Identification of Functionally Relevant Residues of the Rat Ileal Apical Sodium-dependent Bile Acid Cotransporter*

The mechanisms underlying the transport of bile acids by apical sodium-dependent bile acid transporter (Asbt) are not well defined. To further identify the functionally relevant residues, thirteen conserved negatively (Asp and Glu) and positively (Lys and Arg) charged residues plus Cys-270 of rat Asbt were replaced with Ala or Gln by site-directed mutagenesis. Seven of the fourteen residues of rat Asbt were identified as functionally important by taurocholate transport studies, substrate inhibition assays, confocal microscopy, and electrophysiological methods. The results showed that Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt are critical for transport function and may determine substrate specificity. Arg-64 may be located at a different binding site to assist in interaction with non-bile acid organic anions. For bile acid transport by Asbt, Na+ ion movement is a voltage-dependent process that tightly companied with taurocholate movement. Asp-122 and Glu-261 play a critical role in the interaction of a Na+ ion and ligand with Asbt. Cys-270 is not essential for the transport process. These studies provide new details about the amino acid residues of Asbt involved in binding and transport of bile acids and Na+.

The mechanisms underlying the transport of bile acids by apical sodium-dependent bile acid transporter (Asbt) are not well defined. To further identify the functionally relevant residues, thirteen conserved negatively (Asp and Glu) and positively (Lys and Arg) charged residues plus Cys-270 of rat Asbt were replaced with Ala or Gln by site-directed mutagenesis. Seven of the fourteen residues of rat Asbt were identified as functionally important by taurocholate transport studies, substrate inhibition assays, confocal microscopy, and electrophysiological methods. The results showed that Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt are critical for transport function and may determine substrate specificity. Arg-64 may be located at a different binding site to assist in interaction with non-bile acid organic anions. For bile acid transport by Asbt, Na ؉ ion movement is a voltage-dependent process that tightly companied with taurocholate movement. Asp-122 and Glu-261 play a critical role in the interaction of a Na ؉ ion and ligand with Asbt. Cys-270 is not essential for the transport process. These studies provide new details about the amino acid residues of Asbt involved in binding and transport of bile acids and Na ؉ .
Bile acids are the major products of cholesterol catabolism and play a critical role in a multitude of biological processes, including bile secretion and absorption of fat and fat-soluble vitamins. The apical sodiumdependent bile acid transporter (Asbt) 2 is only expressed in apical membranes of ileal enterocytes, renal tubule cells, and cholangiocytes where it plays a pivotal role in maintaining the enterohepatic bile acids through uptake of bile acids from the intestinal lumen, renal tubules, and bile ducts. Asbt (SLC10A2) is part of a subgroup of the sodium-dependent cotransporter superfamily. Asbt is glycosylated at the extracellular N terminus and contains a cytoplasmic C terminus. Its molecular mass is ϳ50 kDa, and the predicted structure that is derived from hydrophobicity analysis contains either seven or nine transmembrane domains (1,2). Naturally occurring mutations in human ASBT gene lead to a genetic disorder, primary bile acid malabsorption (3).
The mechanisms underlying the transport of bile acids by Asbt have not been clearly defined. In the current study, to further identify the functionally relevant residues critical for transport, thirteen conserved negatively (Asp (D) and Glu (E)) and positively (Lys (K) and Arg (R)) charged residues plus Cys-270 of rat Asbt (rAsbt) were selected, and their roles in transport function and membrane localization were examined by a combination of molecular biological, biochemical, and electrophysiological methods. (2.1-3.47 Ci/mmol) was purchased from PerkinElmer Life Sciences. Unlabeled taurocholate was purchased from Sigma. Sulfo-N-hydroxysuccinimide-SS-biotin was from Pierce. Cell culture supplies were obtained from Invitrogen. Subcloning reagents, restriction enzymes, and competent cells were obtained from Stratagene (La Jolla, CA), Invitrogen, and New England BioLabs (Beverly, MA).

Materials-[ 3 H]Taurocholic acid
Generation of GFP-fused Rat Ileal Bile Acid Transporter (rAsbt-GFP) cDNA and Site-directed Mutagenesis-Full-length wild-type rat Asbt cDNAs were inserted in-frame into the HindIII and BamH1 sites of a green fluorescent protein (GFP) vector, pEGFPN2 (Clonetech, Palo Alto, CA), to produce the GFP-fused plasmid constructs as described previously (4). Potential functional determinant residues were mutated by site-specific mutagenesis using rat Asbt-GFP as template. The QuikChange TM site-directed mutagenesis kit (Stratagene) was used to convert codons for potential active determinant residues to alanine or glutamine residues according to the manufacturer's directions with minor modification as described previously (5). After subcloning into expression vectors, fidelity of all the constructs was verified by DNA cycle sequencing using a PerkinElmer GeneAmp 9600, ABI Prism 377 DNA sequencer at the DNA Core Facility, Mount Sinai School of Medicine. PCR amplifications were carried out using a PTC-100 TM Programmable Thermal Controller (MJ Research, Inc., Watertown, MA). For Xenopus laevis oocytes expression, rAsbt-GFP and mutants were subcloned into XhoI and NotI sites of a dual purpose vector, pRAT, for transcript production or cytomegalovirus-based expression as described previously (6). Automated DNA sequencing at WM Keck DNA Core Facility, Yale University School of Medicine, confirmed the constructs. cRNA was synthesized using an mMessage mMachine kit (Ambion, Austin, TX) from T7 promoter in pRAT and quantified by spectroscopy and comparison to control samples separated by electrophoresis.
Cell Culture and Transient Transfection-COS-7 (SV40-transformed monkey kidney fibroblast) cells were maintained in complete Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum, 50 units/ml of penicillin, 50 g/ml of streptomycin, and 2 mM L-glutamine. Cells were transiently transfected with plasmids containing wild-type or mutant rat Asbt-GFP cDNA using Lipofectamine TM reagent (Invitrogen) as described previously (5). Transfected cells were harvested 24 -48 h later for bile acid transport and confocal microscopy analysis.
Confocal microscopy was carried out as described previously (7). Briefly, confocal microscopy was performed on a confluent monolayer of transfected cells cultured on glass coverslips. The cells were fixed and permeabilized for 7 min in 100% methanol at Ϫ20°C, followed by rehydration in phosphate-buffered saline. After being washed with phosphate-buffered saline, the cells on coverslips were inverted onto a drop of VectaShield (Vector Laboratories, Inc., Burlingame, CA). Fluorescence was examined with a Leica TCS-SP (UV) 4-channel confocal laser scanning microscope in the Imaging Core Facility Microscopy Center, Mount Sinai School of Medicine.
Bile Acid Influx Transport Assay and Inhibition Study-The Na ϩdependent taurocholate (TC) influx assay was done as described previously (4,7). Briefly, taurocholate uptake was performed at 37°C for 10 min. The confluent cell monolayers grown on 12-well plates were washed twice with warm uptake buffer (116 mM NaCl (or choline), 5.3 mM KCl, 1.1 mM KH 2 PO 4 , 0.8 mM MgSO 4 , 1.8 mM CaCl 2 , 11 mM D-glucose, 10 mM Hepes, at pH 7.4), and each well was incubated with uptake buffer containing 10 M [ 3 H]taurocholate at the final concentrations. Following 10 min of incubation, the influx assays were terminated by aspirating the medium, and the cells were washed three times with ice-cold uptake buffer. The levels of protein expression of wild-type and various mutated transporters in transfected cells were normalized by total protein concentrations. The protein was determined with the Bio-Rad protein assay kit. In the bile acid and non-bile acid organic anion inhibition studies, taurocholate influx of wild-type or mutant rAsbt-GFP cDNA-transfected cells in the absence of competitor was set as 100%, and all values were graphed relative to this level. The final concentration of each inhibitor was 100 M or as indicated in the figure legends.
Kinetic Characterization of GFP-fused Wild-type and Mutant Rat Asbt-Apparent K m and V max values for taurocholic acid were obtained by measuring initial rates of uptake at varying concentrations (1, 5, 10, 50, 100 M) of taurocholic acid substrate. The sodium concentration was kept constant at 116 mM. The data were fitted to the Michaelis-Menten equation by nonlinear regression with the enzyme kinetic software Enzfitter for MS-DOS.
Membrane Biotinylation Analysis-rAsbt-GFP and mutant cell surface expression was detected essentially as described by Ho et al. (8). Briefly, COS-7 cells were grown on 6-well plates and transfected with rAsbt-GFP and mutants using a similar protocol for transport experiments. Sixteen hours post-transfection, cells were washed with ice-cold phosphate-buffered saline Ca 2ϩ /Mg 2ϩ (138 mM NaCl 2 , 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 9.6 mM Na 2 HPO 4 , 1 mM MgCl 2 , 0.1 mM CaCl 2 , pH 7.3) and then treated with a membrane-impermeable biotinylating agent (sulfo-N-hydroxysuccinimide-SS-biotin, 1.5 mg/ml; Pierce) at 4°C for 1 h. Subsequently the cells were washed three times with ice-cold phosphate-buffered saline Ca 2ϩ /Mg 2ϩ containing 100 mM glycine and then incubated for 20 min at 4°C with the same buffer to remove the remaining labeling agent. After washing, cells were disrupted with 700 l of lysis buffer (10 mM Tris base, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, pH 7.4) containing protease inhibitors (Complete; Roche Applied Science) at 4°C for 1 h with constant agitation. Following centrifugation, 140 l of streptavidin-agarose beads (Pierce) was added to 600 l of cell lysate and incubated overnight in a cold room. Beads were washed four times with ice-cold lysis buffer, and the biotinylated proteins were released by incubation of the beads with 2ϫ Laemmli buffer for 30 min at room temperature. Similar to total cell lysates, samples of the biotinylated fractions (25 l) were subjected to Western analysis for detection of immunodetectable rAsbt-GFP and mutants with polyclonal anti-GFP antibody (1:2000 dilution). The protein concentration was normalized to the amount of actin protein detected by an anti-actin antibody (1:5000).
Oocyte Expression and Electrophysiological Experiments-Oocyte culture and current-voltage analysis were performed as described previously (9). Briefly, oocytes were isolated from Xenopus laevis frogs, defolliculated by collagenase treatment. On the following day, they were microinjected with 46 nl of sterile water containing 5 ng of rAsbt-GFP or mutant cRNA per oocyte in standard experiments. Whole-oocyte currents were measured by two-electrode voltage clamp after 4 days of cRNA injection (Oocyte Clamp; Warner Instruments, Hamden, CT) with constant perfusion (1 ml/min, solution exchange3 ខ s). Data were sampled at 1 kHz and filtered at 0.25 kHz. All experiments were per-  JUNE 16, 2006 • VOLUME 281 • NUMBER 24 formed at room temperature and repeated with at least two branches of oocytes. Standard bath solution was ND-96 (in mM): 96 NaCl, 2 KCl, 1 MgCl 2 , 0.3 CaCl 2 , and 5 HEPES/NaOH, pH 7.5.

Functionally Relevant Residues of Asbt
Statistics Analysis-The results were expressed as mean value Ϯ S.E. and analyzed by using unpaired t test and one-way analysis of variance to test for a difference in means between two groups that may have unequal sizes. The p values were adjusted by Bonferroni correction to deal with multiple comparison. p Յ 0.05 was considered statistically significant.

Effects of Site-directed Mutagenesis on Initial Rate of Taurocholate
Transport by rAsbt-GFP-Previous studies on bile acid-binding proteins and sodium-coupled cotransporters (10,11) indicated that charged amino acids of these proteins may be critical as binding sites for sodium ions and the cotransported substrate. The negatively charged residues Asp-115 (located in the large intracellular loop 3) and Glu-257 (located in the large extracellular loop 6) of rat Ntcp have been identified as being important for bile acid transport activity (10). Recently, Zahner et al. (10) suggested that Cys-266 of rat Ntcp, which is located in the large extracellular loop 6, is involved in taurocholate transport. The importance of the conserved charged residues of Asbt is not fully understood, especially the conserved positively charged residues.
In this study, we selected eleven positively charged residues, two negatively charged residues, Asp-122 (Asp-115 in liver Ntcp) and Glu-261 (Glu-257 in liver Ntcp), and Cys-270 (Cys-266 in liver Ntcp) of rat Asbt to examine their potential functional importance. An alignment ( Fig. 1) of the amino acid sequences of five Asbt proteins from rat, mouse, hamster, rabbit, and human reveals that these fourteen selected residues are conserved in all five species. Because GFP is easily detected and has been shown not to interrupt transport activity, cellular distribution, and protein stability of bile acid transporters (4,12,13), we first constructed a rAsbt-GFP (see "Experimental Procedures"). The QuikChange sitedirected mutagenesis kit was used to convert codons for the charged amino acids of interest and Cys (C) to non-charged alanine (A) or glutamine (Q). The wild type and mutants of rAsbt-GFP were then expressed in COS-7 cells, and the initial rate of taurocholate uptake was examined. Fig. 2 shows that the initial rate of TC transport was significantly reduced in cells transfected with nine of the rAsbt-GFP thirteen mutants. Taurocholate transport activity decreased by 85-90% in the cells transfected with D122A, R256Q, E261A, and double mutated K312Q,K313Q (KK313QQ) mutants. K191Q-and K225Q-transfected cells showed an ϳ80% decrease in transport activity and K57Q, R64Q, and R254A showed ϳ35-55% decrease in transport activity compared with wild-type rat Asbt-GFP. Mutations of Lys-150, Lys-185, and Lys-189 of rAsbt-GFP caused no significant change in transport activity compared with wild-type rat Asbt-GFP. In contrast, replacement of Cys-270 with Ala enhanced the TC transport activity Ͼ20% compared with wild-type rAsbt-GFP-transfected cells.
Kinetic Analysis of rAsbt-GFP and Mutants-A decrease in transport activity after a single amino acid mutation may be caused by a change in substrate binding affinity and/or in membrane distribution of the transport protein (4,5,8). Therefore, we next evaluated the kinetic parameters (apparent K m ) of Na ϩ -dependent taurocholate uptake of wild-type and mutant rAsbt-GFP in transfected COS-7 cells. Initial rates of [ 3 H]taurocholate accumulation in COS-7 cells were measured at varying concentrations of taurocholic acid as described under "Experimental Procedures." The data were fitted by nonlinear regression to the Michaelis-Menten equation. As shown in Table 1 and Fig. 3, the wildtype rAsbt-GFP had a K m value for taurocholate transport of 26.3 Ϯ 8.3 M, which is comparable with the previously reported K m values of 12-37 M in studies of rat ileal brush-border membrane vesicles and Chinese hamster ovary cells transfected with human or hamster Asbt (14 -17). These studies showed that fusion with GFP did not significantly affect taurocholate binding affinity of rAsbt. As shown in Table 1, the K m values of D122A, K191Q, R256Q, E261A, and K312Q,K313Q (KK313QQ) mutants were increased 3ϳ6-fold compared with wildtype Asbt-GFP. The K m values of R64Q and K225Q mutants were increased ϳ2.5-fold compared with wild-type rAsbt-GFP. The apparent K m values for K150Q, K185Q, and K189Q mutants were similar to that of wild-type rAsbt-GFP. Furthermore, corresponding experiments comparing the wild-type and C270A mutant of rAsbt-GFP revealed no significant change in K m value for taurocholate transport. However, the increased initial rate of taurocholate uptake by the C270A mutant appears to be due to in an increased V max (C270A ϭ 164.9 Ϯ 4.8 pmol/   mg/min versus WT ϭ 129.0 Ϯ 6.3 pmol/mg/min) for the transport process. These data suggest that the decrease of initial TC transport activity of the mutants is due to altered substrate binding affinity. Cellular Distribution of Rat Asbt Mutants in Transfected Cells-To define the potential effects of point mutations on the cellular distribution of rAsbt, mutated rAsbt-GFPs were transiently transfected into COS-7 cells. The plasma membrane distributions of these mutants were visualized by confocal microscopy. The confocal images demonstrated that plasma membrane expression of these mutated proteins in transfected COS-7 (Fig. 4) cells was similar to wild-type rAsbt-GFP. Quantitative plasma membrane biotinylation analysis further confirmed these results. Fig. 5 shows no significant differences in cell surface protein expression between the mutants and wild-type rAsbt-GFP-transfected COS-7 cells. Therefore, we conclude that the altered transport activities of these mutants were not due to synthesis and trafficking defects but to mutation-induced changes in the affinity of Asbt for taurocholate.
Effects of Various Bile Acids on Taurocholate Uptake in COS-7 Cells Transfected with rAsbt-GFP Mutants-The charged side chain at the binding cavity of carrier proteins may be critical for ligand binding affinity and/or substrate specificity and may interact with polarized groups of ligands through H-bonding and/or electrostatic interactions. To understand whether these selected charged residues determine substrate binding specificity, cholate, taurodeoxycho-late (TDC), taurochenodeoxycholate (TCDC), taurodehyrocholate (TDHC), and lithocholic acid (LCA) were used as inhibitors in these experiments. These bile acids, differing in the number and position of hydroxyl groups in the steroid nucleus and conjugation with taurine group, were used to map potential ligand-carrier protein-interacting sites in wild-type and mutant transporters (see "Discussion"). As shown in Fig. 6, taurocholate uptake by wild-type rAsbt-GFP was significantly inhibited by 10 min of exposure to cholate, TCDC, TDC, and lithocholic acid, but not by TDHC. These results agree with previous studies by Kramer et al. (18). As seen in Fig. 6, the inhibition by TCDC (absence of 12␣-OH group) on the taurocholate transport was almost abolished by mutation of D122A, R256Q, E261A, and K312Q,K313Q (KK313QQ) and was also significantly decreased by K191Q, K225Q, and R254A mutants compared with wild-type rAsbt-GFP. Similar to the effects of TCDC, the inhibition of taurocholate transport by TDC (absence of 7␣-OH group) was also significantly decreased in the K225Q, R256Q, E261A, and K312Q,K312Q mutants. In contrast, taurocholate transport by all of the mutants was inhibited to the same degree as wild-type rAsbt-GFP by cholate, lithocholic acid, and TDHC. These results suggest that residues Asp-122, Lys-191, Lys-225, Arg-254, Arg-256, Glu-261, and Lys-312,Lys-313 may be important for substrate binding specificity. Asp-122, Lys-191, and Arg-254 may interact with the 7␣-hydroxyl group of bile acids (see "Discussion").

Effects of Non-bile Acid Organic Anions on Taurocholate Uptake by COS-7 Cells Transfected with rAsbt-GFP Mutants-
The ileal Na ϩdependent bile acid carrier exhibits wide substrate specificity and may be inhibited by many cholephilic organic substances of high structural diversity through competitive or non-competitive interactions (19 -22). To further identify functionally relevant residues that may determine substrate specificity, we investigated the effects of five structurally different non-bile acid organic anions on taurocholate transport in COS-7 cells transfected with the cDNAs of rAsbt-GFP and mutated transporters. Fig. 7 shows that bromosulfothalein (BSP), bumetanide (BUM), and diisothiocyanostilbene disulfonate (DIDS) significantly inhibited [ 3 H]taurocholate uptake by wild-type rAsbt-GFP-transfected cells, whereas estron-3-sulfate (E3S) and probenecid had no effect. These results are in agreement with the previous studies except for DIDS, which has been reported to inhibit rabbit Ntcp but not rabbit Asbt (18). Fig. 7 shows the BSP inhibition of taurocholate uptake is enhanced by Ͼ50% in D122A mutant transfected cells. In cells transfected with R64A, K191Q, K225Q, R256Q, E261A, and K312Q,K313Q mutants, the inhibition of taurocholate influx by BSP was also increased by 20 -30%. The inhibition of [ 3 H]taurocholate transport by the other non-bile acid organic anions tested (BUM, DIDS, E3S, and probenecid) was not significantly changed in cells transfected with mutants compared with wild-type rAsbt-GFP-transfected cells.
Electrophysiological Analysis of Substrate-dependent Currents in the Xenopus Oocytes Expressing Wild-type and Mutant rAsbt-GFP-Previous studies indicated that the Na ϩ -dependent bile acid cotransport process is electrogenic with a substrate binding site composed of a closely positioned negatively charged group that interacts with a single sodium ion (10,16). However, because of technical difficulties, so far there has been no direct evidence of electrical currents generated from bile acid and Na ϩ movement by Asbt. To further understand the mech-anism of Na ϩ /bile acid cotransport, we examined the relationship between electrical and chemical driving forces of rAsbt in the Xenopus oocyte expression system, which is widely used to study structure and function of ion channels, receptors, and transporters. First, the surface expression of rAsbt-GFP and mutants in the Xenopus oocytes was examined by confocal microscopy. The results show that the wild-type and mutant rAsbt-GFP proteins were expressed on the cell membrane of oocytes and no GFP fluorescence was observed in non-injected oocytes (data not shown). The whole-oocyte currents were then obtained before and after application of 1 mM taurocholate with a family of test voltage pulses to the oocyte. The taurocholate-induced currents were obtained from the current difference in the absence and presence of taurocholate at each test voltage. In Fig. 8A, whole-oocyte family currents in non-injected oocytes showed no measurable voltage-dependent current changes before and after application of 1 mM taurocholate. In contrast, application of 1 mM taurocholate significantly increased whole-cell currents in oocytes expressing wild-type rAsbt-GFP (Fig. 8B). The taurocholate-induced currents were measured in the same way for the rAsbt-GFP mutants. Interestingly, a small fraction of whole-oocyte current was detected from oocytes expressing rAsbt-GFP even without application of taurocholate (Fig. 8B, left panel), suggesting that Na ϩ ions are able to move and bind to Asbt under the test voltages in the absence of taurocholate. Fig. 8C shows the I-V curves of the taurocholate-induced currents in oocytes expressing rAsbt-GFP wild type or one of its mutants. These results demonstrate that a significant increase of whole-oocyte current only occurred when taurocholate was applied to oocytes injected with rAsbt-GFP or one of its mutant cRNAs. These inward negative currents were induced by taurocholate and were voltage dependent (Fig. 8C). As shown in Fig. 8, C and D, the taurocholateinduced currents from oocytes expressing D122A or E261A mutant were significantly decreased by Ͼ50% compared with those express- ing wild-type rAsbt-GFP. In contrast, the taurocholate-induced currents in oocytes with C270A mutant were increased by Ͼ30% compared with those expressing wild-type rAsbt-GFP. These findings are consistent with our taurocholate transport results described above (see Fig. 2) and suggest that taurocholate uptake is closely accompanied by movement of Na ϩ ions. These data further indicate that the residues of Asp-122 and Glu-261 are critical for both taurocholate and Na ϩ movement and Cys-270 may be located in a sub- strate binding-sensitive domain to optimize both taurocholate and Na ϩ uptake.

DISCUSSION
Early studies using mutagenesis by Hallén and colleagues (27) suggested that the maintenance of a negative charge by a D282E substitu-tion in the mouse Asbt did not affect TC uptake activity. Zhang et al. (2) have recently shown that replacement of Glu-282 with alanine significantly decreased taurocholate transport activity of human ASBT. These data indicated that a negative charge at 282 of Asbt may be critical for interaction with the 12␣-OH group of the bile acids. Previous studies also predicted that positively charged residues of bile acid transporters were important for interaction with substrates (2, 24 -26). So far, there is little experimental evidence reported for the functional relevance of these conserved positively charged residues of Asbt. Studies from topology scanning, putative three-dimensional structure, and computation prediction suggested that Arg-254 and Arg-256 of human ASBT may interact with the 12␣-OH group (2). Our results showed that replacement of the charged Arg-64, Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt with non-charged Ala (A) or Gln (Q) significantly decreased the initial taurocholate transport activity and binding affinity but had no effect on their plasma membrane expression. This indicates that these charged residues of rat Asbt are critical for ligand-carrier protein interaction but not for membrane trafficking.
The 7␣and 12 ␣-hydroxyl groups of natural bile acids are of importance for molecular recognition of a bile acid molecule by Asbt (24). If one of these two hydroxyl groups is removed to yield TCDC (12␣-OH removed) or TDC (7␣-OH removed), the affinity of the bile acid for Asbt is increased (24). The 3␣-hydroxy group of bile acids is also required for optimal transport (24). If all of the three hydroxyl groups are removed, the resulting TDHC had no affinity for Asbt (24).
The data from the inhibition studies using bile acids reveal that mutations at Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues significantly reduced the inhibition of taurocholate uptake by TDC and TCDC but did not have any effect on the inhibition of taurocholate uptake by LCA, TDHC, and cholate. In contrast with the effects of bile acids, mutations at Arg-64, Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues significantly enhanced the inhibition of taurocholate uptake by BSP but did not affect the inhibitions by other non-bile acid organic anions (E3S, BUM, DIDS, and probenecid). These data indicate that these residues have different affinities for bile acids and non-bile acid organic anions and suggest that these residues are important for determination of substrate binding specificity.
Mutations at Asp-122, Lys-191, and Lys-254 residues of rAsbt reduced the inhibition of taurocholate uptake by TCDC, but not by TDC. The D122A mutation almost completely abolished the TCDC inhibition of taurocholate uptake, suggesting that this residue acts as a hydrogen bond acceptor. Therefore, Asp-122 may interact with the 7␣-OH of taurocholate. Based on the work of Zhang et al. (2) and our data, it is suggested that the 12␣-OH and 7␣-OH groups of taurocholate may form H-bonds with Glu-282 and Asp-122 of Asbt, respectively. When the Asp-122 group of Asbt is replaced with Ala (D122A), the 12␣-OH group of TDC still can compete with taurocholate to form an H-bond with the carboxyl group of Glu-282 and reduce the taurocholate uptake activity. The 7␣-OH group of TCDC (which has no 12␣-OH group) cannot form an H-bond with the non-charged Ala group of D122A mutant. Therefore, the inhibition of taurocholate uptake by TCDC was abolished by D122A mutation.
The electrogenicity of Na ϩ -dependent bile acid transporters (human ASBT and rat Ntcp) and other transporters (Na ϩ /glucose and Na ϩ / amino acid transporters) has been reported from several laboratories and suggests that the stoichiometry of transport is two sodium ions and one ligand molecule (10,16,23). Zahner et al. (10) reported that the negatively charged Glu-257 and Asp-115 of rat Ntcp are important for the Na ϩ ion movement and extracellular Glu-257 (as a sodium ion sensor) and cytoplasmic Asp-115 may constitute an appropriate pair of binding sites for sodium ions allowing ion translocation across the cell membrane. Our results for the first time present experimental evidence to show that taurocholate movement by Asbt induces voltage-dependent current change. Consistent with the taurocholate movement, mutation at Asp-122 and Glu-261 of rat Asbt significantly decreased the voltage-dependent current. These findings suggest that taurocholate transport is closely accompanied by movement of a Na ϩ ion and indicate that the residues of Asp-122 and Glu-261 of rat Asbt are critical for both ligand and Na ϩ transport.
Hallén et al. (17) demonstrated that an alanine substitution of cysteine 270 (human ASBT)/266 (human NTCP) significantly decreases the inactivation by thiol reagents but kinetic parameters for sodium activation and taurocholate transport were largely unaffected by the cysteine to alanine substitutions, showing that this residue is nonessential in a functionally important region of the transporter. Our results are consistent with Hallén's findings. The data show that replacement of Cys-270 of rat Asbt with Ala results in enhancement of taurocholate uptake and voltage-dependent currents but there is no change in membrane distribution or K m value compared with wild-type Asbt. Cys-270 may be located in a region near the substrate binding site so that replacement of the Cys with Ala results in a change of the steric environment of the binding site, leading to an increase in V max for the transport process.
Topology studies of Na ϩ /bile acid cotransporter proteins from several laboratories suggested a 7-or 9-transmembrane (TM) configura- FIGURE 8. Mutagenesis of conserved amino acids of rAsbt-GFP alters taurocholate-induced currents. Representative whole-oocyte family currents across the plasma membrane before and after application of 1 mM TC for a noninjected oocyte (A) and an oocyte expressing rAsbt-GFP (B). C, current-voltage relationships of the current difference between the presence and absence of 1 mM TC for rAsbt-GFP and mutants (E122A, D126A, and C271A) representing TC-sensitive current. D, TC-sensitive currents at test voltage Ϫ140 mV. p Ͻ 0.05 between wild type and three mutants.
(2) employed a two-tiered bioinformatics and N-glycosylation-scanning mutagenesis approach to effectively distinguish between a 7-TM and 9-TM topology. Their data strongly support a 7-TM helical model of human ASBT (2). Therefore, on the basis of the 7-TM topology, we built a homology model (Fig. 9) for rat Asbt and positioned the functionally relevant residues examined in this study. As shown in Fig. 9, Lys-225 (at the border of intracellular loop 5 and TM domain VI) and Lys-312,Lys-313 (at the cytoplasmic tail near the border of TM domain VII) may be involved in the release of ligand to the cytoplasm. Arg-256 and Glu-261 (both at extracellular highly conserved loop 6) and Lys-191 (at the border of extracellularloop 4 and TM domain V) may function as substrate recognition sensors and interact with ligands. In contrast with Asp-115 of Ntcp, which is located at an intracellular loop, Asp-122 of Asbt located at extracellular loop 2 may also play a important role in substrate selectivity and may directly interact with the 7␣-OH group of bile acid and the Na ϩ ion. Lys-254 located at highly conserved extracellular loop 6 may only be involved in interaction with bile acids (TCDC). The Arg-64 (located at intracellular loop 1) mutation affected only BSP transport, not transport of bile acids and other non-bile acid organic anions. This suggests that natural bile acids and non-bile acid organic anions may interact with Asbt differently and at more than one tightly related binding site. Cys-270 is located at a highly conserved and functionally important region (the largest extracellular loop 6) and may be involved in optimizing the Na ϩ ion and ligand transport process.
In summary, our data suggest the following. 1) Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt are critical for transport function and determination of substrate specificity. 2) As a hydrogen bond acceptor, Asp-122 may interact with the 7␣-OH group of natural bile acids to form an H-bond and Arg-254 may act as a compensating group for this dynamic interaction. Arg-64 may be located at a tightly related but different binding site important for nonbile acid substrate specificity. 3) Na ϩ ion movement by ASBT is a voltagedependent process that is tightly coupled to taurocholate translocation. Asp-122 and Glu-261 play critical roles in the interaction of ASBT with Na ϩ and ligand and/or their transport processes. 4) Cys-270 is also located at a ligand-and Na ϩ movement-sensitive region but is not essential for transport. Our results provide further insight into understanding the molecular mechanisms underlying the structure and function of Na ϩ -dependent transport proteins and may further suggest strategies for drug design to inhibit intestinal bile acid reabsorption.