Molecular cloning of mouse amino acid transport system B0, a neutral amino acid transporter related to Hartnup disorder.

Resorption of amino acids in kidney and intestine is mediated by transporters, which prefer groups of amino acids with similar physico-chemical properties. It is generally assumed that most neutral amino acids are transported across the apical membrane of epithelial cells by system B(0). Here we have characterized a novel member of the Na(+)-dependent neurotransmitter transporter family (B(0)AT1) isolated from mouse kidney, which shows all properties of system B(0). Flux experiments showed that the transporter is Na(+)-dependent, electrogenic, and actively transports most neutral amino acids but not anionic or cationic amino acids. Superfusion of mB(0)AT1-expressing oocytes with neutral amino acids generated inward currents, which were proportional to the fluxes observed with labeled amino acids. In situ hybridization showed strong expression in intestinal microvilli and in the proximal tubule of the kidney. Expression of mouse B(0)AT1 was restricted to kidney, intestine, and skin. It is generally assumed that mutations of the system B(0) transporter underlie autosomal recessive Hartnup disorder. In support of this notion mB(0)AT1 is located on mouse chromosome 13 in a region syntenic to human chromosome 5p15, the locus of Hartnup disorder. Thus, the human homologue of this transporter is an excellent functional and positional candidate for Hartnup disorder.


Introduction
Epithelial resorption of amino acids across the apical membrane in the kidney and intestine is thought to be carried out by four different transporters (1). Anionic amino acids are taken up by a Na + -dependent aspartate/glutamate transporter, which has been designated system X -AG .
Molecular cloning has identified this transporter as EAAT3 (2). Cationic amino acids are taken up by system b 0,+ ; the molecular correlate of this transporter being the heteromeric amino acid transporter rBAT/b 0,+ AT (3). Proline and glycine are thought to be transported by the IMINO system (4) and it has recently been proposed that the molecular correlate of this transporter may be the proton-dependent amino acid transporter PAT1 (5). Most neutral amino acids are thought to be transported by system B 0 , which has not yet been identified at the molecular level (6,7). System B 0 has been characterized in jejunal brush border vesicles (8)(9)(10), bovine epithelial cells (11) and Caco-2 cells (12). These studies suggest that system B 0 is a Na + -dependent, chloride independent transporter that accepts a wide variety of neutral amino acids. Failures to resorb amino acids in the kidney and intestine underlie a number of inherited transporter diseases, with mutations in either rBAT or b 0,+ AT causing cystinuria (13), mutations of the IMINO system believed to cause iminoglycinuria (14)

Materials and Methods cDNA cloning and plasmids
Total RNA was isolated from mouse kidney by the acid-guanidinium-thiocyanatephenol-chloroform extraction method of Chomczynski and Sacchi (18). For cloning of mouse B 0 AT1, 0.5 µg oligo(dT) 15 was added to 1.5 µg kidney RNA in a total volume of 4.5 µl. The mixture was incubated for 10 min at 65°C and then chilled on ice. Reverse transcription was carried out in a buffer designed to improve the temperature stability of reverse transcriptase (19). The reaction was assembled by adding the following components to the RNA mixture:  into the same sites of the oocyte expression vector pGEM-He-Juel. (20).

Oocytes and injections
Oocyte isolation and management have been described in detail elsewhere (21). For expression in oocytes mouse B 0 AT1 in pGem-He-Juel was linearised with SalI and transcribed in vitro using the T7 mMessage mMachine Kit (Ambion, Austin Texas, USA).
Oocytes were injected with 20 ng of cRNA encoding mouse B 0 AT. Transport measurements were carried out after 3-6 days of expression.

Flux measurements
For each determination, groups of 7

Electrophysiological recordings
Amino acid induced currents were analysed by two-electrode voltage clamp recording.
The recordings were performed with 1 x LU and 10 x MGU headstages connected to a

RT-PCR
Total RNA was isolated from male adult NRMI mouse tissues by the acid-guanidiniumthiocyanate-phenol-chloroform extraction method of Chomczynski and Sacchi (18).
Following washing steps, the slide preparations were dipped in NTB2 emulsion (Kodak, Rochester, NY) and exposed at 4°C for 3 weeks. After development the slides were stained with hematoxylin/eosin and photographed with a Sony DSC digital camera.

Calculations, statistics and computer analysis
Each datapoint or bar in figures and tables represents the mean ± SD activity of m = 7-10 mB 0 AT1 expressing oocytes minus the mean ± SD activity of m = 7-10 non-injected oocytes. Sequence alignments and the tree were calculated using programs of GCG and PHYLIP packages supplied by the Australian National Genomic Information Service (ANGIS).
Sequence alignment was performed using ClustalW (23). Subsequently, protein distance was calculated by using the Dayhoff PAM matrix (24) and converted into a tree diagram using an additive tree model. The peptide sequence of mB 0 AT1 was analysed by hydropathy plotting using the TMHMM

Cloning of mouse B 0 AT1
Hydrophobicity analysis of predicted open reading frames on human chromosome region 5p15 in the NCBI human genome database and the ENSEMBL database revealed the presence of only a limited number of proteins with more than 5 hydrophobic putatively membrane spanning domains (data not shown). Some of these had already been annotated as transporter genes, such as the dopamine transporter (SLC6A3), the Na + /H + exchanger NHE-3 (SLC9A3) and the potassium-chloride cotransporter KCC4 (SLC12A7). These are well characterised transporters and were unlikely to mediate or affect neutral amino acid transport and were not considered valid candidates. Two additional proteins with multiple hydrophobic domains were identified in this region. One of these is XT2, an orphan member of the SLC6 family, which does not appear to have a transport activity (25,26). The second lies distal to LOC340024 in the NCBI database) (27). The twelve coding exons span an area of 18920 bp ( Fig. 1).
Alignment of all mouse members of the SLC6 family reveals that mB 0 AT1, together with XT2 and XT3, another orphan member of the SLC6 family (26), form a subfamily (Fig.   2). mB 0 AT1 has 51% and 43% identical amino acids with XT3 and XT2, respectively; all other SLC6 members having between 21% and 35% identical amino acids with mB 0 AT1. The mouse cDNA has a coding sequence of 1904 bp and encodes a protein of 634 amino acids.
Hydropathy analysis indicates the presence of 12 transmembrane domains (Fig. 3A), which is in agreement with the general structure of transporters in the SLC6 family (28). However, B 0 AT1, XT2 and XT3 are predicted to have a larger loop between helix 7 and 8 compared to other members of the SLC6 family. The predicted structure was supported by the exon/intron boundaries, which in membrane proteins, are usually found in the sequence between transmembrane domain encoding regions (Fig. 3B).

Tissue distribution and cellular distribution of mB 0 AT1
The successful cloning of mB 0 AT1 from mouse kidney cDNA indicated its presence in this tissue. This was confirmed by RT-PCR experiments, which showed significant expression of mouse B 0 AT1 only in kidney and small intestine (Fig. 4) In order to localize the transcriptional activity of mB 0 AT1 in mouse organs we performed radioactive in situ hybridization assays on paraffin-embedded tissue sections. As

Functional properties of mouse B 0 AT1
Expression of mB 0 AT1 in Xenopus laevis oocytes resulted in a significant increase of leucine uptake activity compared to control oocytes. On day 5 of expression, for example, mB 0 AT1 expressing oocytes took up 100 µM [ 14 C]leucine at a rate of 56 ± 10 pmol/15 min.
This was more than five times higher than the activity of non-injected oocytes, which amounted to 10 ± 1 pmol/15 min. Uptake of [ 14 C]leucine was Na + -dependent; replacement of NaCl by NMDG-Cl or LiCl completely abolished the transport activity (Fig. 7A). In contrast to other members of the SLC6 family, leucine uptake via mB 0 AT1 was not significantly affected by replacement of chloride with gluconate (Fig. 7A). Transport of amino acids via by guest on March 23, 2020 http://www.jbc.org/ Downloaded from mB 0 AT1 was driven by the membrane potential. Addition of 50 mM KCl to the transport buffer, a manipulation that reduces the membrane potential of oocytes from -40 ± 7 mV to -16 ± 3 mV, reduced leucine uptake by 52% (Fig. 7B), addition of 50 mM NH 4 Cl reduced transport activity further (68%) consistent with its stronger depolarizing effect (-8 ± 3 mV) as compared to 50 mM KCl (Fig. 7B). Reduction of the transport activity was not caused by the increased osmolarity in these solutions as evidenced by unaltered transport activity observed when 50 mM NMDG-Cl or 100 mM sucrose was added to the ND96 buffer, which contains 96 mM NaCl (Fig. 7B). Transport of leucine via mB 0 AT1 was pH dependent, strongly increasing with alkaline pH (Fig. 7C).
The data from Figure 7A/B suggest that mB 0 AT1 is an electrogenic Na + -dependent transporter, but in contrast to other members of the neurotransmitter transporter family does not appear to be chloride dependent. This suggests that at least 1 Na + is co-transported together with neutral amino acids. An activation analysis of leucine transport as a function of the Na + concentration showed a sigmoidal dependence. Half-maximal transport velocity was reached at a Na + concentration of 54 ± 4 mM (Fig. 8A). Linearisation of the data according to the Hill equation yielded a Hill coefficient of 1.5 ± 0.2 (Fig. 8B), suggesting that either two Na + -ions are cotransported with neutral amino acids or that 1 Na + is cotransported and a second Na + ion binds to a modifier site on the transporter.
In order to determine the substrate specificity of the transporter, uptake of 100 µM partial but significant inhibition was observed with D-leucine and D-serine (Fig. 9B). Some amino acids, however, may be inhibitors of the transporter albeit not being actually translocated. To test for active transport we used a variety of [ 14 C]labelled amino acids (Fig.   10A). Of the tested amino acids, leucine appeared to be the best substrate, followed by isoleucine, glutamine, phenylalanine, alanine and proline. Histidine and glycine transport activity in mB 0 AT1 expressing oocytes was small but significant ( The transporter showed rather low affinity for its substrates. For leucine transport a K m of 630 ± 150 µM was determined (average of n = 3 experiments, representative curve shown in Fig. 11); other neutral amino acids were transported with similar affinity (Table 1). The V max -values varied between oocyte batches and the day of analysis. Nevertheless, we observed a consistently higher V max for leucine transport relative to other substrates (Table 1).
Thus it appears that leucine is the preferred substrate of mB 0 AT1.

Discussion
General properties: amino acid transport in mammalian cells is mediated by a large variety of amino acid transporters with overlapping substrate specificity (1,6,29). The corresponding transport activities have been classified by their substrate specificity (30). The molecular correlates of most of these activities have been identified in recent years (see Ref. (6,31) for recent summaries). In general it has been observed that uptake of neutral amino acids across the apical membrane of epithelial cells is largely mediated by Na + -dependent transport mechanisms (32), whereas Na + -independent transport mechanisms prevail in the plasma membrane other cell types, particularly for large, hydrophobic amino acids (29). The molecular correlate of the major transport activity for neutral amino acids in the brush border membrane of kidney and intestinal epithelial cells (named system B 0 for broad neutral, sometimes also referred to as NBB for neutral brush border) has remained elusive until now.
In this study we have characterized a transporter that has all hallmarks of system B 0 . It transports most neutral amino acids but does not recognise glutamate and arginine, moreover, transport is Na + -dependent, Cl --independent and electrogenic. Mouse B 0 AT1 belongs to the SLC6 family of Na + and Cldependent transporters, which comprises transporters for neurotransmitters, osmolytes and amino acids (28,33). B 0 AT1 forms a small subfamily together with two orphan transporters of the SLC6 family, namely XT2 and XT3 (26), which is characterised by a large loop between helix 7 and 8. Surprisingly, B 0 AT1 is less closely related to amino acid transport system B 0,+ (cDNA ATB 0,+ ), which also is a general amino acid transporter, but transports neutral and cationic amino acids (34). It has to be noticed, however, that the SLC6 family has two major branches. including B 0 AT1 and ATB 0,+ and a number of orphan transporters, the function of which remains elusive. It is tempting to speculate that the orphan transporters of this family, which are all located in the lower branch are amino acid transport related proteins.
Functional properties: The mB 0 AT transporter displays rather low affinity for its substrates.
For the three amino acids tested, K m -values were found to lie between 500 µM and 700 µM.
These figures correspond well with the reported K m -values for system B 0 (9,12,35).
Activation analysis of leucine transport at different Na + concentrations yielded a Hill coefficient of 1.5, suggesting that at least 1 Na + is cotransported together with substrates and that a second Na + either is cotransported or modulating the transport activity. translocation. This has to be seen, for example, in comparison to the amino acid transporter SN1 (SNAT3), which is also Na + -dependent, but transport associated currents are generated by a cation conductance whereas the overall transport process is electroneutral (36). The difference between both transporters becomes apparent when oocytes are depolarised by addition of KCl, which reduces transport activity of mB 0 AT1 but does not affect the activity of SN1 (36). In contrast to most other members of the SLC6 family mB 0 AT1 is not chloride dependent. This feature appears to be unique among the mammalian members of this family.
However, a recently identified insect member cloned from the midgut of the caterpillar Manduca sexta was also found to be unaffected by chloride replacement (37). As a further hallmark of mB 0 AT1, we also observed a strong pH dependence, which has been reported previously for system B 0 in bovine brush-border membranes (38). This pH-dependence might contribute to the strong inhibition observed after addition 50 mM NH 4 Cl. NH 4 Cl at this concentration not only depolarises the oocyte but also causes intracellular acidification (39).

Physiological function:
The functional properties of mB 0 AT1 suggest that it is the molecular  (41). As a result removal of neutral amino acids is thought to provide an additional driving force for the accumulation of cationic amino acids in the kidney (Fig. 12). In agreement with this notion, leucine is the preferred efflux substrate for rBAT/b 0 AT (42) and the preferred uptake substrate for mB 0 AT1.
In addition to the conspicuous expression in kidney and intestine, analysis of the expressed sequence tag database further suggests significant expression of the transporter in the skin.
Pigmentation of melanocytes involves synthesis of melanins. Tyrosine is the major precursor of melanin biosynthesis (43). Thus, B 0 AT1 may play a role to protect skin cells against UV light. Furthermore, enzymes of serotonergic and melatonergic systems have been detected in the skin, which require transport of their precursor tryptophan (44).
Relation of mB 0 AT1 to Hartnup disorder: Hartnup disorder is characterized by a striking increase of neutral amino acids in the urine (15,45). Because of the pattern of amino acid excretion in Hartnup disorder it is generally assumed that mutations in system B 0 underlie its pathogenesis (1). Recently, SLC1A5 (named ASCT2 or ATB 0 ) has been suggested to be the molecular correlate of system B 0 (46,47). Three lines of evidence clearly refute this assignment. First, ASCT2/ATB 0 does not transport phenylalanine or tryptophan (48,49), two well-established substrates of system B 0 , which are transport by mB 0 AT1. Second, ASCT2/ATB 0 is an obligatory amino acid exchanger (50) and as a result cannot mediate net fluxes of neutral amino acids across the apical membrane, whereas mB 0 AT1 mediates net uptake of amino acids. Third, our analysis of Hartnup disorder families did not reveal the presence of potentially causative mutations in the coding sequence or splice donor/acceptor sites in the SLC1A5 gene on chromosome 19 (45). As a result, the name ASCT2 appears to be more appropriate for SLC1A5.
Hartnup disorder has been mapped to human chromosome 5p15 (17), allowing a targeted approach to the molecular identification of the corresponding transporter. A hydropathy screen for putative membrane proteins in 5p15 yielded two uncharacterized members of the neurotransmitter transporter family as candidates. One has been termed XT2 or ROSIT, an orphan member of the SLC6 family (25,26). Several studies and our own unpublished observations indicate that XT2 is expressed in the plasma membrane but does not transport amino acids or other compounds. The second has been annotated as protein XP_291120 in the NCBI database, the mouse homologue of which we have characterized here as mB 0 AT1. In further support of this notion the genomic sequence of mB 0 AT1 is located in an area of mouse chromosome 13 that is synthenic to human chromosome 5p15. Our data suggest that the annotated protein XP_291120 is a likely candidate for mutations associated with Hartnup disorder.         Oocytes were each injected with mB 0 AT1 cRNA or remained uninjected in the controls.

Figure legends
Uptake of [ 14 C]leucine was determined 6 days after injection at concentrations ranging between 3 µM and 3000 µM. The transport activity of non-injected oocytes was subtracted in each case. The experiment was performed n = 3 times, the averaged K m -values for this and other substrates are presented in Table 1.

Figure 12: Resorption of neutral and cationic amino acids in kidney and intestine
Cationic amino acids are transported across the apical membrane by exchange against neutral amino acids via the heteromeric amino acid antiporter rBAT/b 0 AT. Leucine, the preferred neutral amino acid released by rBAT/b 0 AT plus other neutral amino acids already present in the urine are subsequently removed by B 0 AT1. B 0 AT1 accumulates neutral amino acids by cotransport with 1-2 Na + . Cationic amino acids are released on the basolateral side by the heteromeric amino acid transporter 4F2/y + LAT1 in exchange for neutral amino acids, neutral amino acids are released by LAT2 and a yet unidentified uniporter.