Amino Acid Residues Involved in the Substrate Specificity of TauT/ SLC6A6 for Taurine and γ-Aminobutyric Acid

Tohru Yahara,a Masanori Tachikawa,a,b Shin-ichi Akanuma,a Yoshiyuki Kubo,a and Ken-ichi Hosoya*,a a Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama; Toyama 930–0194, Japan: and b Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University; Sendai 980–8578, Japan. Received December 25, 2013; accepted March 4, 2014

In the retina, taurine (2-aminoethanesulfonic acid) play a role in the protection of the retinal neural cells by acting as an osmolyte and antioxidant. [1][2][3][4][5] Recent progress in the research of membrane transporter and the blood-retinal barrier (BRB) has revealed the involvement of taurine transporter (TauT/ SLC6A6) in taurine uptake by the retinal capillary endothelial cells and retinal glial cells (Müller cells) contributing to the regulation of retinal osmolarity. 1,2,[6][7][8][9] TauT is expressed in a variety of tissues, such as the retina, brain, liver, placenta and kidney. 10,11) TauT transports taurine (K m =43 µM) in a Na + -and Cl − -dependent manner. TauT also exhibits the transport activity for β-alanine (K m =56 µM) and γ-aminobutyric acid (GABA) (K m =1.5 mM), and a contribution by TauT has also been suggested to the transport of β-alanine and GABA across the inner BRB. 5,[12][13][14] Studies involving gene knockout mice have shown that TauT controls the concentration of taurine in various tissues since TauT gene knockout mice exhibited severe defects, such as retinal degeneration. 15) The physiological importance of TauT is also supported by evidence that the expression of TauT is regulated by the extracellular taurine concentration and osmolarity. 2,[16][17][18][19] TauT belongs to the SLC6A family that includes Na + -and Cl − -dependent neurotransmitter transporters, 11,20) and it is known that members of SLC6A, such as noradrenaline transporter (NET/SLC6A2), dopamine transporter (DAT/SLC6A3), serotonin transporter (SERT/SLC6A4) and creatine transporter (CRT/SLC6A8), play important roles in the central nervous system. GABA transporters (GATs), including GAT1/SLC6A1, GAT2/SLC6A13, GAT3/SLC6A11 and BGT1/SLC6A12, are involved in the transport of GABA by coupling with Na + and Cl − , and exhibit a very similar amino acid sequence (>50%) to TauT. 11,21) However, TauT exhibits a substrate specificity differing from GATs, and has been described as "honorary" GAT since TauT has a much lower affinity for GABA than GATs (<80 µM). 14,22) Regarding the mechanisms, such as key amino acid residues, involved in their differences in substrate recognition, little is known apart from their well-characterized roles in physiology. Thus, it was our opinion that a study of the crystal structure of TauT and GATs would be helpful for investigating the mechanisms behind their differences in substrate specificity. However, detailed research into their crystal structure was expected to be difficult because of the difficulty in preparing sufficient quantities of mammalian membrane transporter proteins for three-dimensional crystal structure analysis. 23) In 2005, three-dimensional crystal structure of a bacterial homologue of SLC6A, LeuT Aa , was reported to be very useful for the study of SLC6A. 24) LeuT Aa is a bacterial Na +neurotransmitter symporter that transports L-leucine, and has twelve transmembrane (TM) domains. The crystal structure revealed that the substrate pocket of LeuT Aa is formed by TM1, 3, 6 and 8, that exhibit a high degree of similarity to putatively corresponding TM domains of SLC6A members. 24) In LeuT Aa , these TM domains include the twelve amino acid residues, Asn21, Ala22, Leu25, Gly26, Val104, Tyr108, Phe252, Phe253, Ser256, Phe259, Ser355 and Ile359, that contribute to the substrate recognition of LeuT Aa , 24) and the detailed study of LeuT Aa has contributed to updating the structure and functional residues of SLC6A. [25][26][27][28][29] Therefore, it would be helpful to use the information obtained in the study of LeuT Aa to investigate the mechanism for substrate recognition of TauT. In the present study, to investigate this mechanism, the amino acid resides of TauT were substituted with the corresponding residues of GATs, by referring to the functional residues of LeuT Aa , and the uptakes of taurine and GABA by TauT-expressing Xenopus laevis oocytes were determined. Site-Directed Mutagenesis Site-directed mutagenesis was carried out as described previously. 30) The four mutants of TauT (wild type; WT), Gly57Glu (G57E), Phe58Ile (F58I), Leu306Gln (L306Q) and Glu406Cys (E406C), were generated by using the full-length cDNA of TauT (GenBank accession number NM_017206) cloned into the plasmid vector pGEM-HEN (pGEM-HEN/TauT) as a template. The pGEM-HEN plasmid constructs carrying the mutated TauT-cDNA were prepared by polymerase chain reaction (PCR) with a KOD-Plus-Mutagenesis Kit (TOYOBO, Osaka, Japan) and specific primers ( Table 1). The sequences of mutants were confirmed by means of an ABI PRIMS Genetic Analyzer (Life Technologies).
Data Analysis Data analysis was carried out as described before. 32) In brief, the oocyte-to-medium (oocyte/medium) ratio was used to express the radio-labeled compound uptake by Xenopus laevis oocytes, using the Eq. 1: 3 3 oocyte/medium ratio For the kinetic study of [ 3 H] taurine and [ 3 H] GABA transport, uptake data were fitted to a model described by Eq. 2 using the nonlinear least-square regression analysis program, where S, V, V max and K m are the concentration of compound, the uptake rate of the compound for 60 min, the maximal uptake rate and Michaelis-Menten constant, respectively. Unless otherwise indicated, all data represent means± S.E.M. except for kinetic parameters. The data for the kinetic parameters represent means± S.D. For several groups, statistical significance of differences was determined by one-way analysis of variance followed by the modified Fisher's leastsignificant difference method. For two groups, the significance of differences was determined by an unpaired, two-tailed Student's t-test.
Immunohistochemical Analysis The frozen sections (20 µm in thickness) of TauT-expressing oocytes were prepared as described previously. 34,35) In brief, TauT-expressing oocytes were fixed with 4% paraformaldehyde in 10 mM phosphate buffer (8.1 mM Na 2 HPO 4 , 1.9 mM NaH 2 PO 4 , pH 7.2) for 3 h at room temperature, and immersed in 30% sucrose in ND96 solution. Oocytes were embedded in optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan), and cut with a cryostat (Model CM1900; Leica Microsystems, Wetzlar, Germany) and then mounted onto silanized glass slides (Muto Pure Chemicals, Tokyo, Japan). The mounted sections were washed with phosphate-buffered saline containing 0.3% Tween-20, and incubated with 10% goat serum (Nichirei, Tokyo, Japan) for 1 h at room temperature. Immunohistochemistry was performed as described previously, 28,29) and rabbit anti-rat TauT antibody (1 µg/mL) and Alexa 488-labeled goat anti-rabbit IgG antibody (1 : 5000) were used as primary and secondary antibodies, respectively. Vectashield mounting medium (Vector Laboratories, Burlingame, CA, U.S.A.) was used to mount sections on coverslips, and confocal microscopy was performed using a TCS-SP5 instrument (Leica Microsystems).

Multiple Alignment Analysis of TauT, GATs and LeuT Aa
To identify the amino acid residues involved in the substrate specificity of TauT differing from GATs, their amino acid alignments were compared with that of TM1, 3, 6 and 8 in LeuT Aa (Fig. 1). As a result, Gly57, Phe58, Leu61, Gly62, Leu134, Tyr138, Phe299, Phe300, Ala303, Leu306, Ser402 and Glu406 of TauT were shown to correspond to the twelve important residues of LeuT Aa . Furthermore, comparison of the sequences of TauT and BGT1 revealed the four different residues at positions corresponding to Asn21, Ala22, Phe259 and Ile359 of LeuT Aa (Fig. 1), suggesting that Gly57, Phe58, Leu306 and Glu406 of TauT and Glu66, Ile67, Gln313 and Cys413 of BGT1 are possible determinants of their differences in substrate specificity. In addition, multiple alignment analysis revealed that Gly57, Phe58, Leu306 and Glu406 are conserved between human, mouse, rat, dog and monkey orthologs of TauT (data not shown).
Expression Analysis of Mutated TauT Proteins According to the results obtained in the multiple alignments ( Fig. 1), Gly57, Phe58, Leu306 and Glu406 of TauT were suggested as possible residues causing the differences in substrate recognition from GATs. Thus, to investigate the contributions of these residues to the function of TauT, Gly-to-Glu, Phe-to-Ile, Leuto-Gln and Glu-to-Cys substitutions were performed at position 57, 58, 306 and 406 of TauT, respectively. In the confocal microscopy study with an anti-TauT antibody, the fluorescence  signal of TauT (WT, G57E, F58I, L306Q and E406C) proteins were detected at the membrane of oocytes, and there was no marked difference in their amounts of protein expression (Fig.  2). No signal was observed in water-injected oocytes.  (Fig. 3).

DISCUSSION
TauT (SLC6A6), a member of the SLC6A family, is a Na + -and Cl − -dependent taurine transporter, and its physiological relevance has been suggested in a number of in vivo and in vitro studies. [1][2][3][4]15) However, little is known about the mechanism underlying the differences in substrate specificity between TauT and GATs in term of their sequence similarity. 14,22) The study of LeuT Aa , a bacterial homologue of SLC6A, showed the role of amino acid residues in its function, 24) and the sequence analysis suggested that the four residues in TauT, Gly57, Phe58, Leu306 and Glu406, corresponded to Asn21, Ala22, Phe259 and Ile359 in LeuT Aa (Fig. 1), showing their possible roles in the substrate specificity of TauT. In this study, the amino acid substitution and functional analysis were performed to investigate the role of residues in the substrate recognition of TauT (Table 1).
The confocal microscopy suggested that the amino acid substitutions have no marked effect on the expression of TauT-mutant proteins since the specific signals for G57E, F58I, L306Q and E406C proteins were observed at the surface membrane of oocytes (Fig. 2). The [ 3 H] taurine uptake study with TauT-expressing oocytes suggested contributions of Gly57, Phe58, Leu306 and Glu406 to taurine transport by TauT because of the significantly reduced uptake of [ 3 H]taurine by G57E-, F58I-, L306Q-and E406C-expressing oocytes (Figs. 3, 4). The study of [ 3 H] GABA uptake suggested the contribution of Gly57 and Glu406 to GABA transport by TauT since G57E-and E406C-expressing oocytes exhibited significant changes in [ 3 H] GABA uptake (Figs. 3, 4).
G57E lost the activity of [ 3 H] taurine and [ 3 H] GABA uptake (Fig. 4), suggesting the role of Gly57 in the uptake function of TauT. It has been reported that TM1 of LeuT Aa binds to the substrate via the main chain oxygen of Asn21, and that its side chain acts as a volume determinant of the substrate pocket. 24) The model simulation for hGAT1 showed a hydrogen bonding between GABA and the main chain oxygen of Tyr60 that corresponds to the Asn21 of LeuT Aa . 26,27) Therefore, regarding G57E of TauT, the reduced uptakes were caused by the Glyto-Glu substitution that introduced a bulky side chain to the substrate pocket of TauT, suggesting the involvement of Gly57, putatively located in TM1, in providing an appropriate volume of the substrate pocket and in forming a hydrogen bond between the main chain oxygen atom and interacting with the amino group of taurine and the guanidino group of GABA.
The multiple alignments suggest that Phe58 corresponds to Ala22 of LeuT Aa (Fig. 1), the main chain oxygen of which has been reported to interact with the α-amino group of Lleucine. 24) The involvement of the side chain of Phe58 was also supported by the substrate recognition of TauT since the side chain of Ala77 in hDAT1, corresponding to the Ala22 of LeuT Aa , has been reported to be involved in the substrate recognition of hDAT1. 28,29) The kinetic parameters suggest the involvement of Phe58 in the GABA recognition by TauT since F58I showed a higher affinity (K m =103 µM) for GABA than that of WT (K m =564 µM) (Table 2, Fig. 5). The structure formulae suggest a bulkier molecular size of GABA than that of taurine, supporting the hypothesis that the Phe-to-Ile substitution reduced the bulkiness of the side chain, thereby improving the accessibility of GABA to the substrate pocket. Thus, it is suggested that Phe58, putatively located in TM1 of TauT, is involved in the accessibility of the substrate to the substrate pocket. Furthermore, Ala22 of LeuT Aa has been reported to   be a residue forming the Na + -binding site that is essential for the function of LeuT Aa , 24,37) and the insignificant change in [ 3 H] GABA uptake by F58I strongly suggests a minor contribution of Phe58 in the Na + -binding of TauT (Figs. 3D, 4B). In TM6 of LeuT Aa , the side chain of Phe259 has been reported to be involved in a hydrophobic interaction with the isopropyl-group of L-leucine, 24,29) and the multiple alignments suggest that Phe259 of LeuT Aa corresponds to Leu306 of TauT ( Fig. 1), implying a role of the side chain of Leu306 in the substrate recognition of TauT. In actual fact, the uptake study suggests that Leu306 contributes to the taurine transport of TauT since L306Q showed a significant reduction in [ 3 H] taurine uptake by 97% while there was no significant effect on [ 3 H] GABA uptake (Fig. 4). This is also supported by the transport clearance (V max /K m ) of L306Q for taurine that is 60.5-fold lower than that of WT ( Table 2). The kinetic parameters revealed the contribution of Leu306 to the substrate pocket of TauT since the Leu-to-Gln substitution showed lower and higher affinities, respectively, for taurine (K m =41.6 µM) and GABA (K m =22.6 µM) than those of WT ( Table 2, Fig. 5). Leu has a bulkier side chain than that of Gln, and the Leu-to-Gln substitution caused the increased and decreased affinities for GABA and taurine, respectively, showing a minor contribution of the side chain bulkiness at position 306. The structural difference, the branched-side chain of Leu and the straight-side chain of Gln, suggests that the branchedside chain of Leu306 conformationally determines the volume of the substrate pocket of TauT.
Glu406 of TauT is suggested to correspond to Ile359 localized in TM8 of LeuT Aa (Fig. 1), and the side chain of Ile359 in LeuT Aa has been reported to form a hydrophobic interaction with L-leucine, 24) implying the involvement of Glu406 in the substrate recognition of TauT (Fig. 4). This is also supported by the 3.5fold lower and 3.6-fold higher transport clearance (V max /K m ) values of E406C for taurine and GABA, respectively, than those of WT ( Table 2). The kinetic parameters suggested the involvement of Glu406 in the substrate recognition of TauT since E406C showed lower and higher affinities for taurine and GABA, respectively, than those of WT (Table 2, Fig. 5). According to the previous reports, the polar side chain of Glu406 was suggested to make only a minor contribution to the substrate recognition of TauT since the polar side chain of Thr400 in hGAT1, corresponding to Glu406 of TauT, has been reported to play only a minor role in the function of hGAT1. 38,39) Thus, the higher affinity for GABA supports the involvement of Glu406 in determining the volume of the substrate pocket since it is thought that the Glu-to-Cys substitution at position 406 reduced the side chain bulkiness to improve the accessibility of GABA to the substrate pocket. These findings support the hypothesis that the lower affinity of E406C for taurine was caused by an increased volume of the substrate pocket, that weakened the interaction between taurine and the putative TM8 of TauT.
Furthermore, E406C exhibited the highest uptake activity and transport clearance (V max /K m ) for GABA (Table 2), implying that Glu406 is the most important residue among the four residues examined here. The inhibitory effect study revealed differences in transport between E406C and WT. Regarding [ 3 H] taurine transport, E406C showed its GATs-like transport property since the Glu-to-Cys substitution exhibited a lower  sensitivity to taurine and β-alanine, and a higher sensitivity to GABA and nipecotic acid than WT (Table 3). These results suggest the involvement of the amino acid residue at position 406 in determining the substrate specificity of TauT, and this is also supported by the alteration of sensitivities to taurine, β-alanine, GABA and nipecotic acid observed in the transport of [ 3 H] GABA by E406C (Table 4). Although amino acid substitution was performed by referring the sequence of rBGT1, the Glu-to-Cys substitution caused no change in the sensitivity of TauT to betaine (Tables 3, 4). Thus, the minor contribution of Cys413 of rBGT1, corresponding to Glu406 of TauT, was suggested for the recognition of betaine, and this is also supported by the conserved Cys between GATs (Fig. 1).
In conclusion, the sequence analysis showed that Gly57, Phe58, Leu306 and Glu406 of TauT are candidate residues determining the substrate specificity of TauT. The uptake study with TauT-mutants suggested their contribution to the substrate recognition ability of TauT. In particular, it was suggested that Phe58 and Leu306 are involved in the substrate accessibility and recognition of TauT, respectively, and that Glu406 is involved in the substrate specificity of TauT differing from that of GATs. These findings provide helpful information to increase our understanding of the transport function and role of TauT in the body.