XB-ART-59594
Biomolecules
2023 Feb 06;132:. doi: 10.3390/biom13020301.
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Do Amino Acid Antiporters Have Asymmetric Substrate Specificity?
Gauthier-Coles G
,
Fairweather SJ
,
Bröer A
,
Bröer S
.
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Amino acid antiporters mediate the 1:1 exchange of groups of amino acids. Whether substrate specificity can be different for the inward and outward facing conformation has not been investigated systematically, although examples of asymmetric transport have been reported. Here we used LC-MS to detect the movement of 12C- and 13C-labelled amino acid mixtures across the plasma membrane of Xenopus laevis oocytes expressing a variety of amino acid antiporters. Differences of substrate specificity between transporter paralogs were readily observed using this method. Our results suggest that antiporters are largely symmetric, equalizing the pools of their substrate amino acids. Exceptions are the antiporters y+LAT1 and y+LAT2 where neutral amino acids are co-transported with Na+ ions, favouring their import. For the antiporters ASCT1 and ASCT2 glycine acted as a selective influx substrate, while proline was a selective influx substrate of ASCT1. These data show that antiporters can display non-canonical modes of transport.
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Species referenced: Xenopus laevis
Genes referenced: pycard
GO keywords: glutamine transport [+]
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Figure 1. Simulation of symmetric and asymmetric antiporter kinetics in an artificial cell containing LAT2 only. The concentration of cytosolic and extracellular amino acids was set at 1 mM. (a) Intracellular concentrations of all substrate amino acids when LAT2 intracellular KM values were scaled to be 180-fold higher than extracellular KM values. (b) Intracellular concentrations of all substrate amino acids when the intracellular KM value of Gly was scaled to be 0.6-fold higher than the extracellular KM value (all other KM values as in (a)). Gly (beige) is leaving the cell in exchange for all other substrates of the transporter. |
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Figure 2. Analysis of Xenopus laevis oocyte amino acid pools. (a) After expression for five days oocytes were incubated with 12C (black) and 13C amino acids (red) in the order shown. Three samples were analysed for each experiment, namely the preload sample, supernatant sample and extract sample. (b) Cytosolic amino acid concentrations after incubation in a 1 mM 12C amino acid mix of oocytes expressing 4F2hc, 4F2hc + LAT1 or 4F2hc + LAT2 (Preload sample). Isoleucine and leucine cannot be separated by LC–MS and their sum is shown as IL. |
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Figure 3. Analysis of antiport via LAT1 and LAT2. Oocytes expressing 4F2hc–LAT1 (a) or 4F2hc–LAT2 (b) were incubated with labelled and unlabelled amino acids as described in Figure 2a. Net import (positive numbers) and net export (negative numbers) are shown. Isoleucine and leucine cannot be separated by LC–MS and are shown as IL. The activity of oocytes expressing 4F2hc alone was subtracted, resulting in apparent oppositely-directed transport for some amino acids. |
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Figure 4. Analysis of asymmetry and uniport activity of LAT1 and LAT2. (a) Uptake of 0.1 mM [14C]glutamine was measured in the presence and absence of an unlabelled amino acid mix (0.1 mM each) in oocytes expressing 4F2hc, 4F2hc–LAT2 or non-injected oocytes. (b) Oocytes expressing 4F2hc, 4F2hc–LAT2 or non-injected oocytes were preincubated with 0.1 mM [14C]glutamine for 1h. After washing, oocytes were incubated in ND96 in the presence (+Q) and absence (−Q) of 1 mM glutamine and supernatant samples were analysed for radiolabelled glutamine after 15 and 30 min. (c) Oocytes expressing 4F2hc, 4F2hc–LAT1 or 4F2hc–LAT2 were incubated in ND96 for 2 h after which the supernatant was analysed for amino acids to calculate net efflux. Isoleucine and leucine cannot be separated by LC–MS and are shown as IL. *** Significant difference at p < 0.0001. |
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Figure 5. Analysis of antiport via y+LAT1 and y+LAT2. Oocytes expressing 4F2hc–y+LAT1 (a) or 4F2hc–y+LAT2 (b) were incubated with labelled and unlabelled amino acids as described in Figure 2a. Net import (positive numbers) and net export (negative numbers) are shown. Isoleucine and leucine cannot be separated by LC–MS and are shown as IL. |
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Figure 6. Analysis of antiport via asc-1 and xCT. Oocytes expressing 4F2hc–asc-1 (a) or 4F2hc–xCT (b) were incubated with labelled and unlabelled amino acids as described in Figure 2a. Net import (positive numbers) and net export (negative numbers) are shown. Isoleucine and leucine cannot be separated by LC–MS and are shown as IL. |
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Figure 7. Analysis of antiport via ASCT1 and ASCT2. Oocytes expressing ASCT1 (a) or ASCT2 (b) were incubated with labelled and unlabelled amino acids as described in Figure 1a. Net import (positive numbers) and net export (negative numbers) are shown. Isoleucine and leucine cannot be separated by LC–MS and are shown as IL. |
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Figure 8. Analysis of ASCT1-mediated proline and aspartate transport. (a) Uptake of 0.1 mM [14C]proline was measured after 30 min in the presence and absence of an unlabelled amino acid mix (0.1 mM each) in oocytes expressing ASCT1 or in non-injected oocytes. (b) Oocytes expressing ASCT1 or non-injected oocytes were preincubated with 0.1 mM [14C]proline for 1h. After washing, oocytes were incubated in ND96 in the presence (+Thr) and absence (−Thr) of 1 mM threonine and supernatant samples were analysed for radiolabelled proline after 15 and 30 min. Due to the difference in preloading, efflux is shown as % of preloaded [14C]proline. (c) Uptake of 0.025 mM [14C]aspartate was measured after 25 min in oocytes expressing ASCT1 or in non-injected oocytes. (d) Oocytes expressing ASCT1 were preincubated with 0.025 mM [14C]aspartate for 2h. After washing, oocytes were incubated in ND96 in the presence (+Ala) and absence (ND96) of 1 mM alanine and supernatant samples were analysed for radiolabelled aspartate after 15 and 30 min. Due to the difference in preloading, efflux is shown as % of preloaded [14C]aspartate. |
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