Cloning and functional characterization of a new subtype of
the amino acid transport system N
Takeo
Nakanishi1,
Ramesh
Kekuda1,
You-Jun
Fei1,
Takahiro
Hatanaka1,
Mitsuru
Sugawara1,
Robert G.
Martindale2,
Frederick H.
Leibach1,
Puttur D.
Prasad3, and
Vadivel
Ganapathy1
Departments of 1 Biochemistry and Molecular Biology,
3 Obstetrics and Gynecology, and 2 Surgery, Medical
College of Georgia, Augusta, Georgia 30912
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ABSTRACT |
We have cloned a new subtype of the
amino acid transport system N2 (SN2 or second subtype of system N) from
rat brain. Rat SN2 consists of 471 amino acids and belongs to the
recently identified glutamine transporter gene family that consists of
system N and system A. Rat SN2 exhibits 63% identity with rat SN1. It
also shows considerable sequence identity (50-56%) with the
members of the amino acid transporter A subfamily. In the rat, SN2 mRNA is most abundant in the liver but is detectable in the brain, lung,
stomach, kidney, testis, and spleen. When expressed in Xenopus laevis oocytes and in mammalian cells, rat SN2 mediates
Na+-dependent transport of several neutral amino acids,
including glycine, asparagine, alanine, serine, glutamine, and
histidine. The transport process is electrogenic, Li+
tolerant, and pH sensitive. The transport mechanism involves the influx
of Na+ and amino acids coupled to the efflux of
H+, resulting in intracellular alkalization. Proline,
-(methylamino)isobutyric acid, and anionic and cationic amino acids
are not recognized by rat SN2.
system N2; electrogenicity; proton transport; glutamine transporter
family
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INTRODUCTION |
SYSTEM N is a
Na+-dependent amino acid transport system originally
described in rat hepatocytes that mediates the uptake of glutamine,
asparagine, and histidine (15). It is distinct from system
A, another Na+-dependent transport system for neutral amino
acids. A unique characteristic of system N is its Li+
tolerance, meaning that it retains its transport activity even when
Na+ is replaced with Li+. This system also
shows marked pH dependence. Its activity is very low at pH 6.0-6.5
but increases severalfold when the pH is changed from 6.5 to 8.5. Subsequent studies have shown that there may be different subtypes of
system N with distinguishing functional characteristics and with
different tissue distribution patterns (1, 13, 21, 33).
Skeletal muscle expresses a subtype of system N, called Nm,
which shows significantly weaker Li+ tolerance and pH
sensitivity than the hepatic system N (1, 13). Two
different types of system N have been described in the brain (21,
33). The system present in astrocytes is similar to the hepatic
system N, whereas the system present in neurons is distinct from the
hepatic system N and also from the skeletal muscle system
Nm. The neuronal system N, called Nb, also
exhibits weak Li+ tolerance and pH sensitivity, similar to
Nm, but is inhibited by glutamate, a characteristic not
observed with system N and system Nm.
Because all three subtypes of system N mediate active transport of
glutamine, these transport systems are likely to play an important role
in the metabolism of this amino acid in the liver, skeletal muscle, and
brain. Glutamine is the most abundant free amino acid in the
circulation and shuttles carbon and nitrogen between different tissues
in the body (4). A process termed "intercellular
glutamine cycle" has been shown to occur in the liver in which
periportal hepatocytes take up glutamine from the blood, and perivenous
hepatocytes release glutamine into the blood (11, 12).
There is evidence for glutamine uptake as well as glutamine release in
the skeletal muscle, depending on the physiological state
(34). Glutamine also plays an important role in the
skeletal muscle, not only as a substrate for protein synthesis but also as an effective modulator of protein turnover (27, 28). In the brain, glutamine plays an important role in the glutamine-glutamate cycle that occurs between glutamatergic neurons and glial cells (30, 38). A similar glutamine-glutamate cycle is also
known to occur between the liver of the developing fetus and the
placenta (2, 20). In all of these important metabolic
processes involving glutamine uptake or release, the subtypes of system
N are likely to play a significant role.
Recently, Chaudhry et al. (3) reported on the cloning of
the first subtype of system N. This transporter, called SN1, was cloned
from rat brain, but the transporter is also expressed abundantly in the
liver. Functional characteristics of the cloned rat SN1 include
Na+ dependence, Li+ tolerance, pH sensitivity,
and preference for glutamine, asparagine, and histidine as substrates.
This transporter mediates the influx of Na+ and glutamine
into the cells in exchange with intracellular H+. On the
basis of these properties, SN1 is likely to be the hepatic system N. Subsequently, we cloned the human homologue from a human hepatoma cell
line (7). Even though the original report by Chaudhry et
al. (3) claimed that the transport process mediated by rat
SN1 is electroneutral with a Na+:glutamine:H+
stoichiometry of 1:1:1, our studies with human SN1 as well as with rat
SN1 have shown that the transport process is electrogenic (7). This is supported by the inward currents associated
with the transport process in SN1-expressing Xenopus laevis
oocytes under voltage-clamp conditions and also by the findings that
the Na+:glutamine:H+ stoichiometry is 2:1:1. On
the basis of primary structure, SN1 belongs to a distinct gene family.
Three additional members (ATA1, ATA2, and ATA3) of this gene family
have recently been cloned, and they represent three different subtypes
of the amino acid transport system A (9, 10, 26, 31, 32, 35, 37, 40). ATA1, ATA2, and ATA3 mediate Na+-dependent
transport of several neutral amino acids, including the system
A-specific model substrate
-(methylamino)isobutyric acid (MeAIB).
The transport process mediated by ATA1, ATA2, and ATA3 is electrogenic
and highly pH sensitive but Li+ intolerant.
Here we report on the cloning and functional characterization of a new
member of this gene family. We cloned this transporter from rat brain.
This transporter, called SN2, represents a subtype of system N and is
expressed in the liver, brain, lung, stomach, kidney, testis, and spleen.
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MATERIALS AND METHODS |
Materials.
Radiolabeled amino acids were purchased from NEN Life Science Products,
Amersham Pharmacia Biotech, or American Radiolabeled Chemicals.
[14C]glycylsarcosine was custom synthesized by Cambridge
Research Biochemicals (Cleveland, United Kingdom). Restriction enzymes were from either Promega or New England Biolabs. Magna nylon transfer membranes used in library screening were from Micron Separations (Westboro, MA). The Ready-to-go oligolabeling kit was purchased from
Amersham Pharmacia Biotech.
Probe preparation.
The recently cloned rat SN1 is highly homologous to the human cDNA,
designated g17 in the GenBank database (accession no. U49082). This
indicated that g17 most likely represents the human homologue of rat
SN1. This has been confirmed recently in our laboratory by successfully
cloning the human SN1 and establishing its identity with g17
(7). This clone was isolated by screening a human hepatoma
cell line cDNA library with a g17-specific cDNA fragment as a probe.
The same probe was used in the present study to screen a rat brain cDNA
library in an attempt to isolate other subtypes of system N. This probe
was prepared by RT-PCR using primers based on the nucleotide sequence
of g17. The sense primer was 5'-AACATCGGAGCCATGTCCAG-3', which
corresponded to the nucleotide position 581-600 in g17 cDNA
sequence, and the antisense primer was 5'-
AAGGTGAGGTAGCCGAAGAG-3', which corresponded to the nucleotide position 1136-1155 in g17 cDNA sequence. Because Northern blot analysis has shown that rat SN1 mRNA is expressed most abundantly in
the liver (3), we used poly(A)+ mRNA isolated
from Hep G2 cells, a human hepatoma cell line, as a template for
RT-PCR. A single product of expected size (~0.6 kbp) was obtained in
the RT-PCR reaction. This product was subcloned into pGEM-T vector and
sequenced to establish its molecular identity.
cDNA library screening.
The ~0.6-kbp cDNA fragment of g17 was labeled with
[
-32P]dCTP using the Ready-to-go oligolabeling kit.
The rat brain cDNA library (29, 36, 39) was screened with
this probe under low stringency conditions.
DNA sequencing.
Both sense and antisense strands of the cDNAs were sequenced by primer
walking. Sequencing by the dideoxynucleotide chain termination method
was performed by Taq DyeDeoxy terminator cycle sequencing
with an automated Perkin-Elmer Applied Biosystems 377 Prism DNA
sequencer. The sequencer was analyzed using the GCG sequence analysis
software package GCG, version 10 (Genetics Computer Group, Madison, WI).
Functional expression in X. laevis oocytes.
cRNA from the cloned cDNA was synthesized using the mMESSAGE mMACHINE
kit (Ambion) according to the 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/l of gentamicin (17-19). 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
(17-19). Oocytes were perifused 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 8.0)
followed by the same buffer containing different amino acid substrates.
The membrane potential was held steady at
50 mV. For studies
involving the current-voltage 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 amino
acids were calculated using the Michaelis-Menten equation. Data were
analyzed by nonlinear regression and confirmed by linear regression.
When the effects of Na+ on the transport (i.e., amino
acid-induced currents) were evaluated, the oocyte was perifused with buffers containing different concentrations of Na+ and 10 mM glycine. The data for the Na+-dependent activation of
glycine-induced currents were fitted to the Hill equation, and the Hill
coefficient was calculated by nonlinear regression as well as by linear
regression. In some experiments, the perifusion buffer contained LiCl
instead of NaCl to determine whether Na+ was replaceable
with Li+ to support the amino acid-induced currents.
N-methyl-D-glucamine (NMDG) chloride was used in
place of NaCl to serve as negative control. When the influence of
Cl
on the amino acid-induced currents was assessed,
Na+ gluconate was used in place of NaCl. In addition, KCl,
MgCl2, and CaCl2 were replaced with respective
gluconate salts. In experiments dealing with the influence of pH on the
amino acid-induced currents, NaCl-containing buffers of varying pH were
prepared by appropriately adjusting the concentrations of MES, HEPES,
and Tris.
Uptake of [3H]glutamine in control oocytes and in rat
SN2-expressing oocytes was measured at pH 7.5 in the presence of NaCl as described previously (6). The concentration of
amino acids (unlabeled + radiolabeled) was 250 µM. To assess the
role of membrane potential in the rat SN2-mediated glutamine uptake,
uptake measurements were made in control ooctyes and in SN2-expressing
oocytes in the presence of 30 mM Na+, but with low (2 mM)
or high (72 mM) K+. Osmolality was maintained by inclusion
of NMDG at appropriate concentrations. The oocyte membrane was
depolarized with the high concentration of K+. This method
has been used previously in our laboratory to study the dependence of
transport function on membrane potential in the case of several
electrogenic transporters, including SN1 (7, 14, 24).
To determine whether or not the SN2-mediated transport function
involves the efflux of H+ from the oocyte, we coexpressed
rat SN2 and human PEPT1, a H+/peptide cotransporter
(8, 16), in the same oocyte and investigated the
interaction between these two transporters in terms of
transmembrane H+ gradient. The transport function of SN2
was monitored with [3H]glutamine as the substrate while
the transport function of PEPT1 was monitored with
[14C]glycylsarcosine as the substrate. In addition, we
assessed the influence of unlabeled glycylsarcosine on SN2 function and
the influence of unlabeled glutamine on PEPT1 function in these
oocytes. For the assessment of SN2 transport function, the transport of [3H]glutamine (250 µM) was measured in the absence or
presence of 10 mM glycylsarcosine in a NaCl-containing buffer at pH
6.0. For the assessment of PEPT1 transport function, the transport of
[14C]glycylsarcosine (50 µM) was measured in the
absence or presence of 10 mM glutamine in a NaCl-containing buffer at
pH 7.4.
To monitor the H+ efflux associated with the transport
function of rat SN2 directly, we used
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)
as a fluorescent marker of intracellular pH in oocytes expressing the
transporter. We expressed rat SN2 or human PEPT1 individually in
oocytes. Two hours before the measurement of SN2- or PEPT1-mediated
H+ movement, oocytes were injected with BCECF-AM (the
acetoxymethyl ester derivative of BCECF). We then monitored the
fluorescence by confocal microscopy in these oocytes with substrates
specific for SN2 or PEPT1. The excitation wavelength was alternated
between 440 and 490 nm while monitoring emission intensity at 540 nm. The fluorescence of BCECF is expected to decrease with acidification of
intracellular pH and increase with alkalization of intracellular pH
(23). Because PEPT1 is a H+-coupled
transporter that mediates the symport of H+ and its peptide
substrate, we used PEPT1-expressing oocytes as a control to validate
the experimental technique. The fluorescence in PEPT1-expressing
oocytes was monitored in a NaCl-containing buffer (pH 5.5) in the
absence or presence of 10 mM glycylsarcosine. The fluorescence in
SN2-expressing oocytes was monitored in a NaCl-containing buffer (pH
7.5) in the absence or presence of 2.5 mM glutamine.
Functional expression in mammalian cells.
The cloned rat SN2 was functionally expressed in human retinal pigment
epithelial (HRPE) cells using the vaccinia virus expression technique
(7, 9, 10, 31, 32, 37). Uptake measurements were made at
37°C for 15 min with radiolabeled amino acids. The composition of the
uptake buffer in most experiments was 25 mM Tris/HEPES (pH 8.5), 140 mM
LiCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4,
and 5 mM glucose. Because initial experiments showed that rat SN2
expressed in HRPE cells is Li+ tolerant, LiCl was
used in the uptake buffer instead of NaCl. This maneuver suppressed the
constitutively expressed basal amino acid uptake activity in these
cells, which made it ideal to measure the activity of the
heterologously expressed SN2. In addition to this, the uptake buffer
also contained 2 mM leucine to reduce the basal amino acid uptake
activity even further. Leucine is not a substrate for system N, and
most of the constitutive uptake of the amino acids used in the present
study occurs via system L. Therefore, the inclusion of leucine
abolishes the uptake of radiolabeled amino acids through the endogenous
system L without interfering with the transport function of the
heterologously expressed rat SN2. Uptake buffers of different pH were
made by appropriately adjusting the concentrations of Tris, HEPES, and MES. When Li+-activation kinetics were evaluated, the
concentration of Li+ was varied by isoosmotic substitution
of LiCl with NMDG chloride in appropriate concentrations. Even under
these conditions, there was still appreciable endogenous uptake
activity for most amino acids studied. Therefore, the endogenous uptake
was always determined in parallel using cells transfected with vector
alone. cDNA-specific uptake was calculated by adjusting for the
endogenous uptake activity.
Northern blot.
A commercially available, hybridization-ready rat multiple tissue blot
(Origene, Rockville, MD) was used to determine the tissue expression
pattern of SN2. The blot was hybridized with a rat SN2 cDNA probe under
high stringency conditions. The same blot was also hybridized
subsequently with a rat SN1 cDNA probe for comparison of the tissue
expression pattern between SN2 and SN1 and then with a
glyceraldehyde-3-phosphate dehydrogenase cDNA probe for demonstration
of RNA loading in each lane.
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RESULTS |
Structural features of rat SN2.
Screening of a rat brain cDNA library with a human SN1 cDNA fragment
led to the isolation of a clone that is different from the previously
known members of the glutamine transporter gene family. This new clone,
designated SN2, codes for a protein of 471 amino acids. The cDNA is
1,891 bp long with a poly(A)+ tail, and the open reading
frame is flanked by a 115-bp-long 5'-untranslated region and a
360-bp-long 3'-untranslated region (GenBank accession no. AF276870). A
comparison of the amino acid sequence of rat SN2 with that of the other
three members of the glutamine transporter gene family reveals
significant homology (Fig. 1). Rat SN2
exhibits 63% identity with rat SN1 at the amino acid sequence level.
Recently, we cloned the human homologue of SN2 from the Hep G2 liver
cell line (22). The amino acid sequence identity between
rat SN2 and human SN2 is 86%. SN2 is also structurally related to the
members of the amino acid transport system A subfamily. The sequence
identity of rat SN2 with rat ATA1, rat ATA2, and rat ATA3 is 50%,
56%, and 50%, respectively. On the basis of the sequence homology, it
appears that system N and system A form distinct subgroups within the
glutamine transporter gene family, the former consisting of SN1 and SN2
and the latter consisting of ATA1, ATA2, and ATA3. Hydropathy analysis
suggests that rat SN2 possesses 11 putative transmembrane domains. This
membrane topology is similar to that previously described for rat
SN1 (3), rat ATA1 (35), rat ATA2 (26,
40), and rat ATA3 (32).

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Fig. 1.
Comparison of the amino acid sequence of rat SN2 (the second
subtype of system N) with that of the other known members of the
glutamine transporter gene family. GenBank accession nos. used in this
analysis were AF295535 for rATA3, AF249673 for rATA2, and AF075704 for
rATA1. The amino acid sequence of rSN1 was taken from Ref.
15. Dark shading, identical amino acids; light-shading,
conservative substitutions.
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Northern blot analysis with mRNA from various rat tissues shows that
there are two SN2 transcripts, 2.6 kb and 1.9 kb in size (Fig.
2). These two transcripts are expressed
in a differential manner in the brain, lung, stomach, liver, kidney,
spleen, and testis. The transcripts are below detectable levels in the
thymus, heart, skeletal muscle, small intestine, and skin. The size of the transcript in the liver and kidney is 2.6 kb. In contrast, the size
of the transcript in other positive tissues is 1.9 kb. There is a
considerable difference in tissue expression pattern between SN2 and
SN1. SN1 is expressed in the brain, heart, liver, kidney, and skin. SN1
mRNA is not detectable in the lung, stomach, spleen, and testis, the
tissues that express SN2 mRNA. Furthermore, there is only a single
transcript for SN1 in all tissues in which the transcript is detectable
and the size of the transcript is 2.6 kb. The differential
tissue expression pattern and the variation in the size of the
transcripts for SN2 and SN1 in different tissues indicate that the cDNA
probes used in Northern blot hybridize specifically to the respective
mRNA. Because the transcript size in the liver is similar for SN2 and
SN1, we performed RT-PCR with rat liver mRNA using primer pairs
specific for rat SN2 and rat SN1. These studies showed unequivocally
that rat liver expresses SN2 as well as SN1 (data not shown). This
conclusion is also supported by the successful cloning of SN1 as well
as SN2 from the Hep G2 human liver cell line (7, 22).

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Fig. 2.
Tissue expression pattern of SN2 in the rat. A
commercially available hybridization-ready Northern blot was hybridized
sequentially with 32P-labeled rat SN2 cDNA probe,
32P-labeled rat SN1 (the first subtype of system N) cDNA
probe, and 32P-labeled human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA under high stringency conditions.
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Functional expression of rat SN2 in X. laevis oocytes.
The functional characteristics of rat SN2 were studied in the X. laevis oocyte expression system. Because SN1 was found to be
electrogenic (7), we thought that SN2 may also be
electrogenic. Therefore, we tested several amino acids to see whether
any of them induces inward currents under voltage-clamp conditions in oocytes expressing SN2. Several neutral amino acids were found to
induce inward currents in the presence of NaCl at pH 7.5 (Fig. 3). When tested at a fixed concentration
of 10 mM, the magnitude of the currents induced by the amino acids was
in the following order: glycine > asparagine > alanine > serine > glutamine > methionine > histidine. Among
the neutral amino acids tested, proline and MeAIB did not induce any
detectable currents (data not shown). Similarly, the acidic amino acids
glutamate and aspartate and the basic amino acid lysine also failed to
induce detectable currents. MeAIB is a specific model substrate for
system A (5). ATA1, ATA2, and ATA3, which belong to the
system A subgroup, are able to mediate Na+-coupled MeAIB
transport (9, 10, 26, 31, 32, 35, 37, 40). Because the new
clone does not recognize MeAIB as a substrate but is able to transport
asparagine, glutamine, and histidine, we named this clone SN2, the
second member of the system N subgroup to be identified at the
molecular level.

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Fig. 3.
Substrate specificity of rat SN2. Rat SN2 was expressed
heterologously in oocytes, and amino acid-induced currents were
monitored at 50 mV using the two-microelectrode voltage-clamp method.
Oocytes were perifused with different amino acids (10 mM) in a
NaCl-containing buffer (pH 7.5). Because glycine induced the maximal
current among the amino acids tested, data are presented as percent of
glycine-induced current for the rest of the amino acids. The experiment
was done in 3 oocytes from 3 different batches. For each oocyte, the
amino acid-induced currents were normalized based on the glycine
control in the same oocyte. Data represent means ± SE for 3 independent measurements in 3 different oocytes. Gly, glycine; Asn,
asparagine; Ala, alanine; Ser, serine; Gln, glutamine; Met, methionine;
His, histidine.
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Because glycine induced maximal currents in SN2-expressing oocytes, we
used this amino acid as the substrate for further characterization of
the transport function of rat SN2. We first assessed the role of
Na+ and Cl
in the transport process (Fig.
4A). The magnitude of
glycine-induced currents was found to be almost the same in the
presence of NaCl, Na+ gluconate, or LiCl, but the currents
were undetectable in the presence of NMDG chloride. These results show
that Na+ is obligatory for SN2 function and that
Li+ can substitute for Na+ equally well to
support SN2-mediated transport. On the other hand, Cl
does not participate in the transport process. We then assessed the
influence of external pH on the transport function of SN2. The
magnitude of glycine-induced currents was found to be markedly pH
sensitive (Fig. 4B). Acidification of external pH reduced
the transport function, as evidenced from the marked decrease in the currents as the external pH was decreased from 8 and 7 to 6 and 5. These features of SN2, namely Li+ tolerance and
pH sensitivity, are similar to those of SN1 (3, 7).

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Fig. 4.
Ion dependence (A) and pH dependence (B) of
glycine-induced currents in oocytes expressing rat SN2. A:
oocytes were perifused with 20 mM glycine in a buffer (pH 8.0) that
contained 100 mM NaCl, Na+ gluconate (NaGlu), LiCl, or
N-methyl-D-glucamine chloride (NMDGCl), and
glycine-induced currents were monitored using the
two-microelectrode voltage-clamp technique. B: oocytes were
perifused with 20 mM glycine in a NaCl-containing buffer of varying pH.
Similar results were obtained in 3 different oocytes.
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Glycine-induced currents showed a tendency toward saturation with
respect to glycine concentration (Fig. 5,
A and B). The K1/2 for
glycine (i.e., concentration at which the glycine-induced current was
half-maximal) was 15.2 ± 0.6 mM at
70 mV. The
I
(i.e., the maximal
glycine-induced current) was influenced by membrane potential, the
value increasing with hyperpolarization (Fig. 5C). The
K
was also
affected profoundly by membrane potential (Fig. 5D).
Hyperpolarization decreased the value for
K
, whereas depolarization increased the value. The kinetics of Na+
activation were then analyzed by assessing the influence of increasing concentrations of Na+ on glycine-induced currents (Fig.
6, A and B). The
relationship between Na+ concentration and glycine-induced
currents was not clearly hyperbolic (Fig. 6B). The magnitude
of glycine-induced currents measured at maximal Na+
activation (I
) increased markedly with membrane hyperpolarization (Fig. 6C). The
K1/2 for Na+ (i.e., concentration at
which the activation was half-maximal) was 11 ± 1 mM at
50 mV.
This value
(K
) decreased significantly when the membrane was hyperpolarized (Fig. 6D). Even though the sigmoidal relationship was not readily
noticeable in the analysis of Na+-activation kinetics, the
Hill coefficient (nH) for the relationship was
found to be significantly >1. The value was 1.20 ± 0.05 at
50
mV, and it increased to 1.28 ± 0.06 at
150 mV (Fig.
6E).

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Fig. 5.
Saturation kinetics of glycine-induced currents in oocytes
expressing rat SN2. Oocytes were perifused with varying concentrations
of glycine (0.5-40 mM) in a NaCl-containing buffer at pH 8.0. Osmolality was maintained with the appropriate addition of mannitol.
The amino acid-induced currents were monitored at different membrane
potentials. The relationship between glycine concentration and the
magnitude of glycine-induced current was analyzed by the
Michaelis-Menten equation describing a single saturable transport
system. The maximal glycine-induced current
(I ) and the concentration of glycine
needed for the induction of half-maximal current
)
were calculated from this analysis. A: glycine-induced
currents at different membrane potentials and at different glycine
concentrations. B: relationship between glycine
concentration and glycine-induced current at different membrane
potentials. C: influence of membrane potential on
I . D: influence of membrane
potential on
).
Similar results were obtained in 3 different oocytes.
Vtest, testing membrane potential.
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Fig. 6.
Na+-activation kinetics of glycine-induced
currents in oocytes expressing rat SN2. Oocytes were perifused
with 10 mM glycine in a buffer (pH 8.0) that contained varying
concentrations of NaCl (2.5-60 mM). Osmolality was maintained with
the appropriate addition of NMDG chloride. The amino acid-induced
currents were monitored at different membrane potentials. The
relationship between Na+ concentration and glycine-induced
current was analyzed by fitting the data to the Hill equation. The
maximal Na+-activated current
(I ), the
Na+ concentration necessary for the induction of
half-maximal current
(K ),
and the Hill coefficient (nH) were calculated
from this analysis. A: dependence of glycine-induced current
at different membrane potentials and at different Na+
concentrations. B: relationship between Na+
concentration and glycine-induced current at different membrane
potentials. C: influence of membrane potential on
I . D: influence
of membrane potential on
K .
E: influence of membrane potential on
nH. This experiment was repeated in 3 different
oocytes, and the results were similar.
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Because asparagine and glutamine are regarded as preferred substrates
for system N, we determined the affinities of these two amino acids for
SN2-mediated transport by assessing the saturation kinetics of
asparagine- or glutamine-induced currents (data not shown). The
K1/2 for asparagine and glutamine was found to
be 4.2 ± 0.5 and 4.1 ± 0.9 mM, respectively. These data
demonstrate that rat SN2 exhibits much greater affinity for asparagine
and glutamine than for glycine.
Our previous studies on the characterization of human SN1 identified an
interesting feature with regard to the currents induced by glutamine
and glycine (7). With SN1, the glutamine-induced current
reversed when the membrane potential was depolarized beyond
20 to
30 mV, whereas such reversal of the current was not evident with
other substrates of the transporter. To determine whether SN2 also
exhibits this feature, we analyzed the current-membrane potential
relationship for glutamine and glycine (Fig.
7). This analysis showed that the
glutamine-induced current reversed at a membrane potential of about
25 mV, whereas the glycine-induced current did not. The reversal of
the glutamine-induced current was demonstrable in the presence as well
as in the absence of Cl
. Thus both subtypes of system N
possess the interesting feature of current reversal with glutamine. The
mechanism responsible for this phenomenon is unknown.

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Fig. 7.
Dependence of glycine- and glutamine-induced currents on
membrane potential in oocytes expressing rat SN2. Oocytes were
perifused with 1 mM glycine or glutamine in a NaCl-containing buffer
(pH 8.0). The amino acid-induced currents were monitored at different
membrane potentials. Similar results were obtained with 2 different
oocytes.
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SN1 is known to mediate the influx of Na+ and amino acid
coupled to the efflux of H+ (3). The
Na+:amino acid stoichiometry for SN1 is 2:1, and the
transport process is electrogenic (7). Because SN2
exhibits similar characteristics and transport features, we
investigated whether the transport mechanism of SN2 also involves
H+ efflux. For this purpose, we coexpressed rat SN2 and
human PEPT1, a H+-coupled peptide transporter, in oocytes
and used the latter as a reporter of H+ movements. First,
we measured the uptake of glycylsarcosine (a substrate for PEPT1) in
the presence of NaCl at pH 7.4 with or without 10 mM glutamine (a
substrate for SN2). The rationale for this experiment is as follows. If
glutamine transport via SN2 in the presence of an inwardly directed
Na+ gradient is coupled to H+ efflux, the
transport process would lead to intracellular alkalization in the
oocyte. This would create an inwardly directed H+ gradient
(i.e., inside pH > outside pH) that should then stimulate the
transport of glycylsarcosine via PEPT1. Therefore, glutamine should
enhance glycylsarcosine uptake in the oocytes under these conditions.
Water-injected oocytes were used as control, and uptake measured in
these oocytes was subtracted to calculate PEPT1-specific uptake. The
results of these experiments show that PEPT1-specific uptake of
glycylsarcosine was stimulated more than twofold by glutamine (Fig.
8A). We confirmed these
results with another experiment in which we assessed the influence of
PEPT1-mediated H+ influx on SN2-mediated glutamine uptake.
We measured glutamine uptake in oocytes coexpressing SN2 and PEPT1 in
the presence of NaCl at pH 6.0 with or without 10 mM glycylsarcosine.
Again, uptake measured in water-injected oocytes was taken as control
to calculate SN2-specific uptake. Because of the presence of an
inwardly directed H+ gradient under these conditions (i.e.,
outside pH < inside pH), the transport function of PEPT1 should
be optimal, mediating the influx of H+ and glycylsarcosine.
This would lead to intracellular acidification that should then
facilitate the uptake of glutamine and Na+ coupled to
H+ efflux via SN2. Thus glycylsarcosine should enhance
glutamine uptake under these conditions. The results of these
experiments show that SN2-specific glutamine uptake was stimulated
significantly (33 ± 2%) by glycylsarcosine (Fig. 8B).
These studies demonstrate that SN2 mediates the influx of
Na+ and amino acid in exchange for H+ on the
trans side.

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Fig. 8.
Analysis of H+ efflux associated with rat SN2 in
oocytes. Rat SN2 and human PEPT1 were coexpressed in oocytes.
A: influence of glutamine (10 mM) on PEPT1-mediated uptake
of glycylsarcosine (Gly-Sar; 50 µM). The uptake of
[14C]glycylsarcosine was measured for 1 h in a
NaCl-containing buffer (pH 7.4) in the absence ( ) or presence (+) of
10 mM glutamine. B: influence of glycylsarcosine (10 mM) on
SN2-mediated uptake of glutamine (0.25 mM). The uptake of
[3H]glutamine was measured for 1 h in a
NaCl-containing buffer (pH 6.0) in the absence ( ) or presence (+) of
10 mM glycylsarcosine. Uptake measurements were made in parallel under
identical conditions in water-injected oocytes. These uptake values
were subtracted from corresponding uptake values in cRNA-injected
oocytes to calculate PEPT1-specific glycylsarcosine uptake and
SN2-specific glutamine uptake. Data (PEPT1- or SN2-specific uptake) are
presented as the percent of control uptake measured in the absence of
glutamine (A) or glycylsarcosine (B). In each
case, uptake was measured in 10 oocytes, and the results are given as
means ± SE.
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To demonstrate unequivocally the intracellular alkalization associated
with the transport process mediated by SN2, we used a more direct
approach in which BCECF was employed as a fluorescent pH marker (Fig.
9). The fluorescence of this fluorophor
is pH sensitive, and its fluorescence inside the cell increases as the intracellular pH increases. Instead of coexpressing SN2 and PEPT1 in
the same oocyte, we expressed these two transporters individually in
different oocytes. Two hours before the measurement of SN2- or
PEPT1-mediated H+ movement, oocytes were injected with
BCECF-AM. We then monitored the fluorescence by confocal microscopy in
these oocytes with specific transport substrates. In PEPT1-expressing
oocytes, perifusion of the oocytes with glycylsarcosine led to a
significant decrease in fluorescence, indicating intracellular
acidification. Because PEPT1 mediates the cotransport of H+
and the dipeptide substrate into the oocytes, this process is detectable by intracellular acidification, monitored by the decrease in
BCECF fluorescence. In contrast, perifusion of SN2-expressing oocytes
with glutamine led to a significant increase in fluorescence, indicating intracellular alkalization. These data provide direct evidence for H+ efflux associated with SN2-mediated influx
of Na+ and glutamine.

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Fig. 9.
Intracellular alkalization
associated with the transport process mediated by SN2. Rat SN2 and
human PEPT1 were expressed individually in Xenopus laevis
oocytes. Two hours before measurement of SN2- or PEPT1-mediated
H+ measurement, the oocytes were injected with
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM.
Fluorescence was then monitored in SN2-expressing oocytes in the
presence or absence of 2.5 mM glutamine in the presence of NaCl (pH
7.5) by confocal microscopy. Fluorescence in PEPT1-expressing oocytes
was monitored similarly in the presence or absence of 10 mM
glycylsarcosine (GS) in the presence of NaCl (pH 5.5) by confocal
microscopy. This experiment was repeated in 2 additional
oocytes, and the results were similar.
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|
One could argue that SN2-mediated Na+ influx might cause
changes in the activities of other constitutively expressed
transporters such as the Na+/H+ exchanger and
the Na+/HCO
cotransporter in the oocytes and that such changes could explain the observed effects of glutamine on intracellular pH in SN2-expressing oocytes. This alternative explanation is, however, very unlikely. Because the uptake buffer contained Na+, the Na+/H+ exchanger
is expected to mediate the influx of Na+ coupled to the
efflux of H+. Similarly, since there was no
HCO
in the uptake buffer, the
Na+/HCO
cotransporter is expected to mediate the efflux of HCO
under the experimental
conditions. If the Na+/H+ exchanger and/or the
Na+/HCO
cotransporter were involved, the
rise in the intracellular levels of Na+ resulting from
SN2-mediated Na+ influx would be expected to inhibit
H+ efflux via the Na+/H+ exchanger
and/or facilitate HCO
efflux via the
Na+/HCO
cotransporter. In either case, the result would be intracellular acidification rather than
intracellular alkalization. Therefore, we conclude that neither the
Na+/H+ exchanger nor the
Na+/HCO
cotransporter is responsible for
the observed intracellular alkalization associated with the transport
function of SN2.
The amino acid-induced inward currents under voltage-clamp conditions
in SN2-expressing oocytes demonstrate convincingly that the transport
process mediated by SN2 is electrogenic. To provide additional
supporting evidence for the electrogenicity of this process, we
investigated the influence of K+-induced depolarization of
the oocyte membrane on SN2-mediated glutamine uptake. Uptake of
glutamine (250 µM) was measured in water-injected oocytes and in
SN2-expressing oocytes in the presence of 30 mM NaCl (pH 7.5) with
either 2 mM KCl (control) or 72 mM KCl (depolarization). Osmolality was
maintained in control experiments by the addition of 70 mM NMDG
chloride. Uptake in water-injected oocytes was subtracted to calculate
SN2-specific uptake. These experiments showed that
K+-induced depolarization reduced SN2-specific glutamine
uptake significantly (25 ± 2%; control, 250.4 ± 13.5 pmol · oocyte
1 · h
1;
depolarization, 188.5 ± 5.7 pmol · oocyte
1 · h
1). These
results show that the transport function of SN2 is inhibited by
membrane depolarization, confirming the electrogenicity of the
transport process associated with a net transfer of positive charge
into the oocytes.
Previous studies by Tamarapoo et al. (33) showed that rat
brain expresses a distinct subtype of system N, which can be
functionally differentiated from the classic system N described in rat
liver. The classic system N is Li+ tolerant and pH
sensitive, whereas the brain subtype, called Nb, is
comparatively less Li+ tolerant, and, in addition, pH
insensitive. Furthermore, the anionic amino acid glutamate does not
interact with the classical system N but it inhibits glutamine
transport mediated by system Nb. Even though Nb
is sensitive to glutamate inhibition, it is unknown whether glutamate is merely a blocker of glutamine transport or is actually a
transportable substrate. Because we cloned SN2 from rat brain, it is
important to determine whether SN2 represents system Nb.
Our studies with SN2 clearly show that it is Li+ tolerant
and pH sensitive, suggesting that SN2 may not be identical to system
Nb. To support this conclusion with additional studies, we
investigated the sensitivity of the transport function of SN2 to
inhibition by glutamate. If SN2 is, indeed, identical to system
Nb, its transport function should be inhibitable by
glutamate. For this purpose, we measured the uptake of glutamine (0.5 mM) in water-injected oocytes and in SN2-expressing oocytes in a
NaCl-containing medium (pH 7.5) in the absence or presence of 5 mM
glutamate. The results of these experiments show that glutamine uptake
via SN2 is not inhibitable by glutamate (data not shown). It appears from these studies that SN2 may not be identical to system
Nb.
Functional expression of rat SN2 in mammalian cells.
The cloned rat SN2 was expressed heterologously in HRPE cells, and the
functional features of the transporter were examined to compare with
the functional features of the transporter observed in X. laevis
oocytes. Initial experiments showed that the uptake of glycine in
cells transfected with rat SN2 cDNA was approximately twofold higher
than in cells transfected with vector alone. The cDNA-specific uptake
of glycine was pH sensitive and Li+ tolerant as it was in
X. laevis oocytes. Subsequently, we carried out all studies
on the functional characterization of rat SN2 in HRPE cells in the
presence of LiCl instead of NaCl. Substitution of NaCl with LiCl
decreased the basal amino acid uptake activity in vector-transfected
cells, which enhanced the relative increase in the uptake activity in
cDNA-transfected cells. We first examined the substrate specificity of
rat SN2. The uptake of glycine, alanine, serine, glutamine, asparagine,
and histidine was severalfold higher in rat SN2-expressing cells than
in control cells (Table 1). The increase
in uptake was highest for serine (~7.4-fold). There was no increase
in the uptake of the system A-specific model substrate MeAIB. We
confirmed this substrate specificity by cross-inhibition studies in
which the ability of various unlabeled amino acids to compete with
SN2-mediated uptake of radiolabeled serine was assessed (Table 1).
These experiments showed that glycine, alanine, glutamine, asparagine,
and histidine effectively competed with serine for transport via rat
SN2, whereas MeAIB did not. We also investigated the
Li+-activation kinetics of rat SN2-mediated serine uptake
(Fig. 10). The dependence of serine
uptake on Li+ concentration clearly showed a sigmoidal
relationship with a Hill coefficient of 1.4 ± 0.1. Thus the
functional characteristics of rat SN2 are similar in two different
heterologous expression systems.

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Fig. 10.
Li+-activation kinetics of rat SN2-mediated
serine uptake in human retinal pigment epithelial cells. Cells were
transfected with vector or rat SN2 cDNA. Uptake buffer contained LiCl
instead of NaCl. Concentration of Li+ was varied by
substituting LiCl with NMDG chloride isoosmotically. Uptake buffer also
contained 2 mM leucine. Uptake of [3H]serine (5 µM) was
measured in vector-transfected cells and in cDNA-transfected cells.
cDNA-specific uptake was calculated by subtracting the uptake in
vector-transfected cells from the uptake in cDNA-transfected cells, and
the values were used for kinetic analysis. Inset: Hill plot.
Data represent means ± SE for 4 measurements in 2 separate
experiments.
|
|
 |
DISCUSSION |
Successful cloning of SN2 from rat (the present study) and human
(22) tissues/cell lines provide unequivocal evidence for the existence of subtypes within the amino acid transport system N. Even though functional studies have established the expression of
distinct system N subtypes in mammalian tissues (1, 13, 21,
33), these findings have not been corroborated with the identification of these distinct subtypes at the molecular level. The
first subtype of system N was cloned by Chaudhry et al.
(3). This transporter, designated SN1, is expressed in the
hepatocytes uniformly in all regions of the liver. In the brain, the
expression of SN1 is restricted to astrocytes. In the present study, we
cloned the second subtype of system N (SN2) from a rat brain cDNA
library. SN2 mediates the influx of Na+ and amino acid
coupled to the efflux of H+. Thus the transport function of
SN2 involves H+ movement across the membrane, and amino
acid influx into cells via this transporter causes intracellular
alkalization. SN2 represents the newest member of the most recently
identified glutamine transporter gene family. We have recently reported
on the cloning of human SN2 (22).
The substrate specificity of SN2 is interesting. It recognizes not only
glutamine, asparagine, and histidine, but also other neutral amino
acids such as glycine, alanine, and serine as substrates. This is also
true with the recently cloned human SN2 (22). Amino acid
transport system N is traditionally viewed as a transporter specific
for asparagine, glutamine, and histidine. However, both SN1 and SN2,
the two subtypes of system N to be cloned thus far, exhibit
significantly broader substrate specificity. Functional studies have
established the expression of system N in the liver and astrocytes,
system Nm in the skeletal muscle, and system Nb
in neurons. Because SN1 is expressed abundantly in the liver and in
astrocytes, it appears that SN1 represents the classic system N
originally described in the liver. SN2, on the other hand, does not
seem to represent system Nm or system Nb. In
the rat, there is no detectable SN2 mRNA in the skeletal muscle.
Furthermore, system Nm is known to exhibit very little
Li+ tolerance and pH sensitivity. These features of system
Nm directly contrast the features of SN2. Even though SN2
was cloned from the brain, it does not represent system Nb.
The functional features of system Nb include lack of
Li+ tolerance and pH sensitivity. In addition, glutamate
interacts with system Nb to a significant extent. SN2 does
not possess any of these features. Thus SN2 seems to represent a new
subtype of system N that has not been described previously in any
mammalian tissue.
SN2 mRNA is most abundant in the liver, but is expressed at detectable
levels in the brain, lung, stomach, kidney, testis, and spleen. An
interesting feature of SN2 expression is the presence of two different
mRNA transcripts (2.6 and 1.9 kb in size) that are expressed
differentially in different tissues. These transcripts arise most
likely from alternative splicing. The SN2 cDNA described in the present
study is 1,891 bp long and is likely to represent the shorter SN2 mRNA
transcript. The presence of multiple transcripts is also evident in
human tissues where at least three different SN2 mRNA species are
detectable that are expressed in a tissue-specific manner (2.6, 1.9, and 1.4 kb) (22).
The present finding that SN2 is expressed in the stomach is
interesting. Relevant to this finding is our recent observation that
SN2 mRNA is most abundant in the stomach in humans (22). SN1 is not expressed in this tissue, both in the human and the rat.
Because histidine is a good substrate for SN2, we speculate that the
abundant expression of this transporter in the stomach may have
relevance to the synthesis of histamine in specific cell types of this
organ. Histamine produced by enterochromaffin-like cells in the stomach
is a major regulator of parietal cell function (25).
Histidine is the precursor for histamine synthesis. Therefore, the
expression of SN2 in the stomach may be related to histamine synthesis.
 |
ACKNOWLEDGEMENTS |
We thank Vickie Mitchell for excellent secretarial assistance.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants
DA-10045 and HD-33347.
Address for reprint requests and other correspondence: V. Ganapathy, Dept. of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912 (E-mail:
vganapat{at}mail.mcg.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 January 2001; accepted in final form 16 July 2001.
 |
REFERENCES |
1.
Ahmed, A,
Maxwell DL,
Taylor PM,
and
Rennie MJ.
Glutamine transport in human skeletal muscle.
Am J Physiol Endocrinol Metab
264:
E993-E1000,
1993[Abstract/Free Full Text].
2.
Battaglia, FC.
Glutamine and glutamate exchange between the fetal liver and the placenta.
J Nutr
130:
974S-977S,
2000[ISI][Medline].
3.
Chaudhry, FA,
Reimer RJ,
Krizaj D,
Barber D,
Storm-Mathisen J,
Copenhagen DR,
and
Edwards RH.
Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission.
Cell
99:
769-780,
1999[ISI][Medline].
4.
Christensen, HN.
Role of amino acid transport and countertransport in nutrition and metabolism.
Physiol Rev
70:
43-77,
1990[Free Full Text].
5.
Christensen, HN,
Oxender DL,
Liang M,
and
Vatz KA.
The use of N-methylation to direct route of mediated transport of amino acids.
J Biol Chem
240:
3609-3616,
1965[Free Full Text].
6.
Fei, YJ,
Prasad PD,
Leibach FH,
and
Ganapathy V.
The amino acid transport system y+L induced in Xenopus laevis oocytes by human choriocarcinoma cell (JAR) mRNA is functionally related to the heavy chain of the 4F2 cell surface antigen.
Biochemistry
34:
8744-8751,
1995[ISI][Medline].
7.
Fei, YJ,
Sugawara M,
Nakanishi T,
Huang W,
Wang H,
Prasad PD,
Leibach FH,
and
Ganapathy V.
Primary structure, genomic organization, and functional and electrogenic characteristics of human system N1, a Na+ and H+ coupled glutamine transporter.
J Biol Chem
275:
23707-23717,
2000[Abstract/Free Full Text].
8.
Ganapathy, V,
and
Leibach FH.
Proton-coupled solute transport in the animal cell plasma membrane.
Curr Opin Cell Biol
3:
695-701,
1991[Medline].
9.
Hatanaka, T,
Huang W,
Ling R,
Prasad PD,
Sugawara M,
Leibach FH,
and
Ganapathy V.
Evidence for the transporter of neutral as well as cationic amino acids by ATA3, a novel and liver-specific subtype of amino acid transport system A.
Biochim Biophys Acta
1510:
10-17,
2001[ISI][Medline].
10.
Hatanaka, T,
Huang W,
Wang H,
Sugawara M,
Prasad PD,
Leibach FH,
and
Ganapathy V.
Primary structure, functional characteristics and tissue expression pattern of human ATA2, a subtype of amino acid transport system A.
Biochim Biophys Acta
1467:
1-6,
2000[ISI][Medline].
11.
Haussinger, D.
Hepatocyte heterogeneity in glutamine and ammonia metabolism and the role of an intercellular glutamine cycle during ureogenesis in perfused rat liver.
Eur J Biochem
133:
269-275,
1983[Abstract].
12.
Haussinger, D,
Stoll B,
Stehle T,
and
Gerok W.
Hepatocyte heterogeneity in glutamate metabolism and bidirectional transport in perfused rat liver.
Eur J Biochem
185:
189-195,
1989[Abstract].
13.
Hundal, HS,
Rennie MJ,
and
Watt PW.
Characteristics of L-glutamine transport in perfused rat skeletal muscle.
J Physiol
393:
283-305,
1987[Abstract].
14.
Kekuda, R,
Prasad PD,
Wu X,
Wang H,
Fei YJ,
Leibach FH,
and
Ganapathy V.
Cloning and functional characterization of a potential-sensitive, polyspecific organic cation transporter (OCT3) most abundantly expressed in placenta.
J Biol Chem
273:
15971-15979,
1998[Abstract/Free Full Text].
15.
Kilberg, MS,
Handlogten ME,
and
Christensen HN.
Characteristics of an amino acid transport system in rat liver for glutamine, asparagine, histidine, and closely related analogs.
J Biol Chem
255:
4011-4019,
1980[Abstract/Free Full Text].
16.
Liang, R,
Fei YJ,
Prasad PD,
Ramamoorthy S,
Han H,
Yang-Feng TL,
Hediger MA,
Ganapathy V,
and
Leibach FH.
Human intestinal H+/peptide cotransporter. Cloning, functional expression, and chromosomal localization.
J Biol Chem
270:
6456-6463,
1995[Abstract/Free Full Text].
17.
Loo, DDF,
Hazama A,
Supplisson S,
Turk E,
and
Wright EM.
Relaxation kinetics of the Na+/glucose cotransporter.
Proc Natl Acad Sci USA
90:
5767-5771,
1993[Abstract].
18.
Mackenzie, B,
Fei YJ,
Ganapathy V,
and
Leibach FH.
The human intestinal H+/oligopeptide cotransporter hPEPT1 transports differently-charged dipeptides with identical electrogenic properties.
Biochim Biophys Acta
1284:
125-128,
1996[ISI][Medline].
19.
Mackenzie, B,
Loo DDF,
Fei YJ,
Liu W,
Ganapathy V,
Leibach FH,
and
Wright EM.
Mechanisms of the human intestinal H+-coupled oligopeptide transporter hPEPT1.
J Biol Chem
271:
5430-5437,
1996[Abstract/Free Full Text].
20.
Marconi, AM,
Battaglia FC,
Meschia G,
and
Sparks JW.
A comparison of amino acid arteriovenous differences across the liver and placenta of the fetal lamb.
Am J Physiol Endocrinol Metab
257:
E909-E915,
1989[Abstract/Free Full Text].
21.
Nagaraja, TN,
and
Brookes N.
Glutamine transport in mouse cerebral astrocytes.
J Neurochem
66:
1665-1667,
1996[ISI][Medline].
22.
Nakanishi, T,
Sugawara M,
Huang W,
Martindale RG,
Leibach FH,
Ganapathy ME,
Prasad PD,
and
Ganapathy V.
Structure, function, and tissue expression pattern of human SN2, a subtype of the amino acid transport system N.
Biochem Biophys Res Commun
281:
1343-1348,
2001[ISI][Medline].
23.
Negulescu, PA,
and
Machen TE.
Intracellular ion activities and membrane transport in parietal cells measured with fluorescent dyes.
Methods Enzymol
192:
38-81,
1990[Medline].
24.
Prasad, PD,
Srinivas SR,
Wang H,
Leibach FH,
Devoe LD,
and
Ganapathy V.
Electrogenic nature of rat sodium-dependent multivitamin transport.
Biochem Biophys Res Commun
270:
836-840,
2000[ISI][Medline].
25.
Prinz, C,
Zanner R,
Gerhard M,
Mahr S,
Neumayer N,
Hohne-Zell B,
and
Gratzl M.
The mechanism of histamine secretion from gastric enterochromaffin-like cells.
Am J Physiol Cell Physiol
277:
C845-C855,
1999[Abstract/Free Full Text].
26.
Reimer, RJ,
Chaudhry FA,
Gray AT,
and
Edwards RH.
Amino acid transport system A resembles system N in sequence but differs in mechanism.
Proc Natl Acad Sci USA
97:
7715-7720,
2000[Abstract/Free Full Text].
27.
Rennie, MJ,
Khogali SEO,
Low SY,
McDowell HE,
Hundal HS,
Ahmed A,
and
Taylor PM.
Amino acid transport in heart and skeletal muscle and the functional consequences.
Biochem Soc Trans
24:
869-873,
1996[ISI][Medline].
28.
Rennie, MJ,
MacLennan PA,
Hundal HS,
Weryk B,
Smith K,
Taylor PM,
Egan CJ,
and
Watt PW.
Skeletal muscle glutamine transport, intramuscular glutamine concentration, and muscle-protein turnover.
Metabolism
38:
47-51,
1989[ISI][Medline].
29.
Seth, P,
Fei YJ,
Li HW,
Huang W,
Leibach FH,
and
Ganapathy V.
Cloning and functional characterization of a sigma receptor from rat brain.
J Neurochem
70:
922-931,
1998[ISI][Medline].
30.
Sibson, NR,
Dhankhar A,
Mason GF,
Behar KL,
Rothman DL,
and
Shulman RG.
In vivo 13C NMR measurements of cerebral glutamine synthesis as evidence for glutamate-glutamine cycling.
Proc Natl Acad Sci USA
94:
2699-2704,
1997[Abstract/Free Full Text].
31.
Sugawara, M,
Nakanishi T,
Fei YJ,
Huang W,
Ganapathy ME,
Leibach FH,
and
Ganapathy V.
Cloning of an amino acid transporter with functional characteristics and tissue expression pattern identical to that of system A.
J Biol Chem
275:
16473-16477,
2000[Abstract/Free Full Text].
32.
Sugawara, M,
Nakanishi T,
Fei YJ,
Martindale RG,
Ganapathy ME,
Leibach FH,
and
Ganapathy V.
Structure and function of ATA3, a new subtype of amino acid transport system A, primarily expressed in the liver and skeletal muscle.
Biochim Biophys Acta
1509:
7-13,
2000[ISI][Medline].
33.
Tamarapoo, BK,
Raizada MK,
and
Kilberg MS.
Identification of a system N-like Na+-dependent glutamine transport activity in rat brain neurons.
J Neurochem
68:
954-960,
1997[ISI][Medline].
34.
Taylor, PM,
Rennie MJ,
and
Low SY.
Biomembrane transport and interorgan nutrient flows: the amino acids.
In: Biomembrane Transport, edited by Van Winkle LJ.. San Diego, CA: Academic, 1999, p. 295-325.
35.
Varoqui, H,
Zhu H,
Yao D,
Ming H,
and
Erickson JD.
Cloning and functional identification of a neuronal glutamine transporter.
J Biol Chem
275:
4049-4054,
2000[Abstract/Free Full Text].
36.
Wang, H,
Fei YJ,
Ganapathy V,
and
Leibach FH.
Electrophysiological characteristics of the proton-coupled peptide transporter PEPT2 cloned from rat brain.
Am J Physiol Cell Physiol
275:
C967-C975,
1998[Abstract/Free Full Text].
37.
Wang, H,
Huang W,
Sugawara M,
Devoe LD,
Leibach FH,
Prasad PD,
and
Ganapathy V.
Cloning and functional expression of ATA1, a subtype of amino acid transporter A, from human placenta.
Biochem Biophys Res Commun
273:
1175-1179,
2000[ISI][Medline].
38.
Westergaard, N,
Sonnewald U,
and
Schousboe A.
Metabolic trafficking between neurons and astrocytes: the glutamate/glutamine cycle revisited.
Dev Neurosci
17:
203-211,
1995[ISI][Medline].
39.
Wu, X,
Kekuda R,
Huang W,
Fei YJ,
Leibach FH,
Chen J,
Conway SJ,
and
Ganapathy V.
Identity of the organic cation transporter OCT3 as the extraneuronal monoamine transporter (uptake 2) and evidence for the expression of the transporter in the brain.
J Biol Chem
273:
32776-32786,
1998[Abstract/Free Full Text].
40.
Yao, D,
Mackenzie B,
Ming H,
Varoqui H,
Zhu H,
Hediger MA,
and
Erickson JD.
A novel system A isoform mediating Na+/neutral amino acid cotransport.
J Biol Chem
275:
22790-22797,
2000[Abstract/Free Full Text].
Am J Physiol Cell Physiol 281(6):C1757-C1768
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