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INTRODUCTION |
Human placenta expresses a single transport system that transports
the micronutrients pantothenate, biotin, and lipoate (1-3). Studies
using human placental brush border membrane vesicles (1, 2) and human
placental choriocarcinoma cells (JAR) (3) have demonstrated that the
stoichiometry of Na+:substrate for this multivitamin
transporter is 2:1, indicating that for every vitamin molecule entering
the cell, two Na+ ions are cotransported into the cell.
Since the substrates pantothenate, biotin, and lipoate carry one
negative charge at physiological pH, the transport process is
electrogenic. Thus the transport system is energized by both the
Na+ gradient as well as the potential difference that exist
across the cell membrane. Sodium-dependent pantothenate and
biotin transport systems have also been individually characterized in
brush border membrane vesicles isolated from the intestine and kidney
of rat, rabbit, and human (4-13). Although it is unequivocal that the transport system is Na+-dependent, the
electrogenicity of the transport system is highly controversial and
much debated. It is also not known at this time whether a single
transporter, like in placenta, is responsible for the transport of both
biotin and pantothenate in the intestine and kidney. Said et
al. (6-8), characterizing biotin transport in intestinal brush
border membrane vesicles of rat, rabbit, and humans, have shown that
the transport process is electroneutral with a
Na+:substrate stoichiometry of 1:1. An electroneutral
transport system for biotin has also been reported from rat and human
renal brush border membrane vesicles (9, 10). On the other hand, the transport systems for pantothenate and biotin in rabbit renal brush
border membrane vesicles have been reported to be stimulated by an
inside-negative membrane potential indicating that the transport systems are electrogenic (11-13).
Recently, a cDNA clone encoding the rat
sodium-dependent multivitamin transporter
(rSMVT)1 has been isolated
from a rat placental cDNA library in our laboratory (14). The
cloned cDNA, when expressed in HeLa cells, induces Na+-dependent pantothenate and biotin transport
activities. The cDNA-specific uptake of both pantothenate and
biotin is inhibitable by lipoate. The transporter has a
Michaelis-Menten constant (Kt) of ~5
µM for pantothenate and ~15 µM for
biotin. The nucleotide sequence of the cloned transporter predicts a
protein of 68.6 kDa with 634 amino acids and 12 potential transmembrane
domains. Comparison of the protein sequence of SMVT with other proteins entered in the Swiss-Prot data base shows significant sequence homology
with known members of the Na+-dependent glucose
transporter family.
In this study, we describe the molecular cloning and structural and
functional characterization of the human sodium-dependent multivitamin transporter from a placental choriocarcinoma cell line
(JAR). This transporter is homologous to the transporter cloned from
the rat placenta. The cloned transporter has been characterized by
expressing the transporter in human retinal pigmental epithelial (HRPE)
cells and in Xenopus laevis oocytes and by analyzing its
transport function by tracer uptake and electrophysiological methods.
Northern blot analysis shows that the transcript of hSMVT of 3.2 kbp in
size is widely expressed in human tissues. The exon-intron organization
and chromosomal localization of the human SMVT gene are also presented.
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EXPERIMENTAL PROCEDURES |
Materials--
The JAR human placental choriocarcinoma cell line
was purchased from the American Type Culture Collection (Rockville, MD) and routinely cultured in RPMI 1640 medium supplemented with 10% fetal
bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin as
described before (15). The HRPE cell line number 165 originally provided by M. A. Del Monte (W. K. Kellogg Eye Center,
Department of Ophthalmology, Ann Arbor, MI) was routinely maintained in
Dulbecco's modified Eagle's medium/F-12 medium supplemented with 10%
fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin as described before (16, 17). Frogs (Xenopus
sp.) were purchased from Nasco (Fort Atkinson, WI), and MEGAscript cRNA
synthesis kit was obtained from Ambion (Austin, TX). SuperScript System for cDNA cloning, TRIzol reagent, oligo(dT) cellulose, and
Lipofectin were purchased from Life Technologies, Inc. Restriction
enzymes were purchased from New England Biolabs (Beverly, MA). Magna
nylon transfer membranes were purchased from Micron Separations, Inc. (Westborough, MA). D-[14C]Pantothenate (51.5 mCi/mmol) and [3H]biotin (58.2 Ci/mmol) were procured
from NEN Life Science Products. All of the unlabeled vitamins were
purchased from Sigma.
Screening of the JAR Cell cDNA Library--
The
BlpI/StuI fragment of the rat placental SMVT
cDNA was used as the probe. This fragment was 1.45 kbp long and
included 75 bp 5' to the translational start site and most of the
coding region. The probe was labeled with [
-32P]dCTP
by random priming using the Ready-to-go oligolabeling beads. A JAR cell
cDNA library was screened as described earlier (18, 19). Positive
clones were identified and the colonies purified by secondary screening.
DNA Sequencing--
Taq DyeDeoxy terminator cycle
sequencing was performed with an automated Perkin-Elmer Applied
Biosystems 377 Prism DNA Sequencer to sequence both the sense and
antisense strands of the cDNA. The sequence was analyzed using the
GCG sequence analysis software package GCG version 7.B (Genetics
Computer Group, Inc., Madison, WI).
Functional Expression in HRPE Cells--
This was done using the
vaccinia virus expression system (20) using HRPE cells as described
before (14, 21). Subconfluent HRPE cells grown on 24-well plates were
first infected with a recombinant (VTF7-3) vaccinia virus
encoding T7 RNA polymerase and then transfected with the plasmid
carrying the full-length cDNA. After 10-12 h post-transfection,
uptake measurements were made at room temperature with
[14C]pantothenate and [3H]biotin. The
uptake medium was 25 mM Hepes/Tris, pH 7.5, containing 140 mM NaCl, 5.4 mM KCl, 1.8 mM
CaCl2, 0.8 mM MgSO4, and 5 mM glucose. In most experiments, the time of incubation was
30 min. Endogenous transport was always determined in parallel using
cells transfected with empty vector. This transport accounted for
15-25% of the transport measured in cells that were transfected with the vector carrying the cDNA insert. Therefore, the transport values measured in cells transfected with empty vector were always subtracted from the corresponding transport values measured in cells
transfected with vector-cDNA to obtain the cDNA-specific uptake
of the vitamins.
Functional Expression in X. laevis Oocytes--
cRNA from the
cloned hSMVT-cDNA was synthesized using the MEGAscript kit (Ambion)
according to manufacturer's protocol. The cDNA was linearized
using NotI, and the cDNA insert was transcribed in
vitro using T7 RNA polymerase in the presence of an RNA cap analog. The resultant cRNA was purified by multiple extractions with
phenol/chloroform and precipitated with ethanol.
Mature oocytes from X. laevis were isolated by treatment
with collagenase A (1.6 mg/ml), manually defolliculated, and maintained at 18 °C in modified Barth's medium supplemented with 10 mg/liter gentamycin (22). On the following day, oocytes were injected with 50 ng
of cRNA. Oocytes injected with water served as control. The oocytes
were used for electrophysiological studies 6 days after cRNA injection.
Electrophysiological studies were done by the conventional
two-microelectrode voltage clamp method (23-25). Oocytes were
superfused with a NaCl-containing buffer (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 3 mM Hepes, 3 mM Mes, and 3 mM Tris, pH 7.5) followed by the
same buffer containing varying amounts of either pantothenate, biotin,
or lipoate. The membrane potential was held steady at
50 mV. For
studies involving the current-voltage (I-V) relationship, step changes
in membrane potential were applied, each for a duration of 100 ms in
20-mV increments. Kinetic parameters for the saturable transport of the
three substrates were calculated using the Michaelis-Menten equation.
Data were analyzed by nonlinear regression.
Northern Analysis--
Tissue distribution of the hSMVT-specific
transcripts was determined by Northern blot. A commercially available
membrane blot containing size-fractionated poly(A)+
mRNA from several tissues of human origin
(CLONTECH, Palo Alto, CA) was used for this
purpose. The full-length hSMVT cDNA labeled with
[
-32P]dCTP was used as the probe. Hybridization was
performed at 42 °C in 6× SSPE, 50% formamide, 10× Denhardt's
solution, 2% SDS, and 100 µg/ml salmon sperm DNA. Post-hybridization
washing was done at high stringency conditions that involved a final
wash at 60 °C for 30 min in 0.5× SSPE in 0.5% SDS.
Isolation and Characterization of Human SMVT Genomic
Clone--
This was done by screening a commercially available human
placental genomic library (Lambda FIX II, Stratagene, La Jolla, CA)
with a SalI/BglII fragment (~0.65 kbp),
comprising the 5' end of the human SMVT cDNA, as the probe.
Positive clones were isolated and plaque-purified by secondary and
tertiary screening. The size of the genomic insert was determined by
digestion with SalI and size-fractionation of the released
insert on agarose gel. Digestion of the genomic clone with
XbaI released 3 fragments (1.1, 5.0, and ~9.0 kbp) of the
genomic DNA insert. The 1.1- and 5-kbp fragments were subcloned into
pSPORT. The complete nucleotide sequence of the cloned SMVT
gene was obtained by sequencing the subcloned fragments and the phage DNA.
Chromosomal Localization--
The chromosomal localization of
the human SMVT gene was done by somatic cell hybrid
analysis, and the regional assignment of the gene on the chromosome was
accomplished by FISH. In somatic cell hybrid analysis, the mapping
panel number 2 consisting of mouse-human and Chinese hamster-human
somatic cell hybrids, obtained from the Human Genetic Cell Repository
(NIGMS, National Institutes of Health, Camden, NJ) was used. Each
hybrid cell line contained a single human chromosome. DNA was isolated
from the hybrid cells and digested with PstI. The
restriction fragments were size-fractionated, transferred to nylon
filters, and probed with full-length hSMVT cDNA as described
previously (26). The cDNA was labeled with [32P]dCTP
by random priming.
For use in FISH, the entire phage DNA of the hSMVT genomic clone was
labeled by nick translation with biotin-11-dUTP and used for
hybridization to human metaphase chromosome spreads at a concentration of 50 ng/µl. FISH was carried out essentially as detailed earlier (27). The chromosomes were stained by the chromycin
A3/distamycin A/4,6-diamidino-2-phenylindole technique for
fluorescent microscopic analysis.
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RESULTS AND DISCUSSION |
Isolation of Human Placental SMVT cDNA from a JAR Cell cDNA
Library--
Earlier studies using placental membrane vesicles have
demonstrated the presence of SMVT in the brush border membrane of the human placenta (1, 2). We characterized the uptake of pantothenate and
biotin in the choriocarcinoma cell line JAR, and we showed that these
cells express a transporter identical to the transporter that is
expressed in the placental brush border membrane (3). Unlike RNA
isolated from the term placenta that is contaminated with RNA from
other tissues like maternal decidua and non-trophoblast cells of the
placenta, RNA highly specific to the trophoblast can be isolated from
JAR cells in culture. With this in mind, we constructed and screened a
JAR cell cDNA library to clone the human SMVT using the recently
cloned rSMVT as the probe. Upon screening of approximately
3.6 × 105 colonies, several positive colonies were
obtained, one of which was arbitrarily chosen for further
characterization. The cDNA was designated hSMVT for human
sodium-dependent multivitamin transporter.
Structural Features of hSMVT cDNA--
The hSMVT cDNA is
3162 bp long with an open reading frame of 1908 bp (including the
termination codon), encoding a protein of 635 amino acids. The open
reading frame is flanked by a 5'-noncoding sequence 391 bp long and a
863-bp long 3'-noncoding sequence (Fig. 1). The cDNA contains a
poly(A)+ tail and a polyadenylation signal (AATAAA) in the
3'-noncoding region. The predicted molecular mass and pI of the protein
encoded by the open reading frame are 68.7 and 8.6 kDa, respectively. Preceding the putative initiation codon is the Kozak consensus sequence
(GAG GAT) (28). Hydrophobicity analysis of the predicted amino acid
sequence using the algorithm of Kyte and Doolittle (29) with a window
size of 20 amino acid residues indicated that the hSMVT protein
contains 12 putative transmembrane domains. When modeled with both the
amino terminus and the carboxyl terminus of the protein placed on the
cytoplasmic side, there are four potential N-linked
glycosylation sites (Asn138, Asn489,
Asn498, and Asn534), the first site between
transmembrane domains 3 and 4 and the other three sites between
transmembrane domains 11 and 12. The deduced amino acid sequence also
displays two sites (Ser283 and Thr286) with
consensus sequence for protein kinase C-dependent
phosphorylation (30, 31) in the cytoplasmic loop between transmembrane
domains 6 and 7. Amino acid sequence comparison of hSMVT against rSMVT shows a high degree of homology (84% identity and 89% similarity) between the two proteins indicating that the protein is highly conserved across the species. Blast search (32) against known sequences
in the Swiss-Prot data base shows considerable homology with members of
the sodium-dependent glucose transporter family confirming
that the protein is a member of this family. Among the
sodium-dependent glucose transporter family members, the
closest relative to SMVT with respect to amino acid sequence similarity is the sodium-dependent iodide transporter (40% identity
and 64% similarity) (33).

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Fig. 1.
hSMVT cDNA and predicted primary amino
acid sequence. Putative transmembrane domains are
underlined.
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Functional Characterization of hSMVT following Expression in HRPE
Cells--
The cDNA library used in this study was constructed in
pSPORT vector with cDNA inserts unidirectionally ligated in such a way that the sense transcription of the cDNA is under the control of the T7 promoter in the plasmid. This made it possible to employ the
transient vaccinia virus expression system for functional characterization of the clone. Initial experiments involving the time
course of radiolabeled pantothenate uptake into vector- and cDNA-transfected cells indicated that the uptake of pantothenate was higher in cDNA-transfected cells than in vector-transfected cells. The uptake was also found to be linear up to 60 min, and hence
all subsequent experiments were done with a 30-min incubation (data not shown).
The ionic dependence of the cDNA-stimulated pantothenate uptake was
investigated by measuring pantothenate transport in empty vector- and
cDNA-transfected cells in the presence of various inorganic salts
(Table I). Control uptake was measured in
the presence of NaCl. Replacement of Na+ with other cations
such as Li+, K+, choline, or
N-methyl-D-glucamine almost completely abolished the pantothenate uptake in both vector- and cDNA-transfected cells indicating that Na+ is obligatory for the transport
function. Such a marked inhibition was not seen when the
Cl
in the buffer was replaced with gluconate suggesting
the noninvolvement of Cl
in the transport process.
Replacing Cl
with I
on the other hand
reduced the uptake of the radiolabeled pantothenate by nearly 50%. It
is not known at this time whether this inhibition is due to the change
in the potential difference across the plasma membrane brought about by
the prolonged incubation with NaI or if it is due to I
competing with pantothenate for the transport process. The latter hypothesis cannot be ruled out since, as mentioned earlier, hSMVT has
very high sequence homology with the Na+/I
cotransporter.
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Table I
Ionic dependence of hSMVT-induced pantothenate uptake
HRPE cells transfected with either empty vector or with hSMVT cDNA
were incubated with 1 µM [14C]pantothenate for
30 min at room temperature either in the control buffer (25 mM Hepes/Tris, pH 7.5, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 5 mM glucose, and 140 mM NaCl) or in buffer where
NaCl was replaced with 140 mM various inorganic salts. When
the influence of anions was studied, KCl and CaCl2 in the
buffer were replaced with potassium gluconate and calcium gluconate,
respectively. After incubation for 30 min, the cells were washed with
the respective buffer (ice-cold), and the radioactivity associated with
the cells was quantitated. Uptake measured in control cells transfected
with empty vector was subtracted to obtain cDNA-specific uptake.
Values are means ± S.E.
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The substrate specificity of the transporter was evaluated by assessing
the ability of various unlabeled vitamins and vitamin analogs to
inhibit the transport of labeled pantothenate in pSPORT-transfected and
pSPORT-cDNA-transfected cells (Table
II). The cDNA-specific uptake was
determined in these experiments, by subtracting the transport measured
in the corresponding empty vector-transfected cells. Pantothenate,
biotin, and oxidized lipoate were able to inhibit the uptake of
radiolabeled pantothenate almost completely. Desthiobiotin, a biotin
analog, also inhibited the uptake significantly indicating that the
tetrahydrothiophene ring of biotin is not recognized by the
transporter. Iminobiotin and diaminobiotin, two other biotin analogs,
both of which lack the keto group at the second position of the
imidazole ring of biotin, did not inhibit pantothenate uptake
indicating that this group is involved in the interaction of biotin
with the transporter. The two other water-soluble vitamins tested,
namely niacinamide and thiamine, also had no influence on
cDNA-induced pantothenate uptake. These results are similar to that
obtained in pantothenate uptake studies using placental brush border
membrane vesicles and JAR choriocarcinoma cells (2, 3).
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Table II
Substrate specificity of hSMVT
HRPE cells were transfected with hSMVT cDNA as described under
"Experimental Procedures." Transport of radiolabeled pantothenate
(1 µM) was measured for 30 min at room temperature in the
presence or absence of indicated compounds (100 µM).
Uptake measured in control cells transfected with empty vector was
subtracted to obtain cDNA-specific uptake. The data represent
means ± S.E. for three to six determinations.
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The kinetics of pantothenate uptake was determined by measuring the
rate of pantothenate uptake at varying concentrations of pantothenate
(0.25-10 µM) in HRPE cells transfected with the plasmid
vector carrying the cDNA insert. Simultaneous measurements at
identical concentrations of pantothenate were also done in empty
vector-transfected cells. The values obtained were subtracted from the
corresponding values of transport measured in cDNA-transfected cells to obtain cDNA-specific uptake. The data obtained were
analyzed first by nonlinear regression analysis (Fig.
2A) and confirmed by linear
regression (Fig. 2A, inset). The kinetics of biotin uptake
by the cloned transporter was also determined similarly by measuring
the rate of biotin uptake at varying concentrations of biotin (0.5-20
µM) (Fig. 2B). As can be seen from the
figures, the cDNA-induced transport of both biotin and pantothenate
is saturable. The experimental data were found to fit best to a model describing the uptake by a single carrier. The linear regression analysis (Eadie-Hofstee transformation) of the data for the
cDNA-specific uptake yielded a linear plot for pantothenate and
biotin. The kinetic parameters for the carrier-mediated uptake,
Kt (Michaelis-Menten constant) and
Vmax (maximal velocity), were 1.5 ± 0.2 µM and 196.0 ± 12.9 pmol/106 cells/30
min, respectively, for pantothenate and 3.2 ± 0.7 µM and 157.6 ± 18.2 pmol/106 cells/30
min, respectively, for biotin. The Kt values of the
cloned transporter for the transport of pantothenate and biotin are
comparable to the values reported using placental brush border
membranes (1, 2) and JAR cells (3).

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Fig. 2.
Kinetics of pantothenate (A)
and biotin (B) uptake induced by hSMVT cDNA in
HRPE cells. Transport measurements were made simultaneously in
HRPE cells transfected with either empty vector or vector-cDNA
construct. Uptake was measured in the presence of NaCl with a 30-min
incubation at room temperature. The uptake values obtained in cells
transfected with empty vector were subtracted from uptake values
obtained in cells transfected with vector containing cDNA. Values
represent means ± S.E. for four determinations. Inset,
Eadie-Hofstee transformation of the same data. A,
concentration of [14C]pantothenate was varied over a
range of 0.25-10 µM. B, the concentration of
biotin was varied over a range of 0.5-20 µM.
Concentration of [3H]biotin was kept constant at 25 nM. For pantothenate, the endogenous uptake observed in
cells transfected with empty vector was 20-25% of the uptake measured
in cells transfected with plasmid-cDNA construct. For biotin, the
corresponding value was 10-15%.
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The three physiological substrates of SMVT are pantothenate, biotin,
and lipoate. All three substrates possess a long side chain containing
a carboxylate group. In addition, biotin has an imidazole- and
thiazole-fused ring structure. Three-dimensional structural analysis of
the pantothenate molecule indicates that it too exists in a pseudo-ring
conformation with a hydrogen bond between the amino group of the
-alanine moiety and the hydroxyl group in the pantoic acid moiety.
Lipoate can exist in two states. In its oxidized state, it forms a ring
structure by forming a disulfide linkage. In the reduced state with two
sulfhydryl groups, lipoate does not have a ring structure. We
investigated whether hSMVT can differentiate between these two forms of
lipoate. We measured the uptake rate of labeled pantothenate in vector-
and cDNA-transfected cells in the presence of varying
concentrations of oxidized and reduced lipoate. When reduced lipoate
was used, dithiothreitol at a final concentration of 1 mM
was used in the buffer to prevent the lipoate from getting oxidized.
Dithiothreitol by itself at a concentration of 1 mM had no
effect on the uptake of the vitamin (data not shown). The
cDNA-specific transport was determined, and the data were analyzed
by nonlinear regression analysis (Fig.
3). Both oxidized and reduced lipoate
inhibited the uptake of pantothenate; however, the latter was
significantly less potent than the former. The IC50 values
(i.e. the concentration necessary to cause 50% inhibition)
for oxidized lipoate was 2.7 ± 0.2 µM and that for
reduced lipoate 7.3 ± 0.8 µM. Since the concentration of radiolabeled pantothenate used in the assay was 1 µM and the Kt of hSMVT for
pantothenate is 1.5 µM, the KI value
for oxidized lipoate (calculated using the equation
KI = IC50/( 1 + [L]/Kt) where L is the concentration of
pantothenate in the assay) is 1.6 ± 0.2 µM and that
for reduced lipoate is 4.4 ± 0.5 µM. Thus, the
affinity of hSMVT for oxidized lipoate which has the ring conformation is approximately 3-fold higher than that for the reduced lipoate.

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Fig. 3.
Inhibition of pantothenate uptake by lipoate
in HRPE cells transfected with hSMVT cDNA. Uptake of
radiolabeled pantothenate was measured in the presence or absence of
oxidized lipoate ( ) and reduced lipoate ( ). Simultaneous
measurements were made in cells transfected with plasmid alone and
plasmid containing cDNA. The values measured in cells transfected
with empty plasmid were subtracted from values measured in cells
transfected with plasmid-cDNA construct. The concentration of
lipoate was varied from 0.316 to 31.6 µM.
2,3-Dithiothreitol at a concentration of 1 mM was included
in the uptake buffer when the effect of reduced lipoate on pantothenate
uptake was being measured. Values represent means ± S.E. for four
determinations.
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The effect of Na+ on the kinetics of pantothenate and
biotin uptake was investigated by measuring the uptake of radiolabeled pantothenate and biotin in HRPE cells transfected with hSMVT cDNA in the presence of varying concentrations of extracellular
Na+. The concentration of NaCl in the extracellular medium
was varied from 0 to 140 mM. The cDNA-specific uptake
was determined by subtracting the uptake measured simultaneously in
cells transfected with the pSPORT vector. The relationship between the
uptake rate and the Na+ concentration was sigmoidal (Fig.
4), suggesting the involvement of more
than one Na+ per pantothenate molecule transported. The
data were fit to the Hill equation, and the Hill coefficient, which is
the number of Na+ ions interacting with the carrier, was
calculated. The value was 1.9 ± 0.3 for the uptake of
pantothenate (Fig. 4A) and 1.8 ± 0.1 for the uptake of
biotin (Fig. 4B). These values were confirmed from the slope
of the Hill plots (Fig. 4, insets). This indicates that, for
every pantothenate/biotin molecule transported, 2 Na+ ions
are cotransported. Since both pantothenate and biotin are monovalent
anions at physiological pH, the transport process is electrogenic.
Thus, both the Na+ gradient as well as the difference in
the membrane potential across the cell membrane energize the transport
process. This greatly increases the concentrative capacity of the
transport process.

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Fig. 4.
Effect of Na+ on the uptake of
pantothenate (A) and biotin (B) in
HRPE cells expressing the cloned hSMVT. Uptake of pantothenate and
biotin was studied in HRPE cells transiently expressing the cloned
hSMVT with a 30-min incubation in the presence of increasing
concentrations of Na+ (0-140 mM) and a fixed
concentration of Cl (140 mM) in the
extracellular medium. The osmolality of the medium was kept constant by
replacing Na+ with appropriate concentrations of
N-methyl-D-glucamine. The cDNA-specific
transport was calculated by subtracting the uptake measured in
vector-transfected cells from the total uptake measured in cells
transfected with the cloned cDNA. Values are means ± S.E. of
three determinations. Inset, Hill plot of the same
data.
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Functional Characterization of hSMVT following Expression in X. laevis Oocytes--
A more direct way of characterizing a rheogenic
transport system is by electrophysiological means using the
two-microelectrode, voltage clamp technique. Since the transport of a
substrate molecule across the cell membrane mediated by SMVT is
associated with a net transfer of positive charge into cell, the SMVT
function should be associated with inward currents under voltage clamp
conditions in this experimental approach. The results of the
electrophysiological experiments done with SMVT-expressing oocytes are
given in Fig. 5. Perifusion of oocytes
with buffer containing 10 µM either pantothenate, biotin,
or lipoate in the presence of NaCl induced inward currents (~25-35
nA) (Fig. 5A). The inward currents were absent when NaCl in
the buffer was replaced with
N-methyl-D-glucamine chloride and also in
control oocytes injected with water, showing that the substrate-induced
currents are dependent on the presence of Na+ in the uptake
medium and SMVT expression (data not shown). The substrate-induced
inward currents were dependent on the testing membrane potential (Fig.
5B). The currents increased in magnitude as the testing
membrane potential became more hyperpolarized, demonstrating that the
transport activity of SMVT, measured by the magnitude of the
substrate-induced inward currents, is stimulated when the membrane
potential is made more negative. These data demonstrate unequivocally
that SMVT is a potential-sensitive transporter. In addition, since only
transportable substrates can induce the inward currents in
SMVT-expressing oocytes, the above data also demonstrate for the first
time that lipoate is indeed a transportable substrate.

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Fig. 5.
Electrophysiological characteristics of hSMVT
expressed in Xenopus oocytes. A,
representative chart recording of vitamin-induced inward currents. The
measurements were carried out in the presence of 10 µM
substrate (pantothenate, biotin, or lipoate) in NaCl-containing buffer.
B, current-voltage (I-V) relationship for substrate-evoked
inward currents. Currents were measured at different holding membrane
potentials in the presence of either pantothenate ( ), biotin ( ),
or lipoate ( ). The concentration of the substrates was 10 µM, and the measurements were made in the presence of
NaCl.
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Fig. 6 details the kinetics of hSMVT,
characterized electrophysiologically, using pantothenate as the
substrate. The SMVT-induced currents were dependent on the testing
membrane potential as well as on the concentration of pantothenate
(Fig. 6A). The current increased in magnitude with
increasing concentrations of pantothenate and increasing
hyperpolarization of the testing membrane potential. The
substrate-induced currents showed saturation kinetics with respect to
pantothenate concentration at different testing membrane potentials
(Fig. 6B). The kinetic constants Kt and
Imax for the transport of pantothenate at
different testing membrane potentials were calculated by nonlinear
regression analysis. The kinetic constant Kt, a
parameter indicative of the affinity of SMVT for the substrate,
decreased as the testing membrane potential became more hyperpolarized
(Fig. 6C). At a testing membrane potential of
50 mV, the
Kt was 1.9 ± 0.4 µM, whereas the
Kt decreased to 1.0 ± 0.2 µM
when the testing membrane potential was changed to
150 mV. This
suggests that the affinity of SMVT for pantothenate increases at
hyperpolarizing membrane potentials. The kinetic constant
Imax, a parameter indicative of the maximal velocity of the SMVT-mediated transport activity, increased as the
testing membrane potential became more hyperpolarized (Fig. 6D). The value for Imax increased
from 38.7 ± 2.8 nA at
50 mV to 290.4 ± 14.8 nA at
150
mV. This shows that SMVT is activated by an inside-negative membrane
potential.

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Fig. 6.
Characteristics of pantothenate-induced
inward currents in hSMVT cRNA injected oocytes. The composition of
the uptake buffer was 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2,
and 3 mM Mes/Hepes/Tris, pH 7.5. A, influence of
testing membrane potential (Vtest) on
pantothenate-induced currents (I) at various concentrations of
pantothenate. B, influence of pantothenate concentration on
pantothenate-induced currents (I) at various testing membrane
potentials. C, influence of testing membrane potential on
Kt for pantothenate. D, influence of
testing membrane potential on Imax for pantothenate-induced
currents.
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Identical experiments were also carried out using biotin and oxidized
lipoate as the substrates, and similar results were obtained (data not
shown). The affinity constant Kt, calculated for
biotin and oxidized lipoate, when the transport measurements were made
in SMVT-expressing oocytes with membrane potential clamped at
50 mV,
was 19.5 ± 4.6 and 7.3 ± 1.0 µM, respectively. The affinity constants measured using the
electrophysiological technique following expression in
Xenopus oocytes are significantly higher compared with
values obtained using expression in mammalian cells. Although the
reason for this is not clear, it may most probably be due to the
differences in post- translational processing (e.g.
N-linked glycosylation) of the transporter in the two
expression systems.
Northern Blot Analysis--
Tissue distribution of SMVT mRNA
in human tissues was studied using a commercially available membrane
blot containing size-fractionated poly(A)+ mRNA from
eight tissues: heart, brain, placenta, lung, liver, skeletal muscle,
kidney, and pancreas (Fig. 7). A 3.2-kbp
hybridizing signal was detected in all these tissues. Among the tissues
tested, the intensity of the hybridizing signal was the highest in the placenta. Moderate signals were seen in the kidney, liver, and pancreas. The detection of the SMVT-specific transcripts in the kidney
in the present study lends credence to the expression of Na+-dependent, electrogenic transport system
for pantothenate and biotin in the renal tissue. The fact that the
transcripts are detected in all the tissues tested hints at the
possible involvement of SMVT in the uptake of pantothenate, biotin, and
lipoate in all the tissues.

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Fig. 7.
Northern blot analysis of SMVT mRNA
transcripts in human tissues. A commercially available blot
containing 2 µg of poly(A)+ RNA from human heart, brain,
placenta, lung, liver, skeletal muscle, kidney, and pancreas was used.
The poly(A)+ RNA in the blot has been size-fractionated on
a denaturing formaldehyde-agarose gel and transferred onto nylon
membrane. The hybridization and post-hybridization washes were done at
high stringency conditions. The sizes of the hybridizing RNA were
determined using RNA size standards run in parallel in an adjacent
lane.
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Isolation of hSMVT Genomic Clone--
A genomic clone containing
the human SMVT gene was isolated by screening 6 × 105 phage colonies from an amplified human genomic library.
SalI digestion of the DNA isolated from the positive clone
released a single insert of ~15 kbp. Digestion with XbaI
released three fragments of 1.1, 5.0, and ~9.0 kbp. Sequential
Southern hybridization analysis of the SalI and
XbaI restriction-digested fragments of the genomic clone,
first with the SalI/BglII fragment (~0.65 kbp) of the cDNA specific to the 5' end of hSMVT and then with the HincII/XbaI fragment (~0.8 kbp) of the
cDNA, specific to the 3' end of hSMVT, was performed. Both probes
recognized the ~15-kbp SalI fragment, indicating that the
isolated genomic clone contained the complete SMVT gene
(data not shown). Of the three XbaI fragments, the probe
specific to the 5' end hybridized to the 5.0- and ~9.0-kbp fragments,
and the probe specific to 3' end hybridized only to the ~9.0-kbp
fragment (data not shown). The hybridization analysis and subsequent
sequence analysis of the genomic clone led to the conclusion that the
5.0-kbp and the adjacent ~9.0-kbp fragment of the genomic clone
contained the complete sequence of the hSMVT cDNA. The 1.1-kbp
XbaI fragment was 3' to the gene.
Structural Features of hSMVT Gene--
The SMVT gene is
~14.3 kbp in length and contains 17 exons (Fig.
8; GenBankTM accession number
AF116241). The first (184 bp) and the second (67 bp) exons contain only
the 5'-noncoding sequence of the cDNA. The second exon is followed
by a large intron of 3678 bp. None of the other 15 introns exceed more
than 750 bp. Except for the 3rd (532 bp) and 17th exons (989 bp), all
exons are less than 200 bp in length. All exon-intron boundaries
conform to consensus donor-acceptor sequences (gt/ag) for RNA splicing
(Table III).

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Fig. 8.
Exon-intron organization of the human
SMVT gene and its organizational relationship to the
hSMVT cDNA. Black boxes in the gene represent the
protein-coding regions of the exons, and open boxes
represent 5'-noncoding region in exons 1, 2, and 3 and 3'-noncoding
region in exon 17. The numbers in cDNA represent the
position of the first nucleotide of each exon.
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Table III
Exon-intron organization of human SMVT gene
Lowercase letters indicate intron sequences, and uppercase letters
indicate exon sequences. The donor and acceptor nucleotides have been
underlined.
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Chromosomal Localization--
We have mapped the chromosomal
location of the gene encoding the cloned placental SMVT. Southern blot
hybridization of restriction digests of DNA from Chinese hamster-human
and mouse-human hybrid cells with the hSMVT cDNA probe showed six
human-specific fragments of 4.2, 2.5, 1.5, 1.35, 1.3, and 1.25 kbp
(data not shown). Three fragments of 5.5, 2, and 1.6 kbp were detected
in DNA of Chinese hamster and four bands of 4, 2.5, 2.2, and 1.6 kbp
were present in mouse DNA. All six human-specific bands showed perfect
segregation with human chromosome 2. Regional localization of the
SMVT gene was carried out by fluorescent in situ
hybridization (FISH). The human SMVT genomic clone was used as the
probe. In situ hybridization of biotin-labeled genomic clone
to human metaphase chromosome spreads corroborated the initial findings
of Southern blot analysis on the localization of the SMVT
gene to human chromosome 2 (Fig. 9).
Twenty metaphase spreads were scored, and each showed hybridization signal on the short arm of chromosome 2, band p23. Although several inherited metabolic disorders have been mapped to this region of the
chromosome, none of them appears to be due to a defect in SMVT
function. On the other hand, several cases of biotin-responsive multiple carboxylase deficiency have been reported where a defect in
the transport of biotin is suspected (34-37). Unlike the more prevalent forms of multiple carboxylase deficiency where the activity of either holocarboxylase synthetase or biotinidase enzyme is invariably low, the activities of both the enzymes are normal in these
patients. This disorder is characterized with lactic acidosis and
organic aciduria, treatable with pharmacological doses of biotin.
Neither the identity of the gene responsible for the biotin-responsive
multiple carboxylase deficiency nor its chromosomal localization is
known. SMVT may most likely be the defective gene in these
patients. Future studies should be able to establish a role for SMVT in
biotin-responsive multiple carboxylase deficiency.

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Fig. 9.
Fluorescent in situ
hybridization analysis. Human metaphase chromosome spreads
were hybridized to biotin-labeled hSMVT genomic clone. A representative
chromosome spread is shown. Hybridization signals are indicated by
arrows.
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In conclusion, we have isolated a cDNA (hSMVT) from a human
placental trophoblast cell line cDNA library that, when expressed in HRPE cells, induces Na+-dependent transport
of pantothenate, biotin, and lipoate. The functional characteristics of
the induced activity are similar to those of the pantothenate/biotin
transport activity described in human placental brush border membrane
and in JAR cells. The cDNA has also been expressed in X. laevis oocytes and characterized by the electrophysiological
technique. The data presented here not only constitute the first direct
demonstration of the electrogenic nature of the transport process but
also for the first time provide evidence to show that lipoate is a
transportable substrate of this transporter. We have also isolated the
gene that codes for hSMVT, sequenced it, and characterized the
exon-intron organization of the gene. The gene maps to human chromosome 2p23.