Transport of taurocholate by mutants of negatively charged amino acids, cysteines, and threonines of the rat liver sodium-dependent taurocholate cotransporting polypeptide Ntcp Daniel Za
Trang 1Transport of taurocholate by mutants of negatively charged amino acids, cysteines, and threonines of the rat liver sodium-dependent taurocholate cotransporting polypeptide Ntcp
Daniel Zahner, Uta Eckhardt and Ernst Petzinger
Institute of Pharmacology and Toxicology, Justus-Liebig-University Giessen, Germany
The relevance of functional amino acids for taurocholate
transport by the sodium-dependent taurocholate
cotrans-porting polypeptide Ntcp was determined by site-directed
mutagenesis cRNA from 28 single-points mutants of the rat
liver Ntcp clone was expressed in Xenopus laevis oocytes
Mutations were generated in five conserved negatively
charged amino acids (aspartates and glutamates) which were
present in nine members of the SBAT-family, in two
non-conserved negatively charged amino acids, in all eight
Ntcp-cysteines, and in two threonines from a protein kinase C
consensus region of the Ntcp C-terminus Functional amino
acids were Asp115, Glu257, and Cys266, which were found
to be essential for the maintenance of taurocholic acid
transport Asp115 is located in the large intracellular loop
III, whereas Glu257 and Cys266 are located in the large
extracellular loop VI Four mutations of threonines from the C-terminus of the Ntcp by alanines or tyrosines showed no effects on sodium-dependent taurocholate transport Intro-duction of the FLAGmotif into several transport negative point mutations demonstrated that all mutated proteins besides one were present within the cell membrane of the oocytes and provided proof that an insertion defect has not caused transport deficiency by these Ntcp mutants The latter was observed only with the transport negative mutant Asp24Asn In conclusion, loop amino acids are required for sodium-dependent substrate translocation by the Ntcp Keywords: bile acids; P-loop; glutamate; aspartate; mem-brane protein
The sodium-dependent taurocholate cotransporting
poly-peptide Ntcp from rat liver is the major basolateral bile acid
transporter of rat hepatocytes It was the first
sodium-dependent bile acid cotransporter (SBAT), that was
obtained by expression cloning in Xenopus laevis oocytes
[1] It exhibits 77.4% identity and 88.8% similarity on
amino acid level with the human transporter NTCP [2] The
proteins are coded by the Slc/SLC10 gene family in animals
and man SBATs are involved in the maintenance of the
enterohepatic circulation of bile acids and therefore also
participate in the homoeostatis of cholesterol Members of
SBATs are located either in apical membranes of ileum
enterocytes, kidney tubule cells and bile duct cells where
they perform bile acid reabsorption, or in the basolateral
membrane of hepatocytes where they initiate bile acid
secretion [1–9] SBATs constitute a subgroup of the
superfamily of sodium-dependent cotransporters with
about 35% homology among the clones from different
species, e.g from rat, mouse, rabbit, hamster, and human
[10] Their molecular mass is about 50 kDa and the
predicted structure which is derived from hydrophobicity analysis contains either seven or nine transmembrane domains; all SBATs are glycosylated at the extracellular N-terminus and contain a cytoplasmic C-terminus All carriers transport sodium ions together with an organic substrate, e.g a bile acid or an anionic sulfated or glucuroni-dated estrogen conjugate The stoichiometry of this process is electrogenic; two sodium ions are supposed to be trans-located with one taurocholate molecule by the rat Ntcp [11,43,44] Previous reports from Na+/H+proton exchanger [12], Na+/Ca2+exchanger [13], proton pumps [14], sodium-sensitive receptors [15] and sodium-coupled cotransporters [16–18] indicated that negatively charged amino acids in integral membrane channels or carrier proteins are binding sites for sodium ions or other cationic electrolytes Therefore,
an alignment of nine members of the SBAT-family for negatively charged amino acids was made which revealed five conserved glutamates and aspartates The construction of point mutations of all five conserved and two nonconserved negatively charged amino acids into their noncharged counterparts asparagine and glutamine revealed the func-tional importance of two of them for taurocholate transport
In previous studies, we had reported that SH-group reagents with wide varying lipid–water partition values reversibly blocked taurocholate uptake into isolated rat hepatocytes [19,20] We postulated that cysteines from intra-and extramembrane domains of the Ntcp are essential for the transport function of the sodium-dependent bile acid cotransporter Very recently a report on the human NTCP indicated that Cys266, which is located in the final extracellular loop (loop VI as predicted by the seven
Correspondence to E Petzinger, Institute of Pharmacology
and Toxicology, Justus-Liebig-University Giessen,
Frankfurter Str 107, D-35392 Giessen, Germany.
Fax: + 49 641 99 38409, Tel.: + 49 641 99 38400,
E-mail: ernst.petzinger@vetmed.uni-giessen.de
Abbreviations: SBAT, sodium-dependent bile acid cotransporter;
TM, transmembrane.
(Received 19 December 2001, revised 19 December 2002,
accepted 14 January 2003)
Trang 2transmembrane domains model), is involved in taurocholate
transport of the human isoform [21] We therefore looked
for the role of each of the eight cysteines of the rat Ntcp for
taurocholate transport
Finally, threonines, within a protein kinase C consensus
region located in the C-terminus of the Ntcp protein were
analyzed with regard to their role in taurocholate transport
Such threonines might be prone to phosphorylation/
dephosphorylation reactions as it was shown that the Ntcp
is a serine/threonine phosphorylated phosphoprotein which
is dephosphorylated by cAMP [22] Upon phosphorylation
of serine/threonine by a protein kinase A, taurocholate
transport is increased but upon phosphorylation of the Ntcp
by protein kinase C, taurocholate uptake is reduced [23]
Threonines from the C-terminal consensus region were
therefore converted to either tyrosines or alanines to
abrogate any phosphorylation signal by PKC
Materials and methods
Site-directed mutagenesis, cloning procedures,
and DNA sequencing
The cDNA of the Ntcp was a kind gift of B Hagenbuch,
University Hospital, Dept Clinical Pharmacology, Zu¨rich
Point and deletion mutants of the rat liver Ntcp cDNA clone
prLNaBA [1] were generated by site-directed mutagenesis by
the use of the QuikChangeTMkit from Stratagene, La Jolla,
USA The primers were selected for each mutation according
to the manufacturer’s manual and were purchased from
MWG, Biotech AG, Ebersberg, Germany They are shown
in Table 1 Mutants were generated by PCR using 16 cycles
according to the manufacturer’s protocol in a Perkin-Elmer
GenAmp cycler 2400 (Perkin Elmer, U¨berlingen, Germany)
The template DNA prLNaBA was digested with DpnI Each
mutated plasmid was transformed into Epicurian Coli
XL1-Blue Supercompetent cells by heat pulse Bacterial cells were
transferred to LB-ampicillin agar plates and single colonies
were isolated and further cultivated to subconfluency in LB
medium Plasmid DNA was isolated according to the Qiagen
Midi kit instructions (Qiagen, Hilden, Germany) The insert
of 1663 bp length of each clone was upstream and
down-stream sequenced by a dye terminated method using the
ABI-Prism Dye Terminator Cycle Sequencing Ready
Reac-tion kit from Applied Biosystems Inc., Weiterstadt, Germany
in the DNA sequencer 373A from the same company
Alignments
An alignment of nine members of the SBAT-family, five
basolateral (Ntcp rat [1], Ntcp mouse1 and 2 [9], Ntcp rabbit
[24], and NTCP human [2]) and four apical (Isbt rat [6], Isbt
mouse [25], Isbt rabbit [26] and ISBT human [27]) was
performed, using the CLUSTAL W 1.6 program from the
Baylor College of Medicine Search Launcher (Houston,
USA) to identify conserved negatively charged amino acids
and cysteines
Tagging of Ntcp mutants by the FLAGmotif
To determine whether the wild-type and the mutant proteins
are expressed and located on the surface of the oocytes,
the cDNA was extended at the 3¢ end by the sequence GATTACAAGGATGACGACGATAAG coding for the FLAGpeptide Insertion of the sequence was carried out
by site-directed mutagenesis using the QuikChangeTM kit from Stratagene, La Jolla, USA The primers used for the PCR are depicted in Table 1 Here, 18 PCR-cycles were applied Location of the insertion was verified by SeqLab Laboratories, Go¨ttingen, Germany
Immunofluorescence microscopy
X laevis oocytes, prepared and maintained in culture as described [28], were injected with 2.5 ng cRNA coding for the wild-type and mutant Ntcp-protein, both elongated by the FLAG sequence After 2 days of expression, the vitelline membrane was removed by hand and the oocytes were fixed in a solution of 80% methanol/20% dimethyl-sulfoxide Oocytes were washed in decreasing concentra-tions of methanol in phosphate-buffered saline (NaCl/Pi, 0.9%, pH 7.4) and were incubated with the mAb M2-anti-FLAG(Sigma-Aldrich, Taufkirchen, Germany) After a second washing step with NaCl/Pibuffer the oocytes were fixed with 3.7% formaldehyde in NaCl/Pi and incubated with Alexa Fluor 488 goat anti-mouse IgG conjugate (Molecular Probes, Leiden, Netherlands) They were again washed with NaCl/Pi and embedded in Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany) Sections, 5-lm thick, were cut and proteins were detected by reflective fluorescence microscopy at 488 nm (Leitz Diaplan UV Microscope, Wetzlar, Germany)
Heterologous expression of Ntcp-cRNA inX laevis oocytes
Mutated and nonmutated plasmids were linearized by PvuI (MBI Fermentas, Vilnius, Lithuania) Capped mRNA was transcribed in vitro using T7 RNA polymerase (Promega, Madison, USA) in the presence of capping analog
m7G(5¢)ppp(5¢)G from Pharmacia, Freiburg, Germany Unincorporated nucleotides were removed with a Sephadex G-50 spin column (Boehringer, Mannheim, Germany) cRNAs were recovered by ethanol precipitation and resuspended in double distilled water for oocyte injection
X laevisoocytes were prepared and maintained in culture
as described [28] They were microinjected with 2.5 ng of Ntcp/mutant cRNA per oocyte in standard experiments In a series of saturation experiments, 0.46–6.9 ng cRNA per oocyte were injected For expression, oocytes were incubated for 2 days at 18C in modified Barth solution For uptake measurements, 10–15 oocytes were incubated at 25C in a medium containing 5 lM [3H]taurocholate (NEN Life Science Products, Boston, MA, USA; specific activity 2–3.47 CiÆmmol)1), 10 mMHepes/Tris pH 7.5, 2 mMKCl,
1 mM CaCl2, 1 mM MgCl2 and either 100 mM NaCl or
100 mM choline chloride in order to calculate the Ntcp-mediated sodium-dependent taurocholate uptake Hill coefficient analysis of the sodium-coupled taurocholate uptake by wild-type Ntcp and two Ntcp mutants with mutated negatively charged amino acids (Asp115Asn and Glu257Gln) was deduced from [3H]taurocholate uptake experiments in the same Hepes/Tris buffer, however, with sodium chloride concentrations of zero, 30, 50, 100, 150 and
Trang 3Table 1 Primers used for generating the indicated Ntcp-mutations by QuikChangeTM.
Desired mutation Primer name Sequence
Asp24Asn – F Ggccaccgggccacaaacaaggcgcttagcatc
– R Gatgctaagcgccttgtttgtggcccggtggcc
Cys44Ala – F Gctctcactgggcgccaccatggaattcagc
– R Gctgaattccatggtggcgcccagtgagagc
Cys44Trp – F Catgctctcactgggctggaccatggaattcagc
– R gctgaattccatggtccagcccagtgagagcatg
Glu47Gln – F ctgggctgcaccatgcaattcagcaagatcaag
– R cttgatcttgctgaattgcatggtgcagcccag
Glu89Gln – F cacctgagcaacattcaagctctggccatcctc
– R gaggatggccagagcttgaatgttgctcaggtg
Cys96Ala – F ctggccatcctcatcgctggctgctctcccggg
– R cccgggagagcagccagcgatgaggatggccag
Cys96Trp – F ctggccatcctcatctggggctgctctcccggg
– R cccgggagagcagccccagatgaggatggccag
Cys98Ala – F catcctcatctgtggcgcctctcccggggggaac
– R gttccccccgggagaggcgccacagatgaggatg
Cys98Trp – F catcctcatctgtggctggtctcccggggggaac
– R gttccccccgggagaccagccacagatgaggatg
Asp115Asn – F ctggccatgaaggggaacatgaacctcagcatc
– R gatgctgaggttcatgttccccttcatggccag
Cys125Ala – F catcgtgatgaccaccgcctccagcttcagtgcc
– R ggcactgaagctggaggcggtggtcatcacgatg
Cys125Del – F catcgtgatgaccacctccagcttcagtgcc
– R ggcactgaagctggaggtggtcatcacgatg
Asp147Asn – F gcaaaggcatctacaatggagaccttaaggacaagg
– R ccttgtccttaaggtctccattgtagatgcctttgc
Cys170Ala – F gttctcattcctgccaccatagggatcgtcc
– R ggacgatccctatggtggcaggaatgagaac
Cys170Trp – F catagttctcattccttggaccatagggatcgtc
– R gacgatccctatggtccaaggaatgagaactatg
Cys250Ala – F caactcaatccaagcgccagacgcaccatcagc
– R gctgatggtgcgtctggcgcttggattgagttg
Cys250Del – F ccaactcaatccaagcagacgcaccatcagc
– R gctgatggtgcgtctgcttggattgagttgg
Asp257Asn – F gctgcagacgcaccatcagcatgcaaacaggattcc
– R ggaatcctgtttgcatgctgatggtgcgtctgcagc
Cys266Ala – F ccaaaacattcaactcgcttctaccatcctcaatgtg
– R cacattgaggatggtagaagcgagttgaatgttttgg
Cys266Del – F ggattccaaaacattcaactctctaccatcctcaatgtgacc
– R ggtcacattgaggatggtagagagttgaatgttttggaatcc
Asp277Asn – F cctcaatgtgaccttcccccctcaagtcattgggcc
– R ggcccaatgacttgaggggggaaggtcacattgagg
Cys306Ala – F catcattatcttccgggcctatgagaaaatcaagcctcc
– R ggaggcttgattttctcataggcccggaagataatgatg
Cys306Trp – F catcattatcttccggtggtatgagaaaatcaagcctcc
– R ggaggcttgattttctcataccaccggaagataatgatg
Cys306Del – F catcattatcttccggtatgagaaaatcaagcctc
– R gaggcttgattttctcataccggaagataatgatg
Thr317Ala – F gcctccaaaggaccaagcaaaaattacctacaaagc
– R gctttgtaggtaatttttgcttggtcctttggaggc
Thr317Tyr – F atcaagcctccaaaggaccaatacaaaattacctacaaagctgctg
– R cagcagctttgtaggtaattttgtattggtcctttggaggcttgat
Thr320Ala – F ggaccaaacaaaaattgcctacaaagctgctgcaac
– R gttgcagcagctttgtaggcaatttttgtttggtcc
Thr320Tyr – F ccaaaggaccaaacaaaaatttactacaaagctgctgcaactgagg
– R cctcagttgcagcagctttgtagtaaatttttgtttggtcctttgg
FLAG(R)-insert – F Ggtcagatggcaaatgattacaaggatgacgacgataagtagaatgtgaaacttcgaagc
– R Gcttcgaagtttcacattctacttatcgtcgtcatccttgtaatcatttgccatctgacc
Trang 4200 mM The buffers of zero, 25, 50, and 100 mMNaCl were
substituted with the corresponding choline chloride
concen-tration (0/100, 25/75, 50/50, 100/0 NaCl/choline chloride)
The oocyte-associated radioactivity was determined in a
liquid scintillation counter (Wallac 1407, Wallac Inc., Turku,
Finland)
Results
Search for conserved negatively charged amino acids,
cysteines and C-terminal threonines
by sequence identity
An alignment of the amino acid sequence of nine SBAT
proteins, namely five basolateral Ntcp-proteins together
with four apical Isbt-proteins, revealed that the following
glutamates, aspartates, and cysteines in the rat Ntcp are
conserved in all of the nine family members: Cys44, Cys98,
Cys125, and Cys266 as well as Glu47, Asp115, Asp147,
Glu257, and Glu277 The threonines Thr317 and Thr320
are only found in the rat liver Ntcp (Fig 1)
Mutations of negatively charged amino acids residues
The predicted seven transmembrane (TM) structure of rat
liver Ntcp according to [1] and all introduced mutations are
depicted in Fig 2 The organic anion transporting
SBATs are cotransporters with sodium ions as the driving
ion gradient Therefore, in addition to substrate binding
sites, regions for cation binding are also required Earlier
reports have indicated the importance of negatively
charged amino acids for sodium-coupled substrate
cotrans-port or exchange [12,13,16–18] Mutations of all conserved
and two nonconserved negatively charged amino acids to
the noncharged counterparts, i.e Asp to Asn and Glu
to Gln, revealed that the aspartates Asp24 and Asp115 as
well as Glu257 are required for taurocholate transport
(Fig 3)
The negatively charged Glu257 is exposed in an
extracellular loop of the Ntcp and could represent the
sodium ion sensor of sodium-coupled taurocholate
trans-port via Ntcp In order to find out whether and to what
extent this amino acid affects sodium ion dependency of
taurocholate uptake, transport studies were performed in
the presence of varying amounts of extracellular sodium
chloride and Hill analysis was applied (Fig 4) For
comparison the transport-negative Asp115Asn mutant
was investigated in the same manner As a result, the
negative charge in Glu257 is an essential prerequisite for
sodium-dependent taurocholate uptake The Hill
coeffi-cient of this cotransport by wild-type Ntcp is about 2–2.59
[43,44] but dropped to 0.32 (measured at 25–200 mM
NaCl) if Glu257 was converted to Gln (Fig 4) Increase
of the sodium gradient by applying concentrations up to
200 mM NaCl to the outside did not alter the abolished
transport of taurocholate significantly, although at 200 mM
NaCl taurocholate transport slightly increased In contrast,
significant sodium cooperativity was found, however, at a
much lower level, in Ntcp mutant Asp115Asn The Hill
number was 1.15 for the Asp115Asn mutant (Fig 4) which
corresponds to a sodium stoichiometry of one sodium ion
per taurocholate molecule
As taurocholate transport via the carrier mutants Glu257Gln and Asp115Asn was almost nil (2 and 15% of wild-type Ntcp, respectively), tests were carried out to determine whether insufficient expression of the injected cRNA caused this lack of transport Therefore, up to three times the amount of the cRNA compared to the standard amount was injected into oocytes, i.e 6.9 ng instead of 2.3 ng cRNA No improvement of taurocholate transport was observed (Fig 5)
Tests were then performed to determine whether the absence of transport was caused by a sorting defect of these mutant proteins For this reason, the FLAG motif was
Fig 1 Alignment of nine members of the SBAT family Ntcp mouse1, mml1; Ntcp mouse2, mml2; Ntcp rat, rnl; Ntcp rabbit, oclm; NTCP human, hsl; Isbt rat, rni; Isbt mouse, mmi; Isbt rabbit, oci; Isbt human, hsi; conserved cysteines, c; conserved acidic amino acids, a.
Trang 5cloned into each transport-negative mutant clone With this
technique, insertion of the mutants Asp115Asn and
Glu257Gln within the cell membrane of X laevis oocytes
was observed by use of antibodies raised against the
FLAG peptide, and applied to permeabilized oocytes
(Fig 6) An exception was observed, the transport-negative
mutant Asp24Asn, which did not appear in the membrane
(Fig 6), indicating that Asp24 from the extracellular
N-terminus is not essential for transport but for appropriate
cell sorting of the Ntcp protein
Cysteine mutants
Each of the eight cysteines, four in transmembrane
domains, three in cytoplasmic or extracellular loops, and
one at the beginning of the C-terminal tail was altered by site-directed mutagenesis The three cysteines from the nontransmembrane domains and the one from the C-terminus were substituted by alanine or were omitted
to attain deletion mutants All deletion mutants, namely Cys125Del, Cys250Del, Cys266Del, and Cys306Del were transport-negative (Fig 7), indicating that each cysteine per se is required If their alanine counterparts were expressed in X laevis oocytes, all except one showed restored transport activity Only the Cys266Ala mutant remained transport-negative We conclude that Cys266 is the only cysteine of the rat liver Ntcp which appears to
be directly involved in taurocholate uptake into the oocytes
To show whether or not mutant Cys266Ala was present in the cell surface of X laevis oocytes, the corres-ponding cDNA clone was also tagged by the FLAG motif and cRNA from this construct was again injected into oocytes Immunofluorescence pictures confirm that the mutant Cys266Ala protein is present in the cell membrane in a similar amount as the wild-type Ntcp protein (Fig 6)
Eight further cysteine mutations were generated regard-ing the four intramembrane cysteines (Fig 7) Exchanges by alanine or tryptophane were generated With the exception
of Cys306 each tryptophane mutant was either transport negative (Cys98Trp, Cys170Trp, Cys96Trp) or showed decreased uptake (Cys44Trp) However, if these intramem-brane cysteines were substituted by alanines, taurocholate transport was fully regained This indicates that none of the transmembrane cysteines appears to be directly involved in the transport process An exception was the tryptophane substitution of Cys306 This Cys306Trp mutant transported taurocholate more effectively (more than 1.5-fold) than wild-type Ntcp Cys306, however, is located at the beginning
of the C-terminal tail of Ntcp (Fig 7)
Fig 3 Mutations of negatively charged conserved amino acids alters
taurocholate transport via Ntcp Uptake of [3H]taurocholate by
X laevis oocytes two days after microinjection of 50 nL containing
2.5 ng cRNA which was transcribed from wild-type or mutant Ntcp
clones Uptake is given in percentage of wild-type uptake after 30 min
of exposure to 5 l M [3H]taurocholate.
Fig 2 Topology model of rat Ntcp based on hydropathy analysis of the amino acid sequence (according to [1]) Transmembrane domains are symbolized as blocks of amino acids Mutated amino acids are highlighted in gray The resulting mutants are shown in boxes, with deletions indicated (del).
Trang 6Threonine mutants
The threonines Thr317 and Thr320 are located within the
protein kinase C consensus regions LysXXThrLys and
LysXThrXLys of the Ntcp [29] Therefore, both threonines
were substituted by either tyrosine or alanine None of these
mutations significantly altered taurocholate uptake The
transport rate of each mutant was between 80 and 100% of
the wild-type Ntcp (Fig 6)
Discussion
Hepatobiliary transport of the major bile acid taurocholate
in humans and rats begins by uptake across the
baso-lateral membrane of hepatocytes via the high affinity,
Fig 5 The relationship between the amount of injected cRNA and taurocholate uptake into
X laevis oocytes via Ntcp and Ntcp mutants Uptake of [ 3 H]taurocholate by cRNA-injected oocytes after exposure to 5 l M [3 H]taurocho-late for 30 min The amount of cRNA of transport-negative mutants was increased 14-fold; the standard amount of cRNA which was injected for comparison of transport by mutated vs wild-type Ntcp was 2.5 ngÆ oocyte)1.
Fig 4 Sodium dependency of taurocholate uptake by Ntcp mutants Uptake of 5 l M
[ 3 H]taurocholate was measured during 30 min after injection of 2.5 ng cRNA subscribed from wild-type and mutant Ntcp-clones into oocytes The oocytes were incubated in the presence of increasing sodium chloride con-centrations The results obtained by the clones Asp115Asn and Glu257Gln are also given in a Hill plot.
Fig 6 Detection of the presence of Ntcp proteins in the cell membrane
of X laevis oocytes by the reporter FLAG motif The FLAG encoded amino acid sequence was detected by sandwich immuno-fluorescence labeling with monoclonal anti-FLAG Ig and subse-quent labeling with Alexa Fluor 488 goat anti-mouse IgG conjugate
in permeabilized and fixed oocytes after two days of cRNA expression With the exception of Asp24Asn mutated Ntcp, each mutated protein was detected in the cell membrane of oocytes Negative control was oocytes that were injected with water From top left to right: upper, Asp24Asn; Cys266Del; middle, Glu257Gln; Cys266Ala; lower, Asp115Asn; water-injected oocyte (negative control); large picture, wild-type Ntcp (positive control).
Trang 7sodium-dependent and liver-specific basolateral bile acid
carriers NTCP (humans) and Ntcp (rats) Subsequent to
uptake the bile acid is released into the bile canaliculus by the
bile salt export pump BSEP, an ATP-driven ABC-cassette protein related to mdr1 [30,31] Disturbances of the hepatocellular part of the enterohepatic circulation of bile
Trang 8acids causes intrahepatic cholestasis [32–35] Whereas
nat-urally occurring mutations in the BSEP have been described,
causing the rare Byler syndrome in children [36], naturally
occuring mutations of the NTCP-gene locus have not yet
been observed Our study with the rat Ntcp indicates,
however, that several amino acids may be essential for
hepatocellular taurocholate uptake because mutations in
these amino acids caused lack of transport; the amino acids
in question are Asp115, Glu257, and Cys266 All of these are
conserved in SBATs and are found also in the human NTCP
protein Functional mutations of these amino acids in the
human NTCP gene locus would cause hypercholanaemia
but would result in low intrahepatic bile salt levels and
therefore little if any hepatocellular injury This syndrome
has been already described in two children, however,
without mutations of the NTCP gene and therefore
remained unexplained [37] The clinical picture of
nonfunc-tional NTCP carriers would differ from patients with
cholestasis where blockade of bile acid secretion at the
canalicula pole of hepatocytes leads to elevated intracellular
(and extracellular) bile acid concentration and therefore
causes severe liver injury It should be noted that in such
cases of cholestasis, Ntcp expression as a protecting
mechanism decreases dramatically [38], but in the cases of
benign hypercholanemia, Ntcp expression was normal [37]
In the latter syndrome, taurocholate uptake and also bile
acid-dependent bile formation is not expected to cease as bile
acid uptake by liver-type organic anion transporting
poly-peptides OATP 8 and OATP-C continues
All mutations from transport-negative mutants were
located in loop structures of the rat Ntcp; two of them,
Glu257 and Cys266, were located in loop VI, the final
extracellular loop (Fig 2) This region also appears to have
key properties for taurocholate transport in other SBATs,
as it was already reported that a naturally occuring point
mutation of Thr262 in the human intestinal Na+/bile acid
cotransporter ISBT abolished reabsorption of bile acids and
caused primary bile acid malabsorption in patients [38] This
conserved threonine is located in loop VI of ISBT and Ntcp
and is next to Glu257 in the rat Ntcp, which we report here,
is also required for hepatic taurocholate transport
The negatively charged Glu257 is probably a binding site
for extracellular sodium ions The driving force for substrate
transport via all SBATs is the sodium gradient across the cell
membrane Two sodium ions are supposed to be
translo-cated together with one bile acid molecule via ileal Na+-bile
acid cotransporters such as human ASBT [46] or the rat liver
Ntcp [11] Therefore, sodium-driven taurocholate transport
is electrogenic [40,46] This 2 : 1 stoichiometry was altered in the mutant Glu257Gln as revealed by Hill analysis A Hill number of almost zero was calculated indicating that this mutant is unable to translocate sodium ions together with taurocholate Therefore we assume that Glu257 is the extracellular sodium sensor for sodium taurocholate co-transport As taurocholate uptake with this Ntcp protein was almost nil (residual 2% transport compared with wild-type Ntcp), the long established importance of the sodium ion for the translocation step of monoanionic bile acids was reaffirmed The cationic sodium ions are likely to interact with negatively charged amino acid residues at the outer surface of the Ntcp protein, but then need to be translocated through pore-forming transmembrane helices to the cyto-plasmic regions of the protein It has been shown that extracellular loops, containing charged amino acids, can slide between TM domains into the membrane, forming P-loops [40] P-loops allow the introduction of charged molecules into inner parts of the cell membrane from where these can be overtaken by further binding sites originating from the cytoplasmic region of the protein It is tempting to
Fig 7 Taurocholate uptake into X laevis oocytes after injection of cRNA of wild-type and mutated Ntcp Uptake of [3H]taurocholate
in X laevis oocytes which were injected with 2.5 ng wild-type or mutant cRNA and incu-bated for two days as described in Materials and methods The diagram depicts relative uptake in percentage of uptake by wild-type Ntcp (100%) after 30 min incubation with
5 l M [ 3 H]taurocholate.
Fig 8 P-loop model of rat Ntcp depicting Glu257 and Asp115 as putative binding sites for sodium ions The extracellular loop 6 between
TM VI and VII contains a sodium substrate-binding region for tauro-cholate during sodium ion–taurotauro-cholate cotransport.
Trang 9hypothesize that if such a translocation mechanism is active
in the Na+/bile acid cotransporter Ntcp, extracellular
Glu257 and cytoplasmic Asp115 may constitute an
appro-priate pair of binding sites for sodium ions allowing ion
translocation across the cell membrane The cytoplasmic
Asp115 may detract the two sodium ions delivered by
extracellular Glu257 via P-loop formation (Fig 8)
Consis-tent with this suggested model is the observation that charge
modification in the putative cytoplasmic sodium sensor
Asp115 through its conversion into Asn decreased
sodium-dependent taurocholate uptake to 15% of wild-type Ntcp,
probably because the sodium stoichiometry of 2 : 1 declined
to 1 : 1 (Fig 4)
Whereas negatively charged residues are not suspected
to interact with the anionic organic substrates of SBATs
cysteines are It has already been shown that Cys266 is
essential for taurocholate transport by the human NTCP
protein [21] Here we report that the same amino acid in
the corresponding position is also required for
taurocho-late transport by the rat Ntcp This cysteine appears to be
directly involved in taurocholate transport as it is the only
one which remained transport-negative when substituted
by alanine However, this is in contrast to the report by
Halle´n et al 2000 [21], showing that mutant Cys266Ala of
the human NTCP still transported taurocholate without
any marked change in Km and Vmax These authors
obtained evidence for a separate function of that cysteine
by indirect means with SH-group reagents The reason for
this discrepancy is unclear, but might be due to the
different expression systems used for detection (Xenopus
oocytes in this report vs HEK293 cells in Halle´n’s report)
or to different local interactions of this cysteine within
loop VI of the different Ntcps It should be noted that
loop VI of the human NTCP contains four cysteines
whereas in the rat Ntcp only two cysteines (Cys250,
Cys266) are present
Other cysteines (seven out of eight) of the Ntcp may
have indirect effects on taurocholate transport Such effects
were analyzed by cysteine deletion mutants and
trypto-phane substitutions Indirect effects could be space-holding
properties of these cysteines tested by deletion mutants and
lipophilic binding properties other than by SH-groups
tested by tryptophane The deletion mutants of the
cysteines from loops III and VI (Cys125, Cys250 and
Cys266) (Fig 2) were transport-negative Replacement by
alanine, however, restored uptake in the case of Cys125
and Cys250 Therefore, these cysteines appear to have
space holder functions for the loops With respect to the
Cys250Ala mutant of the rat Ntcp, our finding is in full
agreement with the results observed with the human
NTCP, where taurocholate transport by the Cys250Ala
mutant was also not altered [21] Because seven out of
eight cysteine/alanine substitutions were transport-positive
(with the exception of Cys266Ala), we conclude that no
disulfide bonding between cysteines within a monomeric
Ntcp protein has occurred
Among the cysteine/tryptophane substitutions,
Cys306Trp was exceptional in that taurocholate transport
was not abolished but was even enhanced to 150% of
wild-type transport Cys306 is located at the border of TM7 to
the cytoplasmic C-terminus Cysteines in that position
might serve as an anchor for a palmitoyl/isoprenyl residue
which fixes the protein to the plasma membrane If this is true for the Ntcp protein, this could explain why the hydrophobic amino acid tryptophane fully substituted Cys306 only in that protein position and why in contrast
to all other tryptophane substitutions, taurocholate trans-port was not abolished but was even enhanced by this mutation
Cys306 marks the border to a 56 amino acid tail which stretches to the end of the final amino acid, Asn362, of the Ntcp C-terminus It was already shown that this C-terminal tail is not required for transport properties [41]
A truncated rat liver Ntcp protein lacking all amino acids beyond Cys306 transported taurocholate with a Km identical to that of wild-type Ntcp Similarly, a trans-port-positive Ntcp splicing variant which was shortened by
45 amino acids from the end of the C-terminus was cloned from mice [9] Thus the C-terminal tail appears to be unnecessary for taurocholate uptake However, it was required for appropriate basolateral sorting of the protein, because mutations of Tyr307 (following next to Cys306) and Tyr321 (following next to Thr320) accumulated within the cytosol but were absent from the cell membrane [42] Apart from sorting signal motifs, other regulatory func-tions might be phosphorylation/dephosphorylation reac-tions For this reason, two threonines, Thr317 and Thr320, within a protein kinase C-consensus region were mutated
to alanines but also to tyrosines None of these mutations showed any effects on taurocholate transport into X laevis oocytes Our results would not disprove such phosphory-lation reactions being present in mammalian cells, but the localization of receptive serine/threonine residues for a particular protein kinase are unlikely to be expected within that protein kinase C-consensus region, as even the alanine substitutions were without any effect on taurocholate transport
Acknowledgments
The authors wish to acknowledge the receipt of the Ntcp-containing plasmid prLNaBA from Dr Bruno Hagenbuch, Zurich Support of this project was given by Drs Frank and Marita Langewische who helped
to initiate the study by constructing cysteine mutants Technical help was provided by Mrs Elisabeth Ju¨ngst-Carter and Steffi Weghenkel.
Dr Bruce Boschek has provided critical liguistical advice in preparing the manuscript.
References
1 Hagenbuch, B., Stieger, B., Foguet, M., Lu¨bbert, H & Meier, P.J (1991) Functional expression cloning and characterisation of the hepatocyte Na + /bile acid cotransport system Proc Natl Acad Sci USA 88, 10629–10633.
2 Hagenbuch, B & Meier, P.J (1994) Molecular cloning, chromo-somal localization and functional characterisation of a human liver Na + /bile acid cotransporter J Clin Invest 93, 1326–1331.
3 Hagenbuch, B & Meier, P.J (1996) Sinusoidal (basolateral) bile salt uptake systems of hepatocytes Semin Liver Dis 16, 129–136.
4 Wong, M.H., Oelkers, P., Craddock, A.L & Dawson, P.A (1994) Expression cloning and characterization of the hamster ileal sodium-dependent bile acid transporter J Biol Chem 269, 1340– 1347.
5 Wong, M.H., Oelkers, P & Dawson, P.A (1995) Identification of
a mutation in the ileal sodium-dependent bile acid transporter
Trang 10gene that abolishes transport activity J Biol Chem 270, 27228–
27234.
6 Shneider, W.L., Dawson, P.A., Christie, D.-M., Hardikar, W.,
Wong, M.H & Suchy, F.J (1995) Cloning and molecular
char-acterisation of the ontogeny of rat ileal sodium dependent bile acid
transporter J Clin Invest 95, 745–754.
7 Lazarides, K.N., Pham, L., Tietz, P., Marinelli, P.C., Levine, S.,
Dawson, P.A & LaRusso, N.F (1997) Rat cholangiocytes absorb
bile acids at their apical domain via the ileal sodium-dependent
bile acid transporter J Clin Invest 100, 2714–2721.
8 Lazarides, K.N., Tietz, P., Wu, T., Kip, S., Dawson, P.A &
LaRusso, N.F (2000) Alternatic splicing of the rat sodium/bile
acid transporter changes ist cellular localization and transport
properties Proc Natl Acad Sci USA 97, 11092–11097.
9 Cattori, V., Eckhardt, U & Hagenbuch, B (1999) Molecular
cloning and functional characterization of two alternatively
spliced Ntcp isoforms from mouse liver Biochim Biophys Acta
1445, 154–159.
10 Hagenbuch, B (1997) Molecular properties of hepatic uptake
systems for bile acids and organic anions J Membrane Biol 160,
1–8.
11 Weinman, S.A & Weeks, R.P (1993) Electrogenicity of Na +
-coupled bile salt transport in isolated rat hepatocytes Am J.
Physiol 265, G73–G80.
12 Murtazina, R., Booth, B.J., Bullis, B.L., Singh, D.N & Fliegel, L.
(2001) Functional analysis of polar amino acids residues in
membrane associated regions of the NHE1 isoform of the
mam-malian Na + /H + exchanger Eur J Biochem 268, 4674–4685.
13 Nicoll, D.A., Hryshko, L.V., Matsuoka, S., Frank, J.S &
Phi-lipson, K.D (1996) Mutation of amino acid residues in the
putative transmembrane segments of the cardiac sarcolemmal
Na+–Ca++exchanger J Biol Chem 23, 13385–13391.
14 Lanyi, J.K (1997) Mechanism of ion transport across membranes.
Bacteriorhodopsin as a prototype for proton pumps J Biol.
Chem 272, 31209–31212.
15 Martin, S., Botto, J.M., Vincent, J.P & Mazella, J (1999) Pivotal
role of an aspartate residue in sodium sensitivity and coupling to G
proteins ot neurotensin receptors Mol Pharmacol 55, 210–215.
16 Quick, M & Jung, H (1997) Aspartate 55 in the Na+/proline
permease of Escherichia coli is essential for Na + -coupled proline
uptake Biochemistry 36, 4631–4636.
17 Jung, H (2001) Towards the molecular mechanism of Na + /
solute symport in prokaryotes Biochim Biophys Acta 1505,
131–143.
18 Poolman, B., Knol, J., van der Does, C., Henderson, P.J.F.,
Liang, W.-J., Leblanc, G., Pourcher, T & Mus-Veteau, I (1996)
Cation and sugar selectivity determinats in novel family of
trans-port proteins Molec Microbiol 19, 911–922.
19 Blumrich, M & Petzinger, E (1990) Membrane transport of
conjugated and unconjugated bile acids into hepatocytes is
susceptible to SH-blocking reagents Biochim Biophys Acta 1029,
1–12.
20 Blumrich, M & Petzinger, E (1993) Two distinct types of
SH-groups are necessary for bumetanide and bile acid uptake into
isolated rat hepatocytes Biochim Biophys Acta 1149, 278–284.
21 Halle´n, S., Fryklund, J & Sachs, G (2000) Inhibition of the
human sodium/bile acid cotransporters by side-specific
methane-thiosulfonate sulfhydryl reagents: substrate-controlled
accessi-bility of site of inactivation Biochemistry 39, 6743–6750.
22 Mukhopadhayay, S., Ananthanarayanan, M., Stieger, B., Meier,
P.J., Suchy, F.J & Anwer, M.S (1998) Sodium taurocholate
cotransporting polypeptide is a serine, threonine phosphorprotein
and is dephosphorylated by cyclic AMP Hepatology 28, 1629–
1636.
23 Gru¨ne, S., Engelking, L.R & Anwer, M.S (1993) Role of
intracellular calcium and protein kinases in the activation of
hepatic Na+/taurocholate cotransport by cyclic AMP J Biol Chem 268, 17743–17741.
24 Stengelin, S., Becker, W., Maier, M., Noll, R & Kramer, W (1998) Rabbit cDNA encoding hepatic sodium-dependent bile acid transporter GeneBank accession number AJ131361.
25 Saeki, T., Motoba, K., Furukawa, H., Kirifiji, K., Kanamoto, R.
& Iwami, K (1999) Characterisation, cDNA cloning, and func-tional expression of mouse ileal sodium-dependent bile acid transporter J Biochem (Tokyo) 125, 846–851.
26 Stengelin, S., Apel, S., Becker, W., Maier, M., Rosenberger, J., Wess, G & Kramer, W (1997) Ileal sodium/bile acid cotransporter; direct submission, GeneBank accession number Q28727.
27 Craddock, A.L., Love, M.W., Daniel, R.W., Kirby, L.C., Walters, H.C., Wong, M.H & Dawson, P.A (1998) Expression and transport properties of the human ileal and renal sodium-depen-dent bile acid transporter Am J Physiol 274, G157–G169.
28 Eckhardt, U., Horz, J.A., Petzinger, E., Stu¨ber, W., Reers, M., Dickneite, G., Daniel, H., Wagener, M., Hagenbuch, B., Stieger, B.
& Meier, P.J (1996) The peptide-based thrombin inhibitor CRC 220 is a new substrate of the basolateral rat liver organic anion-transporting polypeptide Hepatology 24, 380–384.
29 Pearson, R.B & Kemp, B.E (1991) Proteinkinase phosphoryla-tion site sequences and consensus specificity motifs Tabulaphosphoryla-tions Methods Enzymol 200, 62–82.
30 Gerloff, T., Stieger, B., Hagenbuch, B., Madon, J., Landmann, L., Roth, J., Hofmann, A.F & Meier, P.J (1998) The sister of P-glycoprotein represents the canalicular bile salt export pump
of mammalian liver J Biol Chem 273, 10046–10050.
31 Bahar, R & Stolz, A (1999) Bile acid transport Gastroenterol Clin North Am 28, 27–58.
32 Petzinger, E (1994) Transport of organic anions in the liver Rev Physiol Biochem Pharmacol 123, 47–211.
33 Eckhardt, U., Schroeder, A., Stieger, B., Ho¨chli, M., Landmann, L., Tynes, R., Meier, P.J & Hagenbuch, B (1999) Poly-specific uptake of the hepatic organic anion transporter Oatp1
in stably transfected CHO cells Am J Physiol 276, G1037– G1042.
34 Trauner, M., Meier, P.J & Boyer, J.L (1998) Molecular patho-genesis of cholestasis N Engl J Med 339, 1217–1227.
35 Kullak-Ublick, G.A., Beuers, U & Paumgartner, G (2000) Hepatobiliary transport J Hepatol 32 (Suppl 1), 3–18.
36 Strautnieks, S.S., Kagalwalla, A.F., Tanner, M.S., Knisely, A.S., Bull, L., Freimer, N., Kocoshis, S.H., Gardiner, R.M & Thompson, R.J (1997) Identification of a locus for progressive familial intrahepatic cholestasis PFIC2 on chromosome 2q24.
Am J Hum Genet 61, 630–632.
37 Shneider, B.L., Fox, V.L., Schwarz, K.B., Watson, C.L., Anan-thanarayanan, M., Thevananther, S., Christie, D.M., Hardikar, W., Setchell, K.D.R., Mieli-Vergani, G., Suchy, F.J & Mowat, A.P (1997) Hepatic basolateral sodium-dependent bile acid transporter expression in two unusual cases of hyper-cholanemia and in extrahepatic biliary atresia Hepatology 25, 1176–1183.
38 Gartung, C., Ananthanarayanan, M., Rahman, M., Shuele, S., Nundy, S., Soroka, C & Stolz, A (1996) Down-regulation of expression and function of the rat liver Na/bile acid cotransporter
in extrahepatic cholestasis Gastroeneteology 110, 199–209.
39 Oelkers, P., Kirby, L.C., Heubi, J.E & Dawson, P.A (1997) Primary bile acid malabsorption caused by mutations in the ileal sodium-dependent bile acid transporter gene (SLC10A2) J Clin Invest 99, 1880–1887.
40 Lidofsky, S.D., Fitz, J.G., Weisiger, R.A & Scharschmidt, B.F (1993) Hepatic taurocholate uptake is electrogenic and influenced
by transmembrane potential difference Am J Physiol 264 (Gastrointest Liver Physiol 27), G478–G485.