BACKGROUND
bile acids are synthesized from cholesterol in the liver and secreted into the small intestine as components of bile for the digestion and absorption of lipids and lipid-soluble vitamins.
in addition to the detergent action of bile acids, which aids in the digestion and absorption of lipid and lipid-soluble nutrients by forming micelles with biliary phospholipids and cholesterol, bile acids are now appreciated as signaling molecules that control lipid metabolism and energy production  <cit> .
at the distal end of the ileum, 95%–98% of bile acids are effectively reabsorbed by an ileal sodium-dependent bile acid transporter  and returned to the liver via portal circulation.
among the transporters that are expressed in the liver, intestine, and bile duct and are involved in enterohepatic circulation of bile acids, slc10a <dig> is the key transporter for understanding the in vivo kinetics of bile acids given that reabsorption of bile acids by slc10a <dig> is the first step in enterohepatic circulation.
slc10a <dig> is the second member of the solute carrier family  <dig>  and consists of  <dig> amino acids.
slc10a <dig> is expressed in the ileum, cholangiocytes, and kidney, and contributes to the maintenance of the bile acid pool and cholesterol homeostasis  <cit> .
transport of bile acids by slc10a <dig> is facilitated by sodium symport in an electrogenic process with a 2: <dig> na+/bile acid stoichiometry  <cit> .
given that bile acids are synthesized from cholesterol, inhibition of bile acid reabsorption via slc10a <dig> inhibition has been used as a cholesterol-lowering therapy.
moreover, due to its high transport capacity in the ileum, slc10a <dig> is also an attractive target for the prodrug strategy to enhance drug bioavailability  <cit> .
the membrane topology and detailed transport mechanism of slc10a <dig> have been studied.
hydropathy analysis and membrane insertion scanning revealed that slc10a <dig> has an extracellular n-terminus and a cytoplasmic c-terminus  <cit> .
the exact membrane topology remains controversial: in vitro translation studies using membrane insertion scanning suggested a 9-transmembrane  topology, whereas n-glycosylation scanning mutagenesis and dual-label epitope insertion scanning mutagenesis support a 7-tm topology  <cit> .
the recently published crystal structure of a bacterial homolog of slc10a <dig> from neisseria meningitidis  supports the 9-tm topology  <cit> .
protein regions and amino acid residues of slc10a <dig> involved in membrane trafficking, substrate recognition, and substrate permeation have been identified.
the cytoplasmic tail of rat slc10a <dig> acts as a sorting signal for apical trafficking, and ser <dig> and thr <dig> phosphorylations are crucial for apical targeting  <cit> .
computational analysis based on homology-modeling and remote-threading techniques revealed that asp <dig> and leu <dig> of human slc10a <dig> are involved in hydrogen bond formation with the 12α-hydroxyl group of bile acids  <cit> .
a series of analyses using the substituted-cysteine accessibility method revealed that in the 7-tm model tm <dig>  lines the substrate translocation pathway, tm <dig>  forms part of the pathway, asp <dig> interacts with the 7α-hydroxyl group of bile acids, and the extracellular loop   <dig> corresponding to val99–ser <dig> acts as a na+ sensor  <cit> .
glu <dig> in el <dig> has also been shown to act as a na+ sensor, and el <dig> and el <dig> have been proposed to act as re-entrant loop segments  <cit> .
despite such extensive studies, the mechanisms underlying the binding and transport of bile acids remain unclear.
genes homologous to the mammalian slc <dig> family are widespread in various species  <cit> .
in the alignment of the deduced sequences of these genes, conserved residues are scattered throughout the entire sequences, and some of them are clustered in a region spanning approximately  <dig> residues corresponding to gly104–pro <dig> of slc10a <dig> .
the high-level conservation indicates that this region may play an important role in substrate interaction, conformational change necessary for function, or interaction with cellular cofactors.
as mentioned above, a part of this region corresponding to el <dig> has been proposed to function as a dynamic re-entrant loop, but the importance of this region has not yet been fully elucidated.
to determine the importance of this region, we previously performed mutational analysis of the uncharged polar residues in the distal one-third  of the highly conserved region of mouse slc10a <dig>  and identified residues that may be involved in substrate recognition, transport activity, and cellular localization  <cit> .
in this study, we focused on the uncharged polar residues  and proline  in the remaining part of the highly conserved region.
the hydroxyl group of the side chains could form hydrogen bonds with the substrate and/or other residues and contribute to the transport process and/or formation of higher-order structures, and proline is expected to contribute to the formation of higher-order structure.
to determine the involvement of these residues in substrate recognition, transport, and intracellular sorting and/or stability of mslc10a <dig>  taurocholic acid  transport and cell surface localization were analyzed.
RESULTS
wild-type mslc10a <dig> and pro <dig> mutants  were stably expressed as enhanced green fluorescent protein  fusion proteins in llc-pk <dig> cells, and tca uptake and cellular localization were analyzed.
cells expressing wild-type slc10a <dig> exhibited na+-dependent uptake of tca, but this transport activity was completely abolished in both p107n- and p107l-expressing cells .
western blot analysis of whole cell lysates revealed that wild-type and mutant mslc10a <dig> were abundantly expressed as 55-kda bands, and an additional band with a lower migration rate at approximately  <dig> kda was observed only in the wild type .
because the latter band is considered to represent a fully glycosylated protein that is localized to the plasma membrane, we concluded that the mutant slc10a <dig> proteins were likely detained in the endoplasmic reticulum  and did not reach the cell surface.
this was confirmed by analyzing plasma membrane proteins .
the glycosylated form was detected only in the wild-type plasma membrane fraction.
next, we examined the transient expression of wild-type mslc10a <dig> and uncharged polar residue mutants  in cos- <dig> cells by western blot analysis .
wild-type mslc10a <dig> and all the mutants except s126a were expressed as 55-kda proteins.
a broad band ranging from 60– <dig> kda, the fully glycosylated form, was also detected in each lane.
to evaluate the ability of the mutants to transport bile acids, tca uptake by the wild-type and mutant mslc10a <dig> was compared .
because cellular expression of s126a was not detected, this mutant was omitted from further analysis.
the s128a mutant exhibited uptake levels comparable to that of wild type, and tca uptake by the t110a mutant was significantly higher than that of wild type.
tca uptake by the y117f mutant was approximately half that by the wild type, but the difference was not statistically significant.
s112a did not exhibit na+-dependent tca uptake.
the kinetics of tca transport by the wild type and the  <dig> functional mutants were examined .
the vmax of y117f was approximately half that of the wild type, and those of t110a and s128a were  <dig> - and 5-fold higher than that of the wild type, respectively; interestingly, the km values of the  <dig> mutants were comparable to that of the wild type, suggesting that the differences in the apparent tca transport activities of the polar residue mutants were not due to altered affinity for tca.
data are expressed as mean .
the cellular localization of the expressed transporters was investigated by cell surface biotinylation .
the fully glycosylated form was predominantly detected in the biotinylated fraction.
membrane expression of y117f was similar to that of wild type, and the membrane expression of t110a and s128a was significantly higher than that of wild type, suggesting that removal of the polar hydroxyl group from thr <dig> and ser <dig> improved membrane sorting and/or stability of mslc10a <dig>  although s112a was a loss-of-function mutation, membrane expression of the mutant was clearly detected, indicating that ser <dig> is critical for the activity of mslc10a <dig>
to compare the activities of the mutant transporters, the na+-dependent tca uptake by wild-type and mutant mslc10a <dig> was normalized to the cell surface expression of the corresponding proteins .
the normalized vmax of the t110a mutant  was similar to that of the wild type , indicating that the difference in the apparent tca transport activity of this mutant was mainly due to the different level of its expression at the cell surface.
by contrast, the apparently higher activity of s128a could not be explained by abundant membrane expression because the normalized vmax of the s128a mutant  was remarkably higher than that of wild type.
the normalized vmax value of the y117f mutant  was lower than that of wild type.
the y117c mutation in human slc10a <dig> has been reported to be a loss-of-function mutation due to its impaired membrane expression  <cit> .
this is in striking contrast to our finding that the y117f mutant of mslc10a <dig> was properly expressed on the plasma membrane and that transport activity was preserved even though its apparent activity was lower.
because the tyr to phe mutation used in the present study removed only the polar hydroxyl group from the benzene ring of the side chain, we infer that what is important at this position is not polarity but the bulkiness required for higher-order structure or stability of slc10a2/slc10a <dig>  the complete conservation of bulky residues, i.e., tyr, phe, or leu, at this position in the related genes  supports this view.
therefore, we next examined the cell surface expression and transport activities of mslc10a <dig> mutants in which various residues were substituted for tyr <dig>  for this purpose, a t <dig> tag was attached at the n-terminus of mslc10a <dig>  which is exposed on the cell surface.
western blot analysis revealed that the wild-type and tyr <dig> mutants were expressed as 40-kda bands; additional bands with lower migration rates, ranging from 50– <dig> kda, were also detected in each lane, indicating that all the tyr <dig> mutants as well as the wild type were properly expressed on the plasma membrane .
cell surface expression was confirmed with immunofluorescence.
under nonpermeabilized conditions, the plasma membrane was predominantly stained, whereas additional intracellular compartments were also stained under permeabilized conditions .
cell surface expression levels were examined by surface enzyme-linked immunosorbent assay.
the cell-surface expression of the tyr <dig> mutants was not significantly lower than that of wild type .
na+-dependent tca uptake by all the tyr <dig> mutants, except y117f, was significantly lower than that of wild type .
next, we analyzed the kinetic properties of the y117s mutant, which exhibited remarkably reduced tca uptake compared to that of wild type.
the km for na+-dependent tca uptake by the y117s mutant  was similar to that of wild type , whereas the vmax of y117s  was remarkably lower than that of wild type  .
these results indicate that the apparently decreased activities of the tyr <dig> mutants are probably due to their decreased molecular activities.
discussion
optimal function of slc10a <dig> is required for bile acid reabsorption in the ileum.
impairment of this function not only affects cholesterol homeostasis but may also increase the possibility of colorectal tumorigenesis due to increased flow of bile acids into the large intestine.
indeed, prevention of bile acid reabsorption by surgical removal of the ileum increased colonic tumorigenesis in rats fed deoxycholic acid  <cit> .
the c to t polymorphism at codon  <dig> of the human slc10a <dig> gene is associated with colorectal adenomas, indicating the role of bile acids in the etiology of this disease  <cit> .
a genetic polymorphism associated with primary bile acid malabsorption  or idiopathic intestinal bile acid malabsorption  has been identified, and mutations that abolish transport function  and a haplotype block linked to reduced expression have been reported for human slc10a <dig>  <cit> .
this polymorphism has not been mapped in the highly conserved region, and the importance of the cluster of conserved residues has not yet been fully clarified.
toward the end of the study presented here, the crystal structure of asbtnm was reported, and some of the residues were indicated to form a part of the na+-binding pocket  <cit> .
we have previously reported the importance of pro <dig>  which is located at the distal end of the highly conserved region  <cit> .
substitution of pro <dig> with val completely impaired cell surface localization of mslc10a <dig>  this is consistent with our results from the mutational analysis of pro <dig> showing that asn or leu substitution for pro <dig> impaired cell surface expression of mslc10a <dig>  resulting in the loss of transport activity.
in the 9-tm model, pro <dig> is located in the middle of tm <dig>  proline acts as a “helix breaker” due to its inability to form hydrogen bonds with neighboring residues; therefore, tm <dig> would be bent at pro <dig>  forming hydrophobic and amphipathic half helices.
indeed, the crystal structure of asbtnm suggests that the helix that contains pro <dig> is broken precisely at this residue  <cit> .
in the 7-tm model, pro <dig> is located in the el between tm <dig> and tm <dig>  in the er, immediately after the synthesis of the nascent protein, this loop faces the lumen.
substitution of pro <dig> with asn or leu may have introduced an interaction of this loop with other els or er factors, resulting in detention of the mutant slc10a <dig>  in either case, failure of the pro <dig> mutants to localize to the cell surface suggests that the peculiar nature of proline, an imino acid, and not the hydrophobicity or bulkiness of the side chain is important for intracellular sorting.
given that proline at this position is highly conserved in the related proteins, it is expected to be crucial for function through correct secondary structure formation and cellular localization.
ser <dig> was considered indispensable for the synthesis or stability of human slc10a <dig> because replacement of ser <dig> with cys completely abolished expression  <cit> .
in this study, however, ala substitution of ser <dig> did not impair membrane expression of mslc10a <dig>  whereas tca transport activity was completely lost.
this suggests that ser <dig> is not critical for mslc10a <dig> expression but is essential for tca transport activity.
based on the crystal structure of asbtnm, ser <dig> of mslc10a <dig> is thought to bind na+ with its side chain  <cit> .
ser is conserved at this position in all the related proteins except slc10a <dig> , suggesting that this residue is also critical in all the other members.
expression of the s126a mutant was undetectable even in whole cell lysates.
this is consistent with a previous report showing the failure of expression of the human slc10a <dig> s126c mutant  <cit> .
this residue has been suggested to bind na+ <cit> , and it is likely that ser <dig> plays an important role in the other members as well, given that this residue is conserved in all the genes except those encoding slc10a <dig> and slc10a <dig>
because thr <dig> and ser <dig> are not conserved in the related proteins, particularly in the na+-dependent bile acid transporter slc10a <dig> , which is expressed on the sinusoidal membrane of hepatocytes, it is unlikely that these residues are involved in the interaction with bile acids.
the apparently higher membrane expression of the t110a and s128a mutants suggests that these residues may be involved in the negative regulation of stability or intracellular sorting via formation of higher-order structures or interaction with cellular cofactors.
the phe substitution for tyr <dig>  which removes a polar hydroxyl group, reduced the transport activity of mslc10a2; however, substrate affinity was not affected.
this result was inconsistent with the previous finding that the y117c mutation completely impaired membrane expression of human slc10a <dig>  to resolve this discrepancy, we replaced tyr <dig> of mslc10a <dig> with various amino acids and examined cellular localization and transport activities.
none of the residues substituted for tyr <dig> affected cell surface expression of mslc10a2; however, the apparent transport activities were reduced.
the reason for the contradiction between our results and the results of the y117c mutation in human slc10a <dig> is not clear, but a difference in the amino acid sequence context between mslc10a <dig> and the human counterpart or undefined differences in experimental conditions may have affected the results.
the relationship between the apparent activities and physicochemical properties of the residues were analyzed.
hydrophobicity and polarity did not correlate with activity , but a weak correlation was observed between the van der waals volumes of the residues and the activities of the mutants .
however, the correlation was not statistically significant , because the y117w mutant with the bulkiest side chain exhibited moderate activity, and substitution with ile and leu, which have relatively bulky side chains, elicited low transport activities, suggesting that the volume of the side chain as well as its shape are important at this position.
although the crystal structure of asbtnm indicated that tyr <dig> does not directly interact with na+ or taurocholate, it is obvious that this residue is important for the molecular activity of the transporter.
tyr <dig> may be involved in conformational changes during tca transport.
CONCLUSIONS
in this study, residues critical for transport activity, expression, and stability and/or intracellular trafficking, but not substrate recognition, were identified within the proximal two-thirds of the highly conserved region.
functionally important residues are clustered in the highly conserved region.
due to the specific and high transport capacity of slc10a <dig> in the ileum, bioavailability of drugs may be enhanced by designing them as bile acid-conjugated prodrugs.
the information on functionally critical residues will contribute to the design of prodrugs for efficient drug delivery and slc10a <dig> inhibitors for treatment of hypercholesterolemia.

 bile acids are synthesized from cholesterol in the liver and secreted into the small intestine as components of bile for the digestion and absorption of lipids and lipid-soluble vitamins. in addition to the detergent action of bile acids, which aids in the digestion and absorption of lipid and lipid-soluble nutrients by forming micelles with biliary phospholipids and cholesterol, bile acids are now appreciated as signaling molecules that control lipid metabolism and energy production. at the distal end of the ileum, 95%–98% of bile acids are effectively reabsorbed by an ileal sodium-dependent bile acid transporter and returned to the liver via portal circulation. among the transporters that are expressed in the liver, intestine, and bile duct and are involved in enterohepatic circulation of bile acids, slc10a is the key transporter for understanding the in vivo kinetics of bile acids given that reabsorption of bile acids by slc10a is the first step in enterohepatic circulation. slc10a is the second member of the solute carrier family and consists of amino acids. slc10a is expressed in the ileum, cholangiocytes, and kidney, and contributes to the maintenance of the bile acid pool and cholesterol homeostasis. transport of bile acids by slc10a is facilitated by sodium symport in an electrogenic process with a 2: na+/bile acid stoichiometry. given that bile acids are synthesized from cholesterol, inhibition of bile acid reabsorption via slc10a inhibition has been used as a cholesterol-lowering therapy. moreover, due to its high transport capacity in the ileum, slc10a is also an attractive target for the prodrug strategy to enhance drug bioavailability. the membrane topology and detailed transport mechanism of slc10a have been studied. hydropathy analysis and membrane insertion scanning revealed that slc10a has an extracellular n-terminus and a cytoplasmic c-terminus. the exact membrane topology remains controversial: in vitro translation studies using membrane insertion scanning suggested a 9-transmembrane topology, whereas n-glycosylation scanning mutagenesis and dual-label epitope insertion scanning mutagenesis support a 7-tm topology. the recently published crystal structure of a bacterial homolog of slc10a from neisseria meningitidis supports the 9-tm topology. protein regions and amino acid residues of slc10a involved in membrane trafficking, substrate recognition, and substrate permeation have been identified. the cytoplasmic tail of rat slc10a acts as a sorting signal for apical trafficking, and ser and thr phosphorylations are crucial for apical targeting. computational analysis based on homology-modeling and remote-threading techniques revealed that asp and leu of human slc10a are involved in hydrogen bond formation with the 12α-hydroxyl group of bile acids. a series of analyses using the substituted-cysteine accessibility method revealed that in the 7-tm model tm lines the substrate translocation pathway, tm forms part of the pathway, asp interacts with the 7α-hydroxyl group of bile acids, and the extracellular loop corresponding to val99–ser acts as a na+ sensor. glu in el has also been shown to act as a na+ sensor, and el and el have been proposed to act as re-entrant loop segments. despite such extensive studies, the mechanisms underlying the binding and transport of bile acids remain unclear. genes homologous to the mammalian slc family are widespread in various species. in the alignment of the deduced sequences of these genes, conserved residues are scattered throughout the entire sequences, and some of them are clustered in a region spanning approximately residues corresponding to gly104–pro of slc10a. the high-level conservation indicates that this region may play an important role in substrate interaction, conformational change necessary for function, or interaction with cellular cofactors. as mentioned above, a part of this region corresponding to el has been proposed to function as a dynamic re-entrant loop, but the importance of this region has not yet been fully elucidated. to determine the importance of this region, we previously performed mutational analysis of the uncharged polar residues in the distal one-third of the highly conserved region of mouse slc10a and identified residues that may be involved in substrate recognition, transport activity, and cellular localization. in this study, we focused on the uncharged polar residues and proline in the remaining part of the highly conserved region. the hydroxyl group of the side chains could form hydrogen bonds with the substrate and/or other residues and contribute to the transport process and/or formation of higher-order structures, and proline is expected to contribute to the formation of higher-order structure. to determine the involvement of these residues in substrate recognition, transport, and intracellular sorting and/or stability of mslc10a taurocholic acid transport and cell surface localization were analyzed.  wild-type mslc10a and pro mutants were stably expressed as enhanced green fluorescent protein fusion proteins in llc-pk cells, and tca uptake and cellular localization were analyzed. cells expressing wild-type slc10a exhibited na+-dependent uptake of tca, but this transport activity was completely abolished in both p107n- and p107l-expressing cells. western blot analysis of whole cell lysates revealed that wild-type and mutant mslc10a were abundantly expressed as 55-kda bands, and an additional band with a lower migration rate at approximately kda was observed only in the wild type. because the latter band is considered to represent a fully glycosylated protein that is localized to the plasma membrane, we concluded that the mutant slc10a proteins were likely detained in the endoplasmic reticulum and did not reach the cell surface. this was confirmed by analyzing plasma membrane proteins. the glycosylated form was detected only in the wild-type plasma membrane fraction. next, we examined the transient expression of wild-type mslc10a and uncharged polar residue mutants in cos- cells by western blot analysis. wild-type mslc10a and all the mutants except s126a were expressed as 55-kda proteins. a broad band ranging from 60– kda, the fully glycosylated form, was also detected in each lane. to evaluate the ability of the mutants to transport bile acids, tca uptake by the wild-type and mutant mslc10a was compared. because cellular expression of s126a was not detected, this mutant was omitted from further analysis. the s128a mutant exhibited uptake levels comparable to that of wild type, and tca uptake by the t110a mutant was significantly higher than that of wild type. tca uptake by the y117f mutant was approximately half that by the wild type, but the difference was not statistically significant. s112a did not exhibit na+-dependent tca uptake. the kinetics of tca transport by the wild type and the functional mutants were examined. the vmax of y117f was approximately half that of the wild type, and those of t110a and s128a were - and 5-fold higher than that of the wild type, respectively; interestingly, the km values of the mutants were comparable to that of the wild type, suggesting that the differences in the apparent tca transport activities of the polar residue mutants were not due to altered affinity for tca. data are expressed as mean. the cellular localization of the expressed transporters was investigated by cell surface biotinylation. the fully glycosylated form was predominantly detected in the biotinylated fraction. membrane expression of y117f was similar to that of wild type, and the membrane expression of t110a and s128a was significantly higher than that of wild type, suggesting that removal of the polar hydroxyl group from thr and ser improved membrane sorting and/or stability of mslc10a although s112a was a loss-of-function mutation, membrane expression of the mutant was clearly detected, indicating that ser is critical for the activity of mslc10a to compare the activities of the mutant transporters, the na+-dependent tca uptake by wild-type and mutant mslc10a was normalized to the cell surface expression of the corresponding proteins. the normalized vmax of the t110a mutant was similar to that of the wild type, indicating that the difference in the apparent tca transport activity of this mutant was mainly due to the different level of its expression at the cell surface. by contrast, the apparently higher activity of s128a could not be explained by abundant membrane expression because the normalized vmax of the s128a mutant was remarkably higher than that of wild type. the normalized vmax value of the y117f mutant was lower than that of wild type. the y117c mutation in human slc10a has been reported to be a loss-of-function mutation due to its impaired membrane expression. this is in striking contrast to our finding that the y117f mutant of mslc10a was properly expressed on the plasma membrane and that transport activity was preserved even though its apparent activity was lower. because the tyr to phe mutation used in the present study removed only the polar hydroxyl group from the benzene ring of the side chain, we infer that what is important at this position is not polarity but the bulkiness required for higher-order structure or stability of slc10a2/slc10a the complete conservation of bulky residues, i.e., tyr, phe, or leu, at this position in the related genes supports this view. therefore, we next examined the cell surface expression and transport activities of mslc10a mutants in which various residues were substituted for tyr for this purpose, a t tag was attached at the n-terminus of mslc10a which is exposed on the cell surface. western blot analysis revealed that the wild-type and tyr mutants were expressed as 40-kda bands; additional bands with lower migration rates, ranging from 50– kda, were also detected in each lane, indicating that all the tyr mutants as well as the wild type were properly expressed on the plasma membrane. cell surface expression was confirmed with immunofluorescence. under nonpermeabilized conditions, the plasma membrane was predominantly stained, whereas additional intracellular compartments were also stained under permeabilized conditions. cell surface expression levels were examined by surface enzyme-linked immunosorbent assay. the cell-surface expression of the tyr mutants was not significantly lower than that of wild type. na+-dependent tca uptake by all the tyr mutants, except y117f, was significantly lower than that of wild type. next, we analyzed the kinetic properties of the y117s mutant, which exhibited remarkably reduced tca uptake compared to that of wild type. the km for na+-dependent tca uptake by the y117s mutant was similar to that of wild type, whereas the vmax of y117s was remarkably lower than that of wild type. these results indicate that the apparently decreased activities of the tyr mutants are probably due to their decreased molecular activities. discussion optimal function of slc10a is required for bile acid reabsorption in the ileum. impairment of this function not only affects cholesterol homeostasis but may also increase the possibility of colorectal tumorigenesis due to increased flow of bile acids into the large intestine. indeed, prevention of bile acid reabsorption by surgical removal of the ileum increased colonic tumorigenesis in rats fed deoxycholic acid. the c to t polymorphism at codon of the human slc10a gene is associated with colorectal adenomas, indicating the role of bile acids in the etiology of this disease. a genetic polymorphism associated with primary bile acid malabsorption or idiopathic intestinal bile acid malabsorption has been identified, and mutations that abolish transport function and a haplotype block linked to reduced expression have been reported for human slc10a. this polymorphism has not been mapped in the highly conserved region, and the importance of the cluster of conserved residues has not yet been fully clarified. toward the end of the study presented here, the crystal structure of asbtnm was reported, and some of the residues were indicated to form a part of the na+-binding pocket. we have previously reported the importance of pro which is located at the distal end of the highly conserved region. substitution of pro with val completely impaired cell surface localization of mslc10a this is consistent with our results from the mutational analysis of pro showing that asn or leu substitution for pro impaired cell surface expression of mslc10a resulting in the loss of transport activity. in the 9-tm model, pro is located in the middle of tm proline acts as a “helix breaker” due to its inability to form hydrogen bonds with neighboring residues; therefore, tm would be bent at pro forming hydrophobic and amphipathic half helices. indeed, the crystal structure of asbtnm suggests that the helix that contains pro is broken precisely at this residue. in the 7-tm model, pro is located in the el between tm and tm in the er, immediately after the synthesis of the nascent protein, this loop faces the lumen. substitution of pro with asn or leu may have introduced an interaction of this loop with other els or er factors, resulting in detention of the mutant slc10a in either case, failure of the pro mutants to localize to the cell surface suggests that the peculiar nature of proline, an imino acid, and not the hydrophobicity or bulkiness of the side chain is important for intracellular sorting. given that proline at this position is highly conserved in the related proteins, it is expected to be crucial for function through correct secondary structure formation and cellular localization. ser was considered indispensable for the synthesis or stability of human slc10a because replacement of ser with cys completely abolished expression. in this study, however, ala substitution of ser did not impair membrane expression of mslc10a whereas tca transport activity was completely lost. this suggests that ser is not critical for mslc10a expression but is essential for tca transport activity. based on the crystal structure of asbtnm, ser of mslc10a is thought to bind na+ with its side chain. ser is conserved at this position in all the related proteins except slc10a, suggesting that this residue is also critical in all the other members. expression of the s126a mutant was undetectable even in whole cell lysates. this is consistent with a previous report showing the failure of expression of the human slc10a s126c mutant. this residue has been suggested to bind na+, and it is likely that ser plays an important role in the other members as well, given that this residue is conserved in all the genes except those encoding slc10a and slc10a because thr and ser are not conserved in the related proteins, particularly in the na+-dependent bile acid transporter slc10a, which is expressed on the sinusoidal membrane of hepatocytes, it is unlikely that these residues are involved in the interaction with bile acids. the apparently higher membrane expression of the t110a and s128a mutants suggests that these residues may be involved in the negative regulation of stability or intracellular sorting via formation of higher-order structures or interaction with cellular cofactors. the phe substitution for tyr which removes a polar hydroxyl group, reduced the transport activity of mslc10a2; however, substrate affinity was not affected. this result was inconsistent with the previous finding that the y117c mutation completely impaired membrane expression of human slc10a to resolve this discrepancy, we replaced tyr of mslc10a with various amino acids and examined cellular localization and transport activities. none of the residues substituted for tyr affected cell surface expression of mslc10a2; however, the apparent transport activities were reduced. the reason for the contradiction between our results and the results of the y117c mutation in human slc10a is not clear, but a difference in the amino acid sequence context between mslc10a and the human counterpart or undefined differences in experimental conditions may have affected the results. the relationship between the apparent activities and physicochemical properties of the residues were analyzed. hydrophobicity and polarity did not correlate with activity, but a weak correlation was observed between the van der waals volumes of the residues and the activities of the mutants. however, the correlation was not statistically significant, because the y117w mutant with the bulkiest side chain exhibited moderate activity, and substitution with ile and leu, which have relatively bulky side chains, elicited low transport activities, suggesting that the volume of the side chain as well as its shape are important at this position. although the crystal structure of asbtnm indicated that tyr does not directly interact with na+ or taurocholate, it is obvious that this residue is important for the molecular activity of the transporter. tyr may be involved in conformational changes during tca transport.  in this study, residues critical for transport activity, expression, and stability and/or intracellular trafficking, but not substrate recognition, were identified within the proximal two-thirds of the highly conserved region. functionally important residues are clustered in the highly conserved region. due to the specific and high transport capacity of slc10a in the ileum, bioavailability of drugs may be enhanced by designing them as bile acid-conjugated prodrugs. the information on functionally critical residues will contribute to the design of prodrugs for efficient drug delivery and slc10a inhibitors for treatment of hypercholesterolemia. 
because bile acids act not only as detergents but also as signaling molecules in lipid metabolism and energy production, slc10a is important as the key transporter for understanding the in vivo kinetics of bile acids. slc10a2-mediated reabsorption of bile acids at the distal end of the ileum is the first step in enterohepatic circulation. to elucidate the functional importance of this region, we previously performed mutational analysis of the uncharged polar residues and proline in the distal one-third of the highly conserved region in mouse slc10a in this study, proline and uncharged polar residues in the remaining two-thirds of this region in mouse slc10a were subjected to mutational analysis, and taurocholic acid uptake and cell surface localization were examined. cell surface localization of slc10a is necessary for bile acid absorption. mutants in which asp or leu were substituted for pro were abundantly expressed, but their cell surface localization was impaired. the s112a mutant was properly expressed at the cell surface but transport activity was completely lost. the s126a mutant was completely impaired in cellular expression. the degree of reduction roughly depended on the van der waals volume of the side chains. replacement of tyr with various amino acids resulted in reduced transport activity. bile acidenterohepatic circulationileal sodium-dependent bile acid transporter this information will contribute to the design of bile acid-conjugated prodrugs for efficient drug delivery or slc10a inhibitors for hypercholesterolemia treatment. the t110a and s128a mutants exhibited remarkably enhanced membrane expression. the functional importance of proline and uncharged polar residues in the highly conserved region of mouse slc10a was determined.
 slc10a2-mediated reabsorption of bile acids at the distal end of the ileum is the first step in enterohepatic circulation. because bile acids act not only as detergents but also as signaling molecules in lipid metabolism and energy production, slc10a is important as the key transporter for understanding the in vivo kinetics of bile acids. slc10a family members and the homologous genes of various species share a highly conserved region corresponding to gly104–pro of slc10a the functional importance of this region has not been fully elucidated.  to elucidate the functional importance of this region, we previously performed mutational analysis of the uncharged polar residues and proline in the distal one-third of the highly conserved region in mouse slc10a in this study, proline and uncharged polar residues in the remaining two-thirds of this region in mouse slc10a were subjected to mutational analysis, and taurocholic acid uptake and cell surface localization were examined. cell surface localization of slc10a is necessary for bile acid absorption. mutants in which asp or leu were substituted for pro were abundantly expressed, but their cell surface localization was impaired. the s126a mutant was completely impaired in cellular expression. the t110a and s128a mutants exhibited remarkably enhanced membrane expression. the s112a mutant was properly expressed at the cell surface but transport activity was completely lost. replacement of tyr with various amino acids resulted in reduced transport activity. the degree of reduction roughly depended on the van der waals volume of the side chains.  the functional importance of proline and uncharged polar residues in the highly conserved region of mouse slc10a was determined. this information will contribute to the design of bile acid-conjugated prodrugs for efficient drug delivery or slc10a inhibitors for hypercholesterolemia treatment. bile acidenterohepatic circulationileal sodium-dependent bile acid transporter 