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

ABSTRACT: 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.

SUBMITTER: Wang J

PROVIDER: S-EPMC8773628 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A widely dispersed interference group of retroviruses that includes the feline endogenous virus (RD114), baboon endogenous virus (BaEV), human endogenous virus type W (HERV-W), and type D primate retroviruses uses the human Na(+)-dependent neutral amino acid transporter type 2 (hASCT2; gene name, SLC1A5) as a common cell surface receptor. Although hamster cells are fully resistant to these viruses and murine cells are susceptible only to BaEV and HERV-W pseudotype viruses, these rodent cells both become highly susceptible to all of the viruses after treatment with tunicamycin, an inhibitor of protein N-linked glycosylation. A partial explanation for these results was recently provided by findings that the orthologous murine transporter mASCT2 is inactive as a viral receptor, that a related (ca. 55% identity) murine paralog (mASCT1; gene name, SLC1A4) mediates infections specifically of BaEV and HERV-W, and that N-deglycosylation of mASCT1 activates it as a receptor for all viruses of this interference group. Because the only two N-linked oligosaccharides in mASCT1 occur in the carboxyl-terminal region of extracellular loop 2 (ECL2), it was inferred that this region contributes in an inhibitory manner to infections by RD114 and type D primate viruses. To directly and more thoroughly investigate the receptor active sites, we constructed and analyzed a series of hASCT2/mASCT2 chimeras and site-directed mutants. Our results suggest that a hypervariable sequence of 21 amino acids in the carboxyl-terminal portion of ECL2 plays a critical role in determining the receptor properties of ASCT2 proteins for all viruses in this interference group. In addition, we analyzed the tunicamycin-dependent viral susceptibility of hamster cells. In contrast to mASCT1, which contains two N-linked oligosaccharides that partially restrict viral infections, hamster ASCT1 contains an additional N-linked oligosaccharide clustered close to the others in the carboxyl-terminal region of ECL2. Removal of this N-linked oligosaccharide by mutagenesis enabled hamster ASCT1 to function as a receptor for all viruses of this interference group. These results strongly suggest that combinations of amino acid sequence changes and N-linked oligosaccharides in a critical carboxyl-terminal region of ECL2 control retroviral utilization of both the ASCT1 and ASCT2 receptors.

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

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