Impact of SLC6A Transporters in Physiological Taurine Transport at the Blood–Retinal Barrier and in the Liver

Abstract

Cumulative studies showed that taurine (2-aminoethanesulfonic acid) contributes to a variety of physiological events. Transport study suggested the cellular taurine transport in an Na⁺- and Cl⁻-dependent manner, and the several members of SLC6A family have been shown as taurine transporter. At the inner blood–retinal barrier (BRB), taurine transporter (TauT/SLC6A) is involved in the transport of taurine to the retina from the circulating blood. The involvement of TauT is also suggested in γ-aminobutyric acid (GABA) transport at the inner BRB, and its role is assumed in the elimination of GABA from the retinal interstitial fluid. In the retina, taurine is thought to be a major organic osmolyte, and its influx and efflux through TauT and volume-sensitive organic osmolyte and anion channel (VSOAC) in Müller cells regulate the osmolarity in the retinal microenvironment to maintain a healthy retina. In the liver, hepatocytes take up taurine via GABA transporter 2 (GAT2/SLC6A13, the orthologue of mouse GAT3) expressed at the sinusoidal membrane of periportal hepatocytes, contributing to the metabolism of bile acid. Site-directed mutagenesis study suggests amino acid residues that are crucial in the recognition of substrates by GATs and TauT. The evidence suggests the physiological impact of taurine transporters in tissues.

1. INTRODUCTION

Taurine, also known as 2-aminoethanesulfonic acid, is a physiologically abundant β-amino acid in the human body, and cumulative studies showed its involvement in various physiological events, such as neuroprotection and osmotic regulation.^1–3)

Molecular identification and transport study have shown the cellular transport of taurine mediated by Na⁺- and Cl⁻-dependent transporters, such as taurine transporter (TauT/SLC6A6) and mouse γ-aminobutyric acid (GABA) transporter 3 (GAT3/SLC6A13, the orthologue of rat GAT2), that have high (K_m=43 µM)⁴⁾ and moderate affinity (K_m=540 µM),⁵⁾ respectively (Table 1). A recent report has also shown that proton-coupled amino acid transporter 1 (PAT1/SLC36A1) is assumed to transport taurine with a low affinity (K_m=7.5 mM).⁶⁾ In particular, a functional study revealed that TauT/SLC6A6 transports β-amino acids, such as taurine and β-alanine (β-Ala),⁴⁾ and research in TauT gene knockout mice suggested the critical role of TauT in taurine distribution in mammals since the knockout mice exhibited a reduction in fertility and taurine concentrations in various tissues.⁷⁾

Table 1. Taurine Transporters in SLC6A Family

Gene name	Species	Alias	Km (µM)
			Taurine	GABA
SLC6A1	Human	GAT1		10*
	Rat	GAT1		7.0
	Mouse	GAT1		13
SLC6A6	Human	TauT	6.9
	Rat	TauT	43	1500
	Mouse	TauT	4.5
SLC6A11	Human	GAT3		10*
	Rat	GAT3		2.3–12
	Mouse	GAT4	1400	0.8–12
SLC6A12	Human	BGT1		26*
	Rat	BGT1		300
	Mouse	GAT2		79
SLC6A13	Human	GAT2		8.24–11*
	Rat	GAT2	594	8
	Mouse	GAT3	540	18

K_m values estimated for taurine and GABA transporters in SLC6A were referred from previous reports. Asterisk indicates values calculated as IC₅₀. The table was prepared by reference to Smith et al.,⁴⁾ Liu et al.,⁵⁾ Anderson et al.,⁶⁾ Tomi et al.,¹⁰⁾ Guastella et al.,³²⁾ Borden et al.,³⁴⁾ Lopez-Corcuera et al.,³⁵⁾ Ikeda et al.,⁶¹⁾ Liu et al.,⁸¹⁾ Christiansen et al.,⁸²⁾ Thomsen et al.,⁸³⁾ Al-Khawaja et al.,⁸⁴⁾ Burnham et al.⁸⁵⁾

In the structural and functional appearance of transporters, TauT is classified as a member of SLC6A family, and its representative members are Na⁺- and Cl⁻-dependent neurotransmitter transporters,^8,9) such as noradrenaline transporter (NET/SLC6A2), dopamine transporter (DAT/SLC6A3), serotonin transporter (SERT/SLC6A4) and creatine transporter (CRT/SLC6A8), which are involved in neural transmission in the central nervous system. SLC6A family also includes four GABA transporters (GATs), and it was reported that GAT1 (SLC6A1), GAT2 (SLC6A13), GAT3 (SLC6A11) and betaine/GABA transporter 1 (BGT1/SLC6A12) transport GABA by coupling with Na⁺ and Cl⁻ ions (Table 1). Interestingly, in the analysis of sequences, rat GAT3 (SLC6A11) and rat GAT2 (SLC6A13) were found to have amino acid alignments similar to that of TauT, with much less affinitive for GABA, and this evidence supports the concept of TauT as an “honorary GABA transporter.”^10,11)

This review focuses on the contribution of SLC6A family members, such as TauT and GATs, to the physiological transport of taurine and GABA, referring to recent achievement in the blood–retinal barrier (BRB) and liver.

2. TAURINE TRANSPORT ACROSS THE BRB

The retina is a neural tissue and essential for the vision sense in mammals, including humans. In the retina, the key role of the BRB has been proposed in the homeostatic regulation of low molecular-weight molecules, such as nutrient supply to the retina from the circulating blood and metabolite elimination to the circulating blood from the retina, making an important contribution to maintaining retinal function. In the retina, two barrier structures are known as the inner BRB and outer BRB, which are formed by the retinal capillary endothelial cells and retinal pigment epithelial cells, respectively. Non-specific transport between the circulating blood and neural retina is restricted by the tight junction formed at the inner and outer BRB, and the membrane transporters expressed in these responsible cells are suggested to contribute to selective transport between the circulating blood and neural retina.^12–15)

Biochemical studies showed that taurine is the most abundant free amino acid in the retina (12 mM in rats), and its retinal concentration is much greater than its concentration in the serum (100–300 µM)^16–19) (Fig. 1). Neurochemical studies suggested that taurine has a neuroprotective function, which is achieved by its action as an antioxidant and osmolyte,^1–3) and this is also supported by the retinal dysfunction resulting from a lack of taurine in humans and by severe retinal degeneration in TauT gene knockout mice.^7,20) Interestingly, during the abundance of taurine in the retina, low activity of the rate-limiting enzyme in taurine biosynthesis was reported in the retina,²¹⁾ showing a possible story that the regulation of the retinal taurine concentration is carried out through the transport system for taurine at the BRB. Previously, Törnquist and Alm evaluated taurine transport to the retina from the circulating blood using in vivo analysis, the retinal uptake index (RUI), and suggested the involvement of a selective taurine transport system at the BRB, since the in vivo uptake of [³H]taurine was decreased in the presence of excess amounts of taurine and β-Ala when there was no change by L-phenylalanine (L-Phe) and L-glutamate.²²⁾ RUI is estimated in the experiment of short-term replacement of the blood with a buffer containing [¹⁴C]taurine, and it was markedly reduced in the presence of taurine (1, 10 mM), while it was moderately reduced in the presence of rat serum,²²⁾ suggesting that taurine transport at the BRB is not up to saturation in the presence of serum taurine (100–300 µM). Subsequently, the selective taurine transport system was reinforced by an immunohistochemical trial showing that TauT is expressed at the outer BRB, suggesting the involvement of TauT in taurine transport at the outer BRB.²³⁾

Fig. 1. TauT (SLC6A6) Is Involved in Taurine and GABA Transport across the BRB

TauT is expressed in the retinal capillary endothelial cells and retinal pigment endothelial cells, and contributes to taurine transport at the inner and outer BRB. Furthermore, TauT is assumed to contribute to GABA efflux at the inner BRB.

Furthermore, it is noteworthy that two-thirds of the retina is nourished via the inner BRB,^12,15,24) and this suggests that transport study at the inner BRB is of substantial importance to develop an understanding of taurine regulation in the retina. In in vivo transport study using integration plot, the apparent influx clearance of [³H]taurine [K_{in, retina}=259 µL/(min·g retina)] was shown to be much greater than that of nonpermeable paracellular transport markers [0.26–0.75 µL/(min·g retina)],^25,26) suggesting a carrier-mediated process in taurine transport rather than passive diffusion. These K_{in, retina} values strongly supported the uphill transport of taurine against a concentration gradient from the circulating blood to the retina across the inner and outer BRB because the retinal taurine concentration (12 mM) is much greater than that of the serum (100–300 µM).^17,19,22)

The detailed property of taurine transport at the inner BRB was assessed using TR-iBRB2 cells, an in vitro model cell line derived from rat retinal capillary endothelial cells,^14,15) and the results suggested carrier-mediated taurine transport at the inner BRB, since [³H]taurine uptake was shown in an Na⁺-, Cl⁻-, and concentration-dependent manner with a K_m value of 22.2 µM, which is similar to K_m values (6.5–8.9 µM) previously estimated for the outer BRB.^23,26,27) Considering the much higher serum concentration of taurine (100–300 µM) in rats,^19,22) these K_m values imply more than 80% saturation of taurine transport by endogenous plasma taurine, which in turn indicates a continuous taurine supply from the circulating blood to the retina through the taurine transport system at the inner BRB.

Furthermore, the estimated K_m value was similar to that reported for TauT-expressing COS cells (K_m=43 µM),⁴⁾ suggesting the involvement of TauT in the uptake of taurine at the inner BRB (Fig. 1). The involvement of TauT is supported by the substrate specificity observed in TR-iBRB2 cells, since [³H]taurine uptake was markedly inhibited by typical TauT substrates, such as β-Ala and hypotaurine, with weaker alteration shown by GABA and α-amino acids.^4,26) Such substrate specificity is nicely consistent with the in vivo inhibition profile,²²⁾ and the protein expression of TauT was suggested for the retinal capillary endothelial cells,²⁶⁾ reinforcing the story that TauT plays a role in the blood-to-retina transport of taurine at the BRB. Although the localization of TauT has not been clarified in retinal capillary endothelial cells, it is inferred to be localized at both the luminal and abluminal membranes (Fig. 1), since the studies on the blood–brain barrier (BBB) showed Na⁺- and Cl⁻-dependent taurine transport in the membranes of primary-cultured bovine brain capillary endothelial cells (BCECs).^28,29) This is also supported by the functional features of taurine transport at the inner BRB. In addition, an expression study in TR-iBRB2 cells pretreated with 10 mM taurine suggested that TauT at the inner BRB undergoes transcriptional suppression induced by high concentrations of taurine,²⁶⁾ and similar suppression was also reported for taurine transport at the BBB.²⁹⁾ Thus, at the inner BRB, a high retinal concentration of taurine normally suppresses the expression of TauT, and its expression is induced in the case of insufficient taurine concentration in the retina, showing a possible story that this transcriptional mechanism is essential for keeping the optimal milieu in the retina.

3. INVOLVEMENT OF TAUT IN GABA TRANSPORT AT THE INNER BRB

GABA is an inhibitory neurotransmitter, and its pharmacological action in the synaptic cleft is terminated by the reuptake of GABA mediated by GABA transporters expressed in either neurons or glial cells.³⁰⁾ In SLC6A family, GATs are well known to recognize GABA as a substrate, and a recently reported study utilizing three-dimensional X-ray crystallography of an SLC6A homologue in bacteria showed the functional importance of twelve amino acids located at transmembrane domains forming the substrate binding of SLC6A members.³¹⁾ Furthermore, a comparison of amino acid sequences for rat SLC6A orthologues to determine the close relation of TauT to rat GAT2 and rat GAT3 found that only three of the twelve amino acids in rat TauT differ from those in rat GAT2, rat GAT3 and rat BGT1¹¹⁾ (Fig. 2). Therefore, the crucial role of these three amino acids are suggested by the substrate specificity of TauT, and it was implied that TauT may recognize GABA as a substrate during no enhanced uptake of GABA exhibited by TauT-expressing cells in an earlier study.⁴⁾

Fig. 2. Crucial Amino Acid Residues Reside in TauT

The substrate-interacting residues of LeuT_Aa are indicated by filled circles. Conserved residues between TauT and BGT1 are indicated in grey boxes, and those differing between TauT and rBGT1 are indicated in black boxes. Residues involved in Na⁺ coordination were indicated by the open squares. The figure was prepared with reference to Yahara et al.⁷⁹⁾

In an in vitro uptake study of [³H]GABA by TR-iBRB2 cells, the Na⁺-, Cl⁻-, and concentration-dependent transport of GABA was suggested at the inner BRB, and the estimated K_m value (2.0 mM) was similar to that observed in GABA transport by rat TauT-transfected cells (K_m=1.5 mM), although the value was much higher than those reported for GATs.^10,32–35) The involvement of TauT in GABA transport at the inner BRB was supported by the inhibition profile exhibited by TR-iBRB2 cells, which was consistent with that by rat TauT-transfected cells, since the typical substrates of TauT, such as taurine and β-Ala, had marked inhibitory effects on [³H]GABA uptake, while nipecotic acid showed much less inhibitory effect than previously reported for mouse GAT1, mouse GAT3 and mouse GAT4.^5,10) Furthermore, the competitive inhibition of [³H]GABA and [³H]taurine uptake was exhibited in the presence of taurine and GABA with K_i values of 74 µM and 1.8 mM, respectively, which were similar to the K_m values for taurine (22 µM) and GABA uptake (2.0 mM) by TR-iBRB2 cells, respectively.^10,26) The contribution of TauT to the blood-to-retina transport of GABA across the inner BRB would be minor since TauT is assumed to be saturated with taurine and β-Ala in the circulating blood. Therefore, these in vitro findings revealed that TauT plays another role at the inner BRB, and it is a possible story that an “honorary GABA transporter” is involved in GABA elimination (retina-to-blood transport) from retinal interstitial fluid after GABA releasing³⁶⁾ (Fig. 1).

4. ROLE OF TAURINE TRANSPORT IN CELL VOLUME REGULATION IN THE RETINA

In the neural retina, the release and recovery of various neurotransmitters take place, and the volume of retinal cells is constantly altered due to continuing fluctuations in osmolarity. In particular, cell volume fluctuations have been shown to be associated with severe retinal disorders, including macular edema, neurodegeneration and diabetic retinopathy,³⁷⁾ and the cumulative evidence suggests the importance of cell volume regulation in response to fluctuations in osmolarity in the retina. Earlier studies showed that the volume of cells is controlled through the loss or gain of osmolyte, such as inorganic ions.^38,39) However, it is notable that even slight alterations in cellular inorganic ion levels can lead to changes in membrane potential, the rate of enzymatic reactions and membrane transport.⁴⁰⁾ This suggests the importance of organic molecules in cell volume regulation because of their compatibility or non-perturbation, and taurine has been assumed to be an ideal organic osmolyte in cell volume regulation since taurine is at high concentrations in the retina.³⁷⁾

Exactly, the study in TR-iBRB2 cells suggests that the induction of TauT expression leads to the accumulation of taurine to achieve osmotic equilibrium in the retinal capillary endothelial cells because the induction of [³H]taurine uptake occurs with increased TauT mRNA expression under hypertonic conditions.^26,41) Progress in transcriptional regulation research suggests that the induction of taurine transport involves the tonicity-responsive element (TonE)/TonE-binding protein (TonEBP) pathway that osmosensitively governs the transcription of TauT.⁴²⁾ On the other hand, in response to hypotonic stress, osmolyte extrusion recovers cell volume, and studies in TR-iBRB2 cells suggests that the volume of the retinal capillary endothelial cells is regulated by volume-sensitive organic osmolyte and anion channel (VSOAC) that releases taurine in response to hypoosmotic stress.^41,43) Subsequently, studies under hypotonic conditions suggested the induction of taurine transport of the retinal capillary endothelial cells in the presence of sphingosine-1-phosphate (S1P), a ligand of G protein-coupled receptors (GPCRs),⁴¹⁾ and the induction of volume-sensitive taurine release by retinal capillary endothelial cells through GPCRs is also consistent with a previous report on GPCRs associated with osmolarity regulation.⁴⁴⁾

In the retina, nerve activity is constant, and the various types of neurotransmitters released cause osmolarity fluctuations in the retinal microenvironment, such as the retinal extracellular fluid (ECF). Müller cells are the glial cells in the retina and assumed to have a considerable role in osmolarity regulation of the retinal microenvironment, since they contribute to the homeostasis of low molecular-weight molecules to protect the retinal neurons from excitotoxicity.⁴⁵⁾ Earlier studies showed the transport activity of taurine, the expression of taurine transporters, such as TAUT, GAT1 and GAT3, and high accumulation of taurine in Müller cells,^27,46–48) implying the key role of Müller cells in regulating osmolarity at the retinal ECF. Studies using an in vitro Müller cell model, a conditionally immortalized rat Müller cell line (TR-MUL5 cells),^49,50) suggested that Müller cells are involved in carrier-mediated taurine transport in an Na⁺- and Cl⁻-dependent manner, with a K_m value estimated to be 37.9 µM, similar to that of TauT.^4,26,50) In addition, TauT expression detected in primary cultured Müller cells and TR-NUL5 cells⁵⁰⁾ supports the involvement of TauT in taurine uptake by Müller cells (Fig. 1), and Müller cells are assumed to be involved in the TonE/TonEBP pathway that induces TauT-mediated taurine uptake in response to hyperosmotic stress⁴²⁾ (Fig. 3).

Fig. 3. Taurine Transport in Müller Cells

In Müller cells, TauT contributes to cell volume regulation under isotonic and hypertonic conditions. In response to hypotonic conditions, the volume-sensitive release of taurine is mediated by VSOAC.

Similar to the retinal capillary endothelial cells,⁴¹⁾ further study suggested the induction of a volume-sensitive taurine efflux system in response to hypoosmotic stress (Fig. 3), since greater taurine efflux was exhibited by TR-MUL5 cells under hypotonic conditions than under isotonic conditions.⁵⁰⁾ As reported for the retina capillary endothelial cells,⁴¹⁾ VSOAC is suggested to be responsible for the volume-sensitive release of taurine by Müller cells in response to hypoosmotic stress, since taurine efflux by TR-MUL5 cells was decreased in the presence of 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB), a selective inhibitor of VSOAC,⁵¹⁾ under hypotonic conditions. In addition, taurine release from Müller cells is also suggested to be enhanced through G protein-coupled receptor (GPCR) activated by S1P under hypoosmotic stress,^41,50) supporting the volume-sensitive taurine release by VSOAC, the activity of which is enhanced through GPCRs (Fig. 3). Based on the knowledge of cell signaling, the enhanced efflux of taurine under hypotonic conditions seems to be achieved through a phospholipase C (PLC)-dependent mechanism since S1P increases the intracellular Ca²⁺ content in Müller cells.⁵¹⁾

5. HEPATIC TAURINE UPTAKE MEDIATED BY GAT2

Bile salts have a crucial role in the intestinal absorption of lipid-soluble vitamins and fats, and it is known that taurine is essential for the hepatic synthesis of bile salts. In the liver, the pericentral and periportal lobular periphery hepatocytes are known to be sites where taurine synthesis from cysteine (Cys) and the conjugation of taurine with bile acids preferentially take place, respectively.^52,53) These events were supported by the higher expression of bile acid-CoA-amino acid N-acyltransferase (BAAT) in periportal hepatocytes and the faster Cys incorporation into taurine in pericentral hepatocytes,^53,54) clearly suggesting the heterogeneity of hepatocytes. Immunohistochemistry using taurine antiserum demonstrated that periportal hepatocytes contain abundant taurine and taurine-conjugated bile acids,⁵⁵⁾ while taurine synthesis is largely carried out in pericentral hepatocytes.⁵³⁾ These findings revealed a considerable discrepancy between sites for the synthesis and conjugation of taurine, and it was hypothesized that this could be explained by clarifying the taurine transport system in hepatocytes. Although earlier trials implied the involvement of TauT in Na⁺- and concentration-dependent taurine uptake by hepatocytes,^56,57) the lack of TauT gene resulted in only a 30% reduction in the taurine content of liver parenchymal cells during the marked depletion of taurine shown by nonparenchymal liver cells,⁵⁸⁾ suggesting a minor role of TauT in taurine uptake by hepatocytes.

Studies using rat hepatic plasma membrane vesicles showed Na⁺-dependent taurine uptake, of which the K_m value (174–380 µM) was similar to the K_m value of the mouse orthologue of GAT2 (540 µM, mouse GAT3),^5,59,60) and it is a conceivable that GATs on the sinusoidal membrane of periportal hepatocytes contribute to taurine uptake, followed by conjugation. In addition, the uptake study in isolated rat hepatocytes suggested Na⁺- and Cl⁻-dependent taurine uptake by hepatocytes, and the estimated a K_m value (594 µM) was similar to that of mouse GAT3-mediated taurine transport.^5,61) In rat hepatocytes, the uptake of taurine was inhibited by GABA with an IC₅₀ value (95.0 µM) similar to the K_m value for GABA (22.5 µM) shown by hepatocytes, but not to the value (1.5 mM) shown by TauT.^10,62) A decrease in taurine uptake by rat hepatocytes was demonstrated with decreasing mRNA expression of rat GAT2,⁶¹⁾ and the involvement of rat GAT2 in the hepatic uptake of taurine from the circulating blood is reinforced by in vitro inhibition results that were basically consistent with the in vivo analysis showing the reduction of taurine uptake by guanidinoacetic acid (GAA), nipecotic acid, β-Ala and GABA, but not by L-Ala.⁶¹⁾ The estimation of kinetic parameters underlines the responsibility of rat GAT2 for the carrier-mediated uptake of taurine by hepatocytes, since the saturable component showed uptake clearance of 0.387 µL·min⁻¹·mg protein⁻¹, which corresponds to approximately 50% of the total uptake clearance⁶¹⁾ (Fig. 4A).

Fig. 4. GAT2 in Taurine Transport by Hepatocytes

(A) Schematic diagram of taurine transport in hepatocytes. (B) Preferential distribution of GAT2 in periportal hepatocytes. GAT2 (green) and nuclei (blue) were analyzed in cryosections of rat liver. Single (*) and double (**) asterisks indicate the central vein and portal spaces, respectively. CV, central vein; PV, portal vein; HA, hepatic artery; BD, bile duct. Scale bars: 50 µm.

Previously, rat GAT2 was reported to be involved in the uptake of GAA, γ-butyrobetaine, β-Ala and GABA by hepatocytes, with K_m values of 134, 9.3, 35.3, and 22.5 µM, respectively.^62–64) Because of the blood concentrations of GAA (3.73 µM), γ-butyrobetaine (0.84–6 µM), β-Ala (<0.5 µM), GABA (0.13 µM) and taurine (130 µM),^22,65–69) the K_m values indicate that GAT2-mediated transport in hepatocytes is not completely saturated under physiological conditions, and the uptake of taurine by rat GAT2 seems to depend on the blood concentration of taurine, revealing the possible endorsement that taurine and its conjugated bile acids are increased by the systemic administration of taurine.^70,71) Immunohistochemistry showed that rat GAT2 is preferentially localized on the sinusoidal membrane of the periportal region (Fig. 4B), and such protein localization was also reported for BAAT,⁵⁴⁾ suggesting that rat GAT2 is coupled with BAAT to become a key determinant of bile acid conjugation in periportal hepatocytes. Furthermore, it is known that the intestinal bacteria deconjugate more than 30% of the total bile salt on a daily basis,⁷²⁾ and it seems reasonable to assume that the reconjugation catalyzed by BAAT requires uptake systems for unconjugated bile acid in periportal hepatocytes. Molecular identification and functional studies showed the transport of the unconjugated form of bile acid by bile acid transporters expressed at the sinusoidal membrane.^73–76) The reconjugation of taurine and de novo synthesis of taurine-conjugated bile salts have recently been suggested to occur through the coupling of oatp1a1 (Slco1a1), Ntcp (Slc10a1) and GAT2 in periportal hepatocytes, because of their acinar distribution and pericentral-dominant expression of oatp1b2 (Slco1b2) and oatp1a4 (Slco1a4).^74–76)

6. STRUCTURAL BASIS OF TAURINE AND GABA TRANSPORT BY TAUT

Among Na⁺- and Cl⁻-dependent transporters belonging to SLC6A family, TauT is reported to have a lower affinity (1.5 mM) for GABA than GATs (<80 µM),^10,11) while the amino acid sequence of GATs is similar to that of TauT,^77,78) and the molecular basis of this different affinity has been an important topic in understanding the substrate specificity of SLC6A family members. The crystal structure of LeuTAa, a bacterial SLC6A homologue, suggested the crucial role of three amino acids of TauT, i.e., Gly57, Phe58 and Glu406, in recognizing substrates, and multiple-alignment analysis suggested the significance of Leu306 of TauT.^11,31) These four amino acids, Gly57, Phe58, Leu306 and Glu406, correspond to Asn21, Ala22, Phe259 and Ile359 of LeuTAa, respectively (Fig. 2), and are conserved among the human, monkey, dog, rat and mouse orthologues, supporting the importance of their role.

The role of their function was accessed by amino acid substitution using TauT-expressing Xenopus laevis oocytes, and their involvements in taurine transport by TauT was suggested, since G57E-, F58I-, L306Q- and E406C-expressing oocytes exhibited the lower taurine uptake activity of taurine than that of wild type-expressing oocytes and these mutants showed protein expression comparable to that of the wild type.⁷⁹⁾ Gly57 corresponds to Asn21 on transmembrane region 1 (TM1) of LeuTAa, and the findings in the study of LeuTAa suggested that Gly57, putatively located in the putative TM1 of TauT, is involved in the formation of optimal pocket volume for the substrate pocket, where the main side chain oxygen of Gly57 interacts with GABA via hydrogen bond interaction, since G57E exhibited the marked loss of uptake activities for GABA and taurine.^31,79) On the putative TM1 of TauT, Phe58 corresponds to Ala22 of LeuTAa,³¹⁾ and is suggested to be a determinant in GABA recognition by TauT since F58I exhibited higher affinity for GABA than that of the wild type. In particular, considering that the molecular size of GABA is bulkier than that of taurine, the role of Phe58 is assumed in substrate accessibility to the pocket, since the bulkiness reduction caused by the Phe-to-Ile substitution improves the accessibility of GABA. On the putative TM9 of TauT, Leu306 corresponds to Phe259 of LeuTAa,³¹⁾ and L306Q showed a marked decrease in taurine uptake with much lower transport clearance (V_max/K_m), with no effect on the uptake of GABA,⁷⁹⁾ suggesting the contribution of Leu306 to the taurine transport of TauT. The kinetic parameters showed that the Leu-to-Gln substitution of Leu306 leads to lower affinity for taurine and higher affinity for GABA than those of the wild type,⁷⁹⁾ and the volume of the substrate pocket of TauT is suggested to be conformationally determined by the branched side chain of Leu306 when the structural difference between the straight chain (Gln) and branched chain (Leu) is considered (Table 2).

Table 2. Kinetic Parameters Estimated for TauT Mutants

Substrates	Parameters	Mutants
		Wild-type	G57E	F58I	L306Q	E406C
Taurine	Km (µM)	25.9±8.1	N.D.	27.4±3.2	41.6±13.5	82.4±47.3
	Vmax (pmol/(h·oocyte))	19.8±25	N.D.	115±9	5.27±1.23	183±56
	Vmax/Km (µL/(h·oocyte))	7.68	N.D.	4.21	0.127	2.22
GABA	Km (µM)	564±62	N.D.	103±9	22.6±8.6	292±52
	Vmax (pmol/(h·oocyte))	338±22	N.D.	77.5±3.2	8.27±1.23	628±61
	Vmax/Km (µL/(h·oocyte))	0.599	N.D.	0.749	0.365	2.15

K_m and V_max values were estimated from the data obtained in the study of concentration-dependence, and each value represents the mean±S.D. N.D., not detectable. The table was prepared by reference to Yahara et al.⁷⁹⁾

Glu406 is located on putative TM8, and corresponds to Ile359 of LeuTAa, of which the side chain was reported to have a hydrophobic interaction with L-Leu.³¹⁾ An uptake study showed greater and lower uptake activities for GABA and taurine than those exhibited by the wild type, respectively,⁷⁹⁾ suggesting that Glu406 contributes to GABA and taurine transport by TauT. The kinetic parameters revealed that an amino acid substitution at position 406 caused increased and decreased substrate affinities for GABA and taurine, respectively,⁷⁹⁾ and the increased affinity for GABA suggests that Glu406 can be a determinant of the substrate pocket volume in TauT since the amino acid substitution at position 406 caused the reduction of the side chain bulkiness to improve the accessibility of GABA to the substrate pocket and to weaken the interaction between the putative TM8 and taurine. Furthermore, E406C showed high transport clearance and uptake activity for GABA and weakened sensitivity to β-Ala and taurine and enhanced sensitivity to nipecotic acid and GABA than the wild type.⁷⁹⁾ Based on this GAT-like transport property of E406C, Glu406 is assumed to be the most important residue of TauT to determine the substrate specificity differing from GATs. In particular, rat GAT2, a GABA transporter with a moderate affinity for taurine, contributes to hepatic taurine uptake,⁶¹⁾ and Glu406 is thought to cause the functional difference between rat GAT2 and TauT.

7. CONCLUSION

In the present review, the transport of taurine in the retina, such as at the inner BRB and Müller cells, and the major involvement of TauT was described in the taurine supply and regulation of taurine concentration in the retina, which is essential neural tissue for vision. Among the physiological functions of taurine, its property as an osmolyte is thought to be important, and neural cells are constantly exposed to fluctuations in the osmolarity of the retinal microenvironment. TauT expressed in Müller cells takes up taurine in response to hyperosmotic stress and regulates cell volume to maintain the normal function of the retina. In response to hypoosmotic stress, VSOAC is suggested to contribute to the efflux of taurine. While TauT is also suggested to act as a significant GABA transporter at the inner BRB, the hepatic transport of taurine involves rat GAT2, the localization of which is consistent with the protein zonation shown by taurine-associated enzymes. Their interesting substrate specificity was assessed by amino acid substitution, and the crucial amino acids were revealed in the substrate specificity of TauT and GATs. Furthermore, the contribution of TauT as the influx transporter of β-Ala across the inner BRB has recently been reported in the enzymatic synthesis of L-carnosine in the retina, showing a new insight into peptide supply to the retina.⁸⁰⁾ In addition, the involvement of GAT2 has been also suggested in the uptake of GAA in the liver, which contributes to the biosynthesis of creatine that plays a role in the storage and transmission of phosphate-bound energy.⁶⁴⁾ The study of taurine transport is expected to improve our understanding of taurine functions that would be helpful in the treatment of diseases of the retina and liver, providing a huge opportunity to discover astonishing new physiological and pharmacological mechanisms underlying taurine and its relevant transporters.

Acknowledgments

This work was supported in part by Grants-in-Aids from the Japan Society for the Promotion of Science, and Research Grants on Sensory and Communicative Disorders from the Ministry of Health, Labour, and Welfare of Japan.

Conflict of Interest

The authors declare no conflict of interest.

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