Download Cloning and functional characterization of a new subtype of the

Am J Physiol Cell Physiol
281: C1757–C1768, 2001.
Cloning and functional characterization of a new
subtype of the amino acid transport system N
TAKEO NAKANISHI,1 RAMESH KEKUDA,1 YOU-JUN FEI,1 TAKAHIRO HATANAKA,1
MITSURU SUGAWARA,1 ROBERT G. MARTINDALE,2 FREDERICK H. LEIBACH,1
PUTTUR D. PRASAD,3 AND VADIVEL GANAPATHY1
Departments of 1Biochemistry and Molecular Biology, 3Obstetrics and Gynecology,
and 2Surgery, Medical College of Georgia, Augusta, Georgia 30912
Received 15 January 2001; accepted in final form 16 July 2001
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 glutamineglutamate cycle that occurs between glutamatergic
neurons and glial cells (30, 38). A similar glutamineglutamate 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. Func-
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@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.
system N2; electrogenicity; proton transport; glutamine
transporter family
SYSTEM
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0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society
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Nakanishi, Takeo, Ramesh Kekuda, You-Jun Fei, Takahiro Hatanaka, Mitsuru Sugawara, Robert G. Martindale, Frederick H. Leibach, Puttur D. Prasad, and
Vadivel Ganapathy. Cloning and functional characterization of a new subtype of the amino acid transport system N.
Am J Physiol Cell Physiol 281: C1757–C1768, 2001.—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.
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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
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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 MichaelisMenten 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 pre-
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tional 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.
CLONING OF A NEW SUBTYPE OF AMINO ACID TRANSPORT SYSTEM N
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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, hybridizationready 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.
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 topol-
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pared 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 ␮M. 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 ␮M) was measured in
the absence or presence of 10 mM glycylsarcosine in a NaClcontaining buffer at pH 6.0. For the assessment of PEPT1
transport function, the transport of [14C]glycylsarcosine (50
␮M) 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
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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; lightshading, conservative substitutions.
ogy is similar to that previously described for rat SN1 (3),
rat ATA1 (35), rat ATA2 (26, 40), and rat ATA3 (32).
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
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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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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.
Because glycine induced maximal currents in SN2expressing 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).
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)
Gly
was 15.2 ⫾ 0.6 mM at ⫺70 mV. The I max
(i.e., the
maximal glycine-induced current) was influenced by
membrane potential, the value increasing with hyperGly
polarization (Fig. 5C). The K ⁄ was also affected
profoundly by membrane potential (Fig. 5D). HyperpoGly
larization 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 glycineinduced 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⫹
Na⫹ activation (Imax
) increased markedly with membrane hyperpolarization (Fig. 6C). The K1/2 for Na⫹
(i.e., concentration at which the activation was halfmaximal)
was 11 ⫾ 1 mM at ⫺50 mV. This value
Na⫹
(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).
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
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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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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).
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
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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.
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
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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. 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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CLONING OF A NEW SUBTYPE OF AMINO ACID TRANSPORT SYSTEM N
Fig. 5. Saturation kinetics of glycineinduced 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 glycineGly
induced current (Imax
) and the concentration of glycine needed for the induction of half-maximal current 共K Gly
⁄ ) were
calculated from this analysis. A: glycineinduced 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
Gly
potential on Imax
. D: influence of membrane potential on 共K Gly
⁄ ). Similar results were obtained in 3 different oocytes. Vtest, testing membrane potential.
12
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.
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⫹/HCO3⫺ 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 conAJP-Cell Physiol • VOL
tained Na⫹, the Na⫹/H⫹ exchanger is expected to
mediate the influx of Na⫹ coupled to the efflux of H⫹.
Similarly, since there was no HCO3⫺ in the uptake
buffer, the Na⫹/HCO3⫺ cotransporter is expected to
mediate the efflux of HCO3⫺ under the experimental
conditions. If the Na⫹/H⫹ exchanger and/or the Na⫹/
HCO3⫺ 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 HCO3⫺ efflux via the Na⫹/HCO3⫺ 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⫹/HCO3⫺
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 ␮M) 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 SN2specific glutamine uptake significantly (25 ⫾ 2%; con-
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CLONING OF A NEW SUBTYPE OF AMINO ACID TRANSPORT SYSTEM N
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12
trol, 250.4 ⫾ 13.5 pmol 䡠 oocyte⫺1 䡠 h⫺1; depolarization,
188.5 ⫾ 5.7 pmol 䡠 oocyte⫺1 䡠 h⫺1). These results show
that the transport function of SN2 is inhibited by
membrane depolarization, confirming the electrogenic-
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.
AJP-Cell Physiol • VOL
ity 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,
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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
Na⫹
by fitting the data to the Hill equation. The maximal Na⫹-activated current (I max), the Na⫹ concentration necessary for
Na⫹
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
Na⫹
Na⫹
membrane potential on Imax
. 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.
CLONING OF A NEW SUBTYPE OF AMINO ACID TRANSPORT SYSTEM N
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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
AJP-Cell Physiol • VOL
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 PEPT1expressing 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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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 PEPT1mediated uptake of glycylsarcosine (Gly-Sar; 50 ␮M).
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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CLONING OF A NEW SUBTYPE OF AMINO ACID TRANSPORT SYSTEM N
Table 1. Substrate specificity of rat SN2 expressed
heterologously in HRPE cells
Uptake of Radiolabeled
Amino Acid, pmol 䡠 106
cells⫺1 䡠 15 min⫺1
Radiolabeled
or Unlabeled
Amino Acid
Vector
SN2 cDNA
cDNA-Specific Uptake of
[3H]Serine, pmol 䡠 106
cells⫺1 䡠 15 min⫺1
Control
Glycine
Alanine
Serine
Glutamine
Asparagine
Histidine
MeAIB
46 ⫾ 1
127 ⫾ 15
53 ⫾ 3
82 ⫾ 2
63 ⫾ 2
27 ⫾ 1
43 ⫾ 1
145 ⫾ 8 (3.2)*
238 ⫾ 10 (1.9)
391 ⫾ 9 (7.4)
274 ⫾ 12 (3.4)
279 ⫾ 15 (4.5)
56 ⫾ 1 (2.1)
37 ⫾ 2 (0.9)
309 ⫾ 11 (100)†
95 ⫾ 4 (31)
89 ⫾ 3 (29)
37 ⫾ 3 (12)
74 ⫾ 5 (24)
87 ⫾ 7 (28)
26 ⫾ 1 (8)
291 ⫾ 15 (94)
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.
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).
AJP-Cell Physiol • VOL
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 ␮M) 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.
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Values are means ⫾ SE for 6 determinations from 2 separate
experiments. Human retinal pigment epithelial (HRPE) cells were
transfected with vector or rat SN2 cDNA. Uptake of radiolabeled
amino acids was measured in the presence of LiCl and leucine as
described in MATERIALS AND METHODS. Concentration was 5 ␮M for all
amino acids except for MeAIB, in which case concentration was 15
␮M. * Values in first column of parentheses represent fold increase in
the uptake of each radiolabeled amino acid in cDNA-transfected cells
compared with vector-transfected cells. In cross-inhibition studies,
uptake of [3H]serine (5 ␮M) was measured in vector-transfected cells
and in cDNA-transfected cells in the absence or presence of unlabeled amino acids (10 mM). cDNA-specific uptake was calculated by
subtracting uptake in vector-transfected cells from corresponding
uptake in cDNA-transfected cells. † Values in second column of
parentheses represent percent of control uptake measured in the
absence of competing unlabeled amino acids. SN2, second subtype of
system N; MeAIB, ␣-(methylamino)isobutyric acid.
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
CLONING OF A NEW SUBTYPE OF AMINO ACID TRANSPORT SYSTEM N
We thank Vickie Mitchell for excellent secretarial assistance.
This work was supported by National Institutes of Health Grants
DA-10045 and HD-33347.
12.
13.
14.
15.
16.
17.
18.
19.
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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.
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