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. 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. Monocarboxylate transporters (MCTs) belong to the solute carrier family 16A (SLC16A) that comprises 14 members. MCTs (MCT1–4) catalyze H+-coupled transport of metabolically important monocarboxylates such as lactate and contribute to maintenance of intracellular pH (1Halestrap A.P. The SLC16 gene family - structure, role and regulation in health and disease.Mol. Aspects Med. 2013; 34: 337-349Crossref PubMed Scopus (367) Google Scholar). Typical MCTs are known to mediate the bidirectional transport of substrates and protons depending on their concentration gradients across the plasma membrane (2Dimmer K.S. Friedrich B. Lang F. Deitmer J.W. Bröer S. The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells.Biochem. J. 2000; 350: 219-227Crossref PubMed Scopus (429) Google Scholar, 3Draoui N. Feron O. Lactate shuttles at a glance: From physiological paradigms to anti-cancer treatments.Dis. Model. Mech. 2011; 4: 727-732Crossref PubMed Scopus (197) Google Scholar). MCT1 and MCT4 in cancer cells are related to tumor growth and proliferation by mediating the influx and efflux of lactate and protons (4Halestrap A.P. Wilson M.C. The monocarboxylate transporter family--role and regulation.IUBMB Life. 2012; 64: 109-119Crossref PubMed Scopus (427) Google Scholar). Functional expression of MCTs is supported by several ancillary proteins such as basigin/CD147 and embigin/GP70 (5Felmlee M.A. Jones R.S. Rodriguez-Cruz V. Follman K.E. Morris M.E. Monocarboxylate transporters (SLC16): Function, regulation, and role in health and disease.Pharmacol. Rev. 2020; 72: 466-485Crossref PubMed Scopus (71) Google Scholar). These ancillary proteins interact with MCTs at the plasma membrane and regulate membrane localization and function. Apart from prototypical MCTs, other members transport not only monocarboxylates but also zwitterionic metabolites as substrates. For instance, MCT12/SLC16A12 transports intracellular creatine across the plasma membrane in a H+-independent manner (6Abplanalp J. Laczko E. Philp N.J. Neidhardt J. Zuercher J. Braun P. Schorderet D.F. Munier F.L. Verrey F. Berger W. Camargo S.M.R. Kloeckener-Gruissem B. The cataract and glucosuria associated monocarboxylate transporter MCT12 is a new creatine transporter.Hum. Mol. Genet. 2013; 22: 3218-3226Crossref PubMed Scopus (43) Google Scholar, 7Takahashi M. Kishimoto H. Shirasaka Y. Inoue K. Functional characterization of monocarboxylate transporter 12 (SLC16A12/MCT12) as a facilitative creatine transporter.Drug Metab. Pharmacokinet. 2020; 35: 281-287Crossref PubMed Scopus (11) Google Scholar). MCT9/SLC16A9 also functions as a H+-independent carnitine efflux transporter (8Suhre K. Shin S.Y. Petersen A.K. Mohney R.P. Meredith D. Wägele B. Altmaier E. Deloukas P. Erdmann J. Grundberg E. Hammond C.J. de Angelis M.H. Kastenmüller G. Köttgen A. Kronenberg F. et al.Human metabolic individuality in biomedical and pharmaceutical research.Nature. 2011; 477: 54-60Crossref PubMed Scopus (721) Google Scholar). However, the substrates of several MCTs have not been identified, and the physiological role has not been fully understood. Mammalian monocarboxylate transporter 7 (MCT7/SLC16A6) is an orphan transporter of the SLC16A family whose substrates and transport mechanisms are yet to be characterized. An ortholog of mammalian MCT7 in zebrafish (encoded by Slc16a6a) is reported to transport a ketone body, β-hydroxybutyrate, and the loss of gene function was shown to cause steatosis of the liver (9Hugo S.E. Cruz-Garcia L. Karanth S. Anderson R.M. Stainier D.Y.R. Schlegel A. A monocarboxylate transporter required for hepatocyte secretion of ketone bodies during fasting.Genes Dev. 2012; 26: 282-293Crossref PubMed Scopus (83) Google Scholar). Exogenous gene expression of human SLC16A6 in the liver, instead of Slc16a6a, enabled liver recovery from the steatosis. Based on this report, MCT7 is classified as a transporter for ketone bodies; however, the physiological substrates of mammalian MCT7 remain unclarified. In humans, MCT7 is primarily expressed in the liver, brain, and endocrine pancreas (10Jones R.S. Morris M.E. Monocarboxylate transporters: Therapeutic targets and prognostic factors in disease.Clin. Pharmacol. Ther. 2016; 100: 454-463Crossref PubMed Scopus (59) Google Scholar). In addition, intestine and colon are the expression sites of MCT7 in rodents (11Cedernaes J. Olszewski P.K. Almén M.S. Stephansson O. Levine A.S. Fredriksson R. Nylander O. Schiöth H.B. Comprehensive analysis of localization of 78 solute carrier genes throughout the subsections of the rat gastrointestinal tract.Biochem. Biophys. Res. Commun. 2011; 411: 702-707Crossref PubMed Scopus (15) Google Scholar). Interestingly, MCT7 is highly expressed in melanoma cells, and single nucleotide polymorphisms are related to cutaneous melanoma survival (12Dai W. Liu H. Chen K. Xu X. Qian D. Luo S. Amos C.I. Lee J.E. Li X. Nan H. Li C. Wei Q. Genetic variants in PDSS1 and SLC16A6 of the ketone body metabolic pathway predict cutaneous melanoma-specific survival.Mol. Carcinog. 2020; 59: 640-650Crossref PubMed Scopus (6) Google Scholar). Considering that ketogenesis occurs primarily in the mitochondria of hepatocytes, such wide expression of MCT7 implies multiple physiological roles in the body. Therefore, identification of mammalian MCT7 substrate(s) is crucial in understanding functionality. The purpose of this study was to identify MCT7 substrates and characterize transport mechanisms. Substrate identification focused on efflux profiles of endogenous amino acids from the MCT7-transfected cells. Taurine emerged as a novel substrate of MCT7. We then examined MCT7-dependent taurine transport characteristics and localization using HEK293T cells and polarized Caco-2 cells. Furthermore, MCT7 regulation by ancillary proteins CD147 and GP70 was also investigated. Since some MCTs recognize zwitterionic metabolites and amino acid derivatives (7Takahashi M. Kishimoto H. Shirasaka Y. Inoue K. Functional characterization of monocarboxylate transporter 12 (SLC16A12/MCT12) as a facilitative creatine transporter.Drug Metab. Pharmacokinet. 2020; 35: 281-287Crossref PubMed Scopus (11) Google Scholar, 8Suhre K. Shin S.Y. Petersen A.K. Mohney R.P. Meredith D. Wägele B. Altmaier E. Deloukas P. Erdmann J. Grundberg E. Hammond C.J. de Angelis M.H. Kastenmüller G. Köttgen A. Kronenberg F. et al.Human metabolic individuality in biomedical and pharmaceutical research.Nature. 2011; 477: 54-60Crossref PubMed Scopus (721) Google Scholar, 13Ramadan T. Camargo S.M.R. Summa V. Hunziker P. Chesnov S. Pos K.M. Verrey F. Basolateral aromatic amino acid transporter TAT1 (Slc16a10) functions as an efflux pathway.J. Cell Physiol. 2006; 206: 771-779Crossref PubMed Scopus (74) Google Scholar), we first examined efflux profiles of amino acids in HEK293T cells expressing enhanced green fluorescent protein (EGFP)-tagged MCT7. Efflux of endogenous amino acids was measured using Na+-free buffer to suppress the Na+-dependent reuptake activity. After more than 30 min of incubation, levels of alanine, asparagine, aspartic acid, proline, and taurine in MCT7-transfected cells were lower than mock-transfected cells (Fig. 1). In comparison to the other amino acids, only taurine content showed a significant decrease in MCT7-transfected cells, whereas not in mock-transfected cells. This efflux profile suggests that MCT7 is involved in the regulation of intracellular taurine levels. To clarify whether taurine is directly transported by MCT7, we performed uptake and efflux experiments using HEK293T cells expressing EGFP-tagged MCT7. For efflux experiments, [3H]taurine was loaded in the cells using a buffer containing Na+, and then, the cells were incubated in Na+-free buffer. The loaded [3H]taurine amount of MCT7-transfected cells (398 ± 18 μl/30 min/mg protein) was similar to levels in mock-transfected cells (371 ± 13 μl/30 min/mg protein). After incubation in Na+-free buffer for 120 min, [3H]taurine amount in the MCT7-transfected cells was reduced by 95% of the initial amount, whereas no changes were observed in the mock-transfected cells (Fig. 2A). Next, we examined whether MCT7 mediates taurine uptake into cells. In Na+-free buffer, [3H]taurine was taken up into the MCT7-transfected cells with time (Fig. 2B). The uptake in MCT7-transfected cells increased in a linear fashion for a duration of 30 min, whereas uptake in mock-transfected cells plateaued at 5 min. We also examined the ability of untagged MCT7 to transport taurine via uptake and efflux studies (Fig. S1). However, the transport activities of untagged MCT7 were lower than those of EGFP-tagged MCT7 (Fig. 2, A and B). Therefore, we used the EGFP-tagged transporter for evaluating the transport function of MCT7. Furthermore, the uptake and efflux of [3H]taurine by MCT7 did not change under acidic (pH 6.5) and alkaline pH (pH 8.5) buffer conditions (Fig. 2, C and D). The concentration dependence of the taurine uptake was examined, and we observed no saturation in concentrations between 1 and 270 mM (Fig. 2E). These data suggest that MCT7 can directly transport taurine as an ultra-low affinity substrate in a Na+- and pH-independent manner and mediate the influx and efflux of taurine. We also determined the intracellular and extracellular concentrations of taurine in MCT7- and mock-transfected cells at equilibrium (Fig. 2F). The intracellular taurine concentration was calculated, assuming that the intracellular volume is protein B. Functional of the with the in J. Physiol. PubMed Google Scholar). After of incubation, at the taurine concentration to the taurine concentration in MCT7-transfected cells was whereas that in mock-transfected cells was The in MCT7-transfected cells that the taurine concentration in the was with the extracellular concentration at that a of intracellular taurine is in such as mitochondria R. localization of taurine in types of the and J. 2000; PubMed Scopus Google Scholar). These data suggest that MCT7 mediates the of taurine. To whether ancillary proteins CD147 and GP70 MCT7 we performed efflux and uptake studies using HEK293T cells expressing EGFP-tagged MCT7 and the ancillary [3H]taurine in the cells MCT7 and ancillary protein with the MCT7-transfected cells (Fig. In with the [3H]taurine uptake via MCT7 was increased by and in with expression of CD147 and (Fig. Interestingly, CD147 and GP70 on taurine uptake was not observed in the mock-transfected min/mg cells min/mg and cells min/mg protein). Next, we examined the of MCT7 with ancillary proteins by Y. An with a PubMed Scopus Google Scholar). was in the cells MCT7 and ancillary proteins and but not in the cells and ancillary protein (Fig. The was also observed using the cells MCT7 and ancillary proteins and These data suggest MCT7 can be by the with ancillary proteins CD147 and To whether MCT7 a role in taurine we Caco-2 cells in EGFP-tagged MCT7 expression was by the We first to the [3H]taurine efflux of MCT7 in cells with only but we observed no (Fig. Next, we cells with is known to H. M. and characterization of the rat transporter in an in and rat 2012; Scopus Google Scholar, Y. for in Commun. 2016; PubMed Scopus (6) Google Scholar), in with a MCT7 expression levels in cells were increased the (Fig. and [3H]taurine efflux from the cells was than that from the cells (Fig. Next, we examined the of MCT7 expression on the taurine across the polarized cells. The transport of taurine was enhanced by induction of MCT7 whereas that of a of (Fig. the MCT7 was localized at basolateral membrane of cells (Fig. These data suggest that MCT7 may function as a taurine at basolateral membrane of polarized cells and contribute to taurine taurine transporter and H+-coupled amino acid transporter 1 are known to function as Na+- and H+-coupled taurine in the intestine A. V. Taurine uptake across the human membrane is via transporters: H+-coupled and Na+- and Physiol. PubMed Scopus Google Scholar). Therefore, we examined the of for these transporters on MCT7-mediated taurine uptake 1). We also the of other substrates and several amino acids on the uptake. For only acid inhibited MCT7-mediated taurine uptake of but and are substrates of did not the uptake. For amino acids, and the uptake to and an on the uptake. Interestingly, the of a prototypical was than that of lactate only In substrates of other MCTs, also a on the uptake. We also examined the of ketone bodies and on MCT7-mediated taurine uptake. showed a whereas showed no bodies were to be a substrate of of MCTs, on [3H]taurine uptake by of ± ± ± ± ± ± ± ± with the by with multiple ± ± ± with the by with multiple ± ± ± ± with the by with multiple ± with the by with multiple ± cells were transfected with of [3H]taurine by the cells was measured by Na+-free buffer for 30 min. The of transporters were used at mM for acid at 30 mM. MCT7-mediated uptake was by the uptake amount of mock-transfected cells from that of MCT7-transfected cells. the ± with the by with multiple in a new HEK293T cells were transfected with of [3H]taurine by the cells was measured by Na+-free buffer for 30 min. The of transporters were used at mM for acid at 30 mM. MCT7-mediated uptake was by the uptake amount of mock-transfected cells from that of MCT7-transfected cells. the ± Mammalian MCT7 is to be a ketone body whose function has not been in mammalian cells. We first observed that MCT7 mediates facilitative taurine transport in mammalian cells. Taurine is a involved in not only volume regulation but also in and that these functions on the of to those be important in the Taurine is by the membrane transport. Therefore, of a novel taurine transport mechanism may be related with the physiological Slc16a6a, an ortholog of transports in the has been in steatosis (9Hugo S.E. Cruz-Garcia L. Karanth S. Anderson R.M. Stainier D.Y.R. Schlegel A. A monocarboxylate transporter required for hepatocyte secretion of ketone bodies during fasting.Genes Dev. 2012; 26: 282-293Crossref PubMed Scopus (83) Google Scholar). In this we found that MCT7 transports taurine (Fig. 2, A and B). Taurine is known to decrease the of and and the of steatosis in the liver T. S.Y. Y. K. H. S. Taurine the secretion of and in PubMed Scopus Google Scholar, S. A. A. T. T. Taurine the of steatosis the of in a of liver in and in PubMed Scopus Google Scholar, M. The of taurine on steatosis by in zebrafish 2012; PubMed Scopus Google Scholar). However, whether MCT7-mediated taurine transport to the of steatosis. studies the between taurine transport and steatosis are Taurine is throughout the body and highly in such as concentrations are and to H. Taurine and and membrane of and in Scholar). Therefore, affinity transport is for an efflux of taurine from cells. In experiments, MCT7-mediated taurine transport was (Fig. 2E). The transport is for efflux of taurine from cells taurine is highly Taurine efflux from cells is known to be by of of family of proteins J. B. K. J. A.P. A. a plasma membrane is an of PubMed Scopus Google Scholar, F. J. K. D. N. M.A. T. of as an of the PubMed Scopus Google Scholar). The efflux of taurine via is by and under physiological taurine Furthermore, mediates efflux of not only taurine but other such as acid and aspartic acid H. A. L. C. V. A. F. M. M. R. of in J. 2016; PubMed Scopus Google Scholar). However, other to taurine efflux were not fully understood. study showed MCT7 mediates efflux of taurine from cells under (Fig. The MCT7-mediated taurine transport was also and to and (Fig. and 1). These transport characteristics of MCT7 to be similar to those of is not whether MCT7 is by However, taurine is taken up and in the cells as an under is from the cells under during recovery from the this the facilitative MCT7-mediated transport of taurine might be for the rapid recovery of the This is MCT7 can as a facilitative taurine transporter under Taurine is from intestine and into the The of and in the PubMed Google Scholar, K.S. of of taurine across rat Res. Scopus Google Scholar), membrane is due to In cells, and are known to contribute to taurine at the A. V. Taurine uptake across the human membrane is via transporters: H+-coupled and Na+- and Physiol. PubMed Scopus Google Scholar, M. N. R. and of taurine in The of and the of the amino acid in taurine Scopus Google Scholar). However, to the of the mechanism of taurine to the We showed that MCT7 enhanced taurine across Caco-2 cells and was localized at the basolateral membrane of the cells (Fig. and MCT7 is reported to be expressed in the intestine of rat (11Cedernaes J. Olszewski P.K. Almén M.S. Stephansson O. Levine A.S. Fredriksson R. Nylander O. Schiöth H.B. Comprehensive analysis of localization of 78 solute carrier genes throughout the subsections of the rat gastrointestinal tract.Biochem. Biophys. Res. Commun. 2011; 411: 702-707Crossref PubMed Scopus (15) Google Scholar). We also the expression (Fig. Considering facilitative transport MCT7 function in taurine uptake as as taurine efflux in cells. Taurine is and are involved in taurine in the to the intracellular of taurine in such that MCT7 taurine efflux than taurine uptake in Typical substrates of and and and did not MCT7-mediated taurine uptake 1). uptake was inhibited by monocarboxylates and and acidic amino acids and MCT7 expression enhanced the decrease of intracellular amino acids (Fig. 1). These data suggest that MCT7 has substrate for not only taurine but other acidic In this we did not the of by acidic metabolites acid, and to the affinity of MCT7 for taurine. we examined whether these are directly transported by MCT7 via uptake and efflux studies and found that acid and were transported by MCT7 (Fig. However, no were in the transport of between MCT7- and mock-transfected cells, the of taurine uptake by Therefore, MCT7 may function as of taurine at the of rat cells than a of However, in the human whether MCT7 can contribute to taurine are some the expression of MCT7 in the human intestine M. S. expression profiles of human solute carrier transporter Metab. Pharmacokinet. PubMed Scopus Google Scholar, A. H. H. gastrointestinal of the and at 34: PubMed Scopus Google Scholar). In of the human the of MCT7 was by and the expression was than that in the liver M. S. expression profiles of human solute carrier transporter Metab. Pharmacokinet. PubMed Scopus Google Scholar). analysis showed MCT7 expression in the cells of the but the expression in the of the was with that in the A. H. H. gastrointestinal of the and at 34: PubMed Scopus Google Scholar). on the expression of MCT7 in the human intestine is It is known that and MCT4 interact with with We showed MCT7 interactions with CD147 and GP70 using the (Fig. MCT7, to interact with GP70 than with as the regulation of MCT7 was shown (Fig. A and B). Furthermore, we observed localization of EGFP-tagged MCT7 ancillary proteins (Fig. in the MCT7 to be localized at the plasma membrane and a of the showed a the were by of ancillary is not the MCT7 The interactions between MCT7 and ancillary proteins the MCT7 proteins that showed in the of MCT7-mediated taurine transport. However, has whether these interactions under a physiological and are for the expression of MCT7. a study reported that MCT1 amino acids and for in the amino acid N. X. S. A. Y. Xu H. J. C. of human monocarboxylate transporter 1 by anti-cancer PubMed Scopus Google Scholar). The amino acids are prototypical MCTs and However, MCT7 the amino acid to the amino acids to and are It is reported that the of in in transport B. Q. Xu L. Li N. Y. S. X. J. Chen Y. D. N. X. F. J. S. transport mechanism of human monocarboxylate transporter Commun. 2020; PubMed Scopus Google Scholar). the pH-independent taurine transport of MCT7 may be by the amino acid required for in the MCT7 In summary, study findings that MCT7 is a novel facilitative taurine transporter in mammalian cells. MCT7 be a taurine at the plasma membrane of cells polarized expression of MCT7 is by the with ancillary protein CD147 and GP70 under in findings suggest a novel physiological role of MCT7 as a taurine transporter. and were from other were of Cell and were from and and were from and T. T. Y. protein in human cells by with 2016; PubMed Scopus Google Scholar). and were from and Y. An with a PubMed Scopus Google Scholar, Liu H. X. of new fluorescent protein for analysis under physiological 2006; PubMed Scopus Google Scholar). The was from rat using The of and was from the by The was into To a MCT7 expressing with the of was by and into HEK293T cells and Caco-2 cells were used to transport cells were in with at a concentration of and with amino acids Caco-2 cells were transfected with the of EGFP-tagged and by using and then in a containing to Caco-2 cells expressing MCT7 under the of the Caco-2 cells were used for transport The expression of EGFP-tagged MCT7 in the cells was by and by HEK293T cells were transfected with by using and for The cells were incubated in the Na+-free buffer of mM (pH mM mM mM mM mM mM mM mM and mM for The amino acid of the cells were by The cells were with and the were by The were a and the were used for The analysis was in using a with the was performed at a on an acid The was by using a The was of A mM buffer in and as A for to min, to A for to min, A for to min, and A for to min. The was at was used to the and to and efflux of taurine were measured using of cells in using Na+-free buffer buffer The were in a at The was and the cells were by buffer. For uptake Na+-free buffer containing [3H]taurine was to the cells. incubation for the was by and the cells were with Na+-free buffer. In the efflux [3H]taurine was loaded into cells using buffer for 30 min. After the cells were by Na+-free buffer and then incubated in Na+-free buffer for After incubation, the Na+-free buffer was The cells were in The was used for the of and protein was performed using was on The cells were in a for and then with and mM for to MCT7 The of [3H]taurine and across the cells was in an buffer (pH were in more than and the experiments were with In data are expressed as ± and were performed in between and were by analysis analysis of by for multiple was For these the was using the data are the This The that have no of with the of this K. H. and K. K. K. and R. T. K. H. and K. data K. H. K. R. and H. K. H. K. and K. and K. This was supported in by a from for and

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