ASCT1 and 2 show different substrate selectivities, where ASCT1 does not respond to extracellular glutamine. Finally, in both transporters, we measured rapid, capacitive charge movements upon application and removal of amino acid, due to rearrangement of the translocation equilibrium. This charge movement decays rapidly, with a time constant of 4–5 ms and recovers with a time constant in the 15 ms range after substrate removal. This places a lower limit on the turnover rate of amino acid exchange by these two transporters of 60–80 s−1.

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Section: Discussionsupporting

confidence: 91%

Functional and Kinetic Comparison of Alanine Cysteine Serine Transporters ASCT1 and ASCT2

2022

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“…Furthermore, the use of the single-protein proteoliposome approach shed new light on some controversial aspects of the ASCT2 function. The transport stoichiometry was established as 2Na + ex :1aa 0 ex :1aa 0 in [18]; this finding, obtained by combining radiometric and fluorometric assays, corroborated the previous observations on the electrogenic nature of the ASCT2 transport reaction, which is slightly stimulated by an imposed membrane potential across membrane [17]. Interestingly, the low extent of stimulation by membrane potential is shared with the hEAAT3 [19] and with the bacterial homologue Glt Ph [20].…”

Section: Introductionsupporting

confidence: 86%

Cysteine 467 of the ASCT2 Amino Acid Transporter Is a Molecular Determinant of the Antiport Mechanism

Scalise

Pappacoda

Mazza

et al. 2022

IJMS

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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.

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“…After the incubation under rotatory stirring (30°C 15 min 750 rpm), the solution was dried using the rotavapor. The lipid film was resuspended in 1.5 ml water (10% final concentration), and single bilayer liposomes were prepared by two sonication cycles of 1 min (1 pulse ON and 1 pulse OFF, 40 W) with a Vibracell VCX-130 Sonifier, as previously suggested ( Mazza et al, 2021 ).…”

Section: Methodsmentioning

confidence: 99%

The Nutraceutical Alliin From Garlic Is a Novel Substrate of the Essential Amino Acid Transporter LAT1 (SLC7A5)

et al. 2022

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The plasma membrane transporter LAT1 (SLC7A5) is a crucial player for cell homeostasis because it is responsible for providing cells with essential amino acids and hormones. LAT1 forms a functional heterodimer with the cell surface antigen heavy chain CD98 (also known as 4F2hc and SLC3A2), a type II membrane glycoprotein, which is essential for LAT1 stability and localization to the plasma membrane. The relevance of LAT1 for human metabolism is also related to its altered expression in human diseases, such as cancer and diabetes. These features boosted research toward molecules that are able to interact with LAT1; in this respect, the recent resolution of the LAT1-CD98 3D structure by Cryo-EM has opened important perspectives in the study of the interaction with different molecules in order to identify new drugs to be used in therapy or new substrates of natural origin to be employed as adjuvants and food supplements. In this work, the interaction of LAT1 with alliin, a garlic derivative, has been investigated by using a combined approach of bioinformatics and in vitro transport assays. Alliin is a nutraceutical that has several beneficial effects on human health, such as antidiabetic, anticarcinogenic, antioxidant, and anti-inflammatory properties. The computational analysis suggested that alliin interacts with the substrate binding site of LAT1, to which alliin was docked. These data were then confirmed by the competitive type inhibition measured in proteoliposomes. Interestingly, in the same experimental model, alliin was also revealed to be a substrate of LAT1.

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The involvement of sodium in the function of the human amino acid transporter ASCT2

Cited by 13 publications

References 44 publications

Functional and Kinetic Comparison of Alanine Cysteine Serine Transporters ASCT1 and ASCT2

Functional and Kinetic Comparison of Alanine Cysteine Serine Transporters ASCT1 and ASCT2

Cysteine 467 of the ASCT2 Amino Acid Transporter Is a Molecular Determinant of the Antiport Mechanism

The Nutraceutical Alliin From Garlic Is a Novel Substrate of the Essential Amino Acid Transporter LAT1 (SLC7A5)

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