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. 2016 Jul;30(7):796-808.
doi: 10.1210/me.2016-1037. Epub 2016 May 31.

Few Amino Acid Exchanges Expand the Substrate Spectrum of Monocarboxylate Transporter 10

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Free PMC article

Few Amino Acid Exchanges Expand the Substrate Spectrum of Monocarboxylate Transporter 10

Jörg Johannes et al. Mol Endocrinol. .
Free PMC article

Abstract

Monocarboxylate transporters (MCTs) belong to the SLC16 family within the major facilitator superfamily of transmembrane transporters. MCT8 is a thyroid hormone transporter mutated in the Allan-Herndon-Dudley syndrome, a severe psychomotor retardation syndrome. MCT10 is closely related to MCT8 and is known as T-type amino acid transporter. Both transporters mediate T3 transport, but although MCT8 also transports rT3 and T4, these compounds are not efficiently transported by MCT10, which, in contrast, transports aromatic amino acids. Based on the 58% amino acid identity within the transmembrane regions among MCT8 and MCT10, we reasoned that substrate specificity may be primarily determined by a small number of amino acid differences between MCT8 and MCT10 along the substrate translocation channel. Inspecting the homology model of MCT8 and a structure-guided alignment between both proteins, we selected 8 amino acid positions and prepared chimeric MCT10 proteins with selected amino acids changed to the corresponding amino acids in MCT8. The MCT10 mutant harboring 8 amino acid substitutions was stably expressed in Madin-Darby canine kidney 1 cells and found to exhibit T4 transport activity. We then successively reduced the number of amino acid substitutions and eventually identified a minimal set of 2-3 amino acid exchanges which were sufficient to allow T4 transport. The resulting MCT10 chimeras exhibited KM values for T4 similar to MCT8 but transported T4 at a slower rate. The acquisition of T4 transport by MCT10 was associated with complete loss of the capacity to transport Phe, when Tyr184 was mutated to Phe.

Figures

Figure 1.
Figure 1.. Substrate spectrum of MCT10.
Competition experiments were performed by coincubating 10nM 125I-T3 with 1μM various iodothyronines or 1mM Trp in MDCK1 cells stably expressing MCT10. Cell associated radioactivity was measured after 3 minutes. 125I-T3 uptake of cells transfected with pcDNA3 was considered as background and subtracted. Uptake without test compound was set to 0% inhibition. ***, P < .001; NS, not significant vs Dimethyl Sulfoxide.
Figure 2.
Figure 2.. Amino acid substitutions in MCT10.
A, Schematic representation of MCT10 based on the model of MCT8 (15). The 8 target amino acids of MCT10x8 are represented in boldface. Known interactions in MCT8 (salt bridge, His-Arg clamp) are indicated for orientation. B, Overview of all amino acid exchanges of the relevant MCT10MCT8 chimeras. The colors correspond to A.
Figure 3.
Figure 3.. Biochemical characterization of MCT10MCT8 chimeric proteins.
A, Uptake of 10nM 125I-T3 after 30 minutes in stably transfected MDCK1 cells expressing various MCT10MCT8 chimeras and the wild-type MCT8 and MCT10, respectively. Uptake of cells transfected with pcDNA3 was considered as background and subtracted. B, T3 time-course experiment in Xenopus oocytes. Oocytes injected with the appropriate cRNA were exposed to 10nM 125I-T3 for 3–20 minutes, mock-injected oocytes served as background control. T3 uptake over time is not changed in MCT10x4.1 and MCT10x8 compared with MCT10. C, Uptake of 10nM 125I-T4 after 30 minutes in stably transfected MDCK1 cells expressing various MCT10MCT8 chimeras and the wild-type MCT8 and MCT10, respectively. Uptake into mock-transfected cells was subtracted. MCT10x8 and MCT10x6 gained the ability to transport T4. D, Determination of KM values for T4 of MCT10MCT8 chimeras in relation to MCT8. Uptake into mock-transfected cells was subtracted as background. Measurements were performed with increasing concentrations of T4 for 3 minutes. Uptake of MCT8 at highest concentration (12.5μM) was set to 100%. E, Eadie-Hofstee plot of data from D, revealing similar KM values for MCT10x8 and MCT10x6 compared with MCT8. F, Western blotting for anti-HA tag with 10-μL biotinylated and affinity-purified plasma membrane protein fraction of MDCK1 stably expressing MCT10MCT8 chimeras MCT10 and MCT8 wild type. β-Actin served as loading control. All chimeras reached the cell surface and were expressed in a similar manner. *, P < .5; **, P < .01; ***, P < .001; NS, not significant.
Figure 4.
Figure 4.. Efflux of substrates by MCT10 chimeras compared with MCT10 and MCT8.
MDCK1 cells expressing various MCT10MCT8 chimeras and the wild-type MCT8 and MCT10, respectively, were incubated for 30 minutes with 10nM substrate (A) T3, or (B) T4 After substrate-loading, cells were washed briefly with PBS and incubated for 5 minutes with uptake buffer w/o substrate. Substrates from cell lysate and supernatant were extracted and measured by LC-MS2. The background of empty-vector transfected cells was subtracted. MCT10x6 and MCT10x8 mirror the uptake and efflux of MCT8, whereas MCT10x4.1 behaved like MCT10. *, P < .5; **, P < .01; ***, P < .001; NS, not significant.
Figure 5.
Figure 5.. Substrate spectra of MCT10MCT8 chimeras compared with MCT10 and MCT8.
MDCK1 cells expressing various MCT10MCT8 chimeras and the wild-type MCT8 and MCT10, respectively, were incubated for 30 minutes with 100nM each of the indicated substrates. Substrates from cell lysate was extracted and measured by LC-MS2. The background of empty-vector transfected cells was subtracted. MCT10x3, MCT10x4.2, MCT10x6, and MCT10x8 mirror the substrate spectrum of MCT8, whereas MCT10x4.1 shows the same substrate specificity as MCT10. **, P < .01; ***, P < .001.
Figure 6.
Figure 6.. Determination of rT3 KM values.
A, MDCK1 cells stably transfected with MCT10x4.1, MCT10x8, MCT10, and MCT8 were incubated with increasing concentrations of rT3 for 5 minutes. rT3 from cell lysate was extracted and measured by LC-MS2. The background of empty-vector transfected cells was subtracted as background. B, Eadie-Hofstee plots of data from A, revealing very similar KM values for MCT8 and MCT10x8. *, P < .05; **, P < .01; ***, P < .001; NS, not significant.
Figure 7.
Figure 7.. Biochemical characterization of additional MCT10MCT8 chimeric proteins.
All experiments were performed in stably transfected MDCK1 cells expressing various MCT10MCT8 chimeras and the wild-type MCT8 and MCT10, respectively. Uptake into mock-transfected cells was subtracted as background. A, Western blotting for anti-HA tag with 10-μL biotinylated and affinity-purified plasma membrane protein fraction of MDCK1 cells. β-Actin served as loading control. All chimeras reached the cell surface and were expressed at a similar level. B, Uptake of 10nM 125I-T4 after 30 minutes. MCT10x4.2, MCT10x3.1, MCT10x2.2, and MCT10x2.3 gained the ability to transport T4, whereas the chimeras with a single mutation behaved like MCT10. C, Determination of KM values of T4 transporting MCT10MCT8 chimeras in relation to MCT8. Measurements were performed with increasing concentrations of T4 for 10 minutes (MCT8 3 min). Uptake of MCT8 at the highest concentration (12.5μM) was set to 100%. D, Eadie-Hofstee plot of data from C, revealing similar KM but decreased vmax values compared with MCT8. E, T4 time-course experiment. MDCK1 cells were exposed to 10nM 125I-T4 for 1–30 minutes. MCT10x2.2 and MCT10x2.3 show a slower uptake of T4 compared with MCT8.
Figure 8.
Figure 8.. Effect of amino acid exchanges on amino acid uptake in MDCK1 cells and Xenopus oocytes.
A, Competition uptake of 125I-T3 (10nM) and Trp (1mM) after 10 minutes in MCT10 chimeras compared with wild-type MCT8 and MCT10. All chimeras including the Y184F mutation show no significant inhibition. B, 3H-Phe uptake activity of MCT10 chimera expressed in Xenopus oocytes after 20 minutes. Only the oocytes injected with MCT10-cRNA, which did not include a Tyr184 mutation, showed a clear accumulation of 3H-Phe. *, P < .5; **, P < .01; ***, P < .001; NS, not significant.

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Grant support

This work was supported by Deutsche Forschungsgemeinschaft Grants SCHW914/3-1 and SCHW914/4-1 (to U.S.) and KO922/17-1 (to J.K.) and by grants from Universitätsklinikum Bonn (U.S.).
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