Mammalian monocarboxylate transporter 7 (MCT7/Slc16a6) is a novel facilitative taurine transporter

Abstract

Monocarboxylate transporter 7 (MCT7) is an orphan transporter expressed in the liver, brain, and in several types of cancer cells. It has also been reported to be a survival factor in melanoma and breast cancers. However, this survival mechanism is not yet fully understood due to MCT7's unidentified substrate(s). Therefore, here we sought to identify MCT7 substrate(s) and characterize the transport mechanisms by analyzing amino acid transport in HEK293T cells and polarized Caco-2 cells. Analysis of amino acids revealed significant rapid reduction in taurine from cells transfected with enhanced green fluorescent protein-tagged MCT7. We found that taurine uptake and efflux by MCT7 was pH-independent and that the uptake was not saturated in the presence of taurine excess of 200 mM. Furthermore, we found that monocarboxylates and acidic amino acids inhibited MCT7-mediated taurine uptake. These results imply that MCT7 may be a low-affinity facilitative taurine transporter. We also found that MCT7 was localized at the basolateral membrane in polarized Caco-2 cells and that the induction of MCT7 expression in polarized Caco-2 cells enhanced taurine permeation. Finally, we demonstrated that interactions of MCT7 with ancillary proteins basigin/CD147 and embigin/GP70 enhanced MCT7-mediated taurine transport. In summary, these findings reveal that taurine is a novel substrate of MCT7 and that MCT7-mediated taurine transport might contribute to the efflux of taurine from cells.

Keywords: amino acid transport; efflux; facilitative transport; intestinal epithelium; membrane transport; monocarboxylate transporter 7; protein–protein interaction; taurine; transporter.

Figure 1

MCT7 causes a rapid decrease of intracellular taurine level. HEK293T cells were transfected with EGFP-tagged MCT7 (○) or empty-vector (mock) (•). The transfected cells were incubated in Na⁺-free buffer (pH 7.4) at 37 °C for designed time. After the incubation, the amino acids contents were measured by LC-MS/MS and the obtained chromogram area is correlated by protein amount. The values were expressed as % of initial value. Each point represents the mean ± S.D. (n = 7). ∗∗ p < 0.01 and ∗ p < 0.05, compared with the corresponding mock-transfected cells by two-way ANOVA with Sidak's multiple comparisons test. EGFP, enhanced green fluorescent protein; MCT7, monocarboxylate transporter 7.

Figure 2

MCT7 mediates a bidirectional taurine transport.A and C, efflux of [³H]taurine by HEK293T cells expressing EGFP-tagged MCT7 (MCT7) or empty-vector (mock). [³H]taurine was loaded to the cells by incubation in NaCl buffer for 30 min. The cells were incubated in Na⁺-free buffer (pH 7.4) for designed time or in the pH-modified buffer (pH 6.5 or pH 8.5) for 30 min. The residual [³H]taurine in the cells was measured. B and D, [³H]taurine uptake (5 μM) was measured in Na⁺-free buffer (pH 7.4) for designed time or in the pH modified buffer (pH 6.5 or pH 8.5) for 30 min. E, concentration dependence of [³H]taurine uptake. The uptake was measured at designed concentration (1–270 mM). Velocity of MCT7-mediated uptake was calculated by subtracting the taurine uptake in mock-transfected cells from EGFP-tagged MCT7 transfected cells. F, intracellular and extracellular concentration of taurine in MCT7- or mock-transfected cells. The cells were incubated in Na⁺-free buffer (pH 7.4) for designed time. The residual taurine in the cells and effluxed taurine in the buffer were measured by LC-MS/MS. Each point represents the mean ± S.D. (n = 3). ∗∗ p < 0.01, compared with the corresponding mock-transfected cells by two-way ANOVA with Sidak's multiple comparisons test or one-way ANOVA with Tukey’s multiple comparison test. EGFP, enhanced green fluorescent protein; MCT7, monocarboxylate transporter 7.

Figure 3

Ancillary proteins CD147 and GP70 interact with MCT7 and regulate the function. HEK293T cells were transfected with expressing EGFP-tagged MCT7 and CD147 or GP70. A, MCT7-mediated [³H]taurine efflux was measured. [³H]taurine was loaded into those cells by incubation with NaCl buffer. Those cells were incubated in Na⁺-free buffer (pH 7.4) for designed time. The residual [³H]taurine in the cells was measured. Each point represents the mean ± S.D. (n = 3). B, [³H]taurine uptake (5 μM) was measured in Na⁺-free buffer (pH 7.4) for 2 min. MCT7-mediated uptake was calculated by subtracting the uptake amount of mock-transfected cells from that of MCT7-transfected cells. Each bar represents the mean ± S.D. (n = 6). ∗∗ p < 0.01, compared with the corresponding only MCT7-transfected cells by one-way ANOVA with Tukey’s multiple comparison test. C, Bi-FC assay of interaction between MCT7 and ancillary proteins. VN and VC are the N- and C-terminal fragments of Venus fluorescent protein, respectively. HEK293T cells were transfected with MCT7-VN or MCT7-VC and ancillary proteins-VN or -VC. EGFP-tagged MCT7 was used as a positive control. After 48-hr transfection, Venus fluorescent signals were detected by a fluorescence microscopy. The obtained fluorescent images were merged with phase contrast images. EGFP, enhanced green fluorescent protein; MCT7, monocarboxylate transporter 7; VC, C-terminal Venus-fragment; VN, N-terminal Venus-fragment.

Figure 4

MCT7 enhances taurine penetration across Caco-2 cells.A, efflux of [³H]taurine by Caco-2-Tet-MCT7 cells inducing MCT7. Caco-2-Tet-MCT7 cells were cultured on 48-well plate and then treated with doxycycline at 5 μg/ml and sodium butyrate at 10 mM (Dox [+]) or sodium butyrate at 10 mM (Dox [-]) for 48 h to induce MCT7 expression. [³H]taurine was loaded into those cells by incubation with NaCl buffer. Those cells were incubated in Na⁺-free buffer (pH 7.4) for designed time. The residual [³H]taurine in the cells was measured. B, apical to basal (A-to-B) transport of [³H]taurine and lucifer yellow (LY). Caco-2-Tet-MCT7 cells were cultured on a Transwell membrane. After 21 days of culture, the cells were treated with doxycycline as description above. Transport of [³H]taurine (5 μM) and LY (500 μM) from apical to basal chamber were measured. C, localization of induced MCT7 in Caco-2-Tet-MCT7 cells cultured on a Falcon cell culture insert membrane. The nuclei were stained by Hoechst33342. MCT7 (green) and nuclei (blue) were observed by a confocal fluorescence microscopy. Each point represents the mean ± SD (n = 3). ∗∗ p < 0.01 and ∗ p < 0.05, compared to the corresponding Caco-2-Tet-MCT7 cells treated with only sodium butyrate by two-way ANOVA with Sidak's multiple comparisons test. MCT7, monocarboxylate transporter 7.