Serine 318 Is Essential for the Pyrimidine Selectivity of the
N2 Na+-Nucleoside Transporter*
Juan
Wang and
Kathleen M.
Giacomini
From the Departments of Biopharmaceutical Sciences and Cellular and
Molecular Pharmacology, University of California,
San Francisco, California 94143
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ABSTRACT |
Molecular cloning has isolated two subtypes of
Na+-nucleoside transporters; one is
pyrimidine-selective (N2), and the other is purine-selective (N1).
Using chimeric rat N2/N1 transporters, we previously demonstrated that
transmembrane domains (TM) 8 and 9 are the major sites for substrate
binding and discrimination. Interestingly, when TM8 of N2 was replaced
by that of N1, the resulting chimera, T8, lost the pyrimidine
selectivity of N2 and accepted both purine and pyrimidine nucleosides.
Five residues differ between rat N2 and N1 in TM8. To identify the
critical residues responsible for transport selectivity, the five
residues in N2 were systematically changed to their equivalents in N1. Replacing the serine residue at position 318 to its equivalent N1
residue, glycine, caused N2 to lose its selectivity for pyrimidine nucleosides and accept purine nucleosides as substrates. In contrast, replacing the other four residues did not change the pyrimidine selectivity of N2. Furthermore, when glycine 318 in chimera T8 was
changed back to serine, the chimeric transporter regained pyrimidine
selectivity. These observations suggest that serine 318 is located in
the nucleoside permeation pathway and is responsible for the substrate
selectivity of N2. An adjacent residue, glutamine 319, was found to be
important in modulating the apparent affinity for nucleosides.
 |
INTRODUCTION |
In mammalian cells, transmembrane flux of purine and pyrimidine
nucleosides is mediated by both facilitated and
Na+-dependent nucleoside transporters (1-3).
These transporters also play important roles in the cellular uptake of
many therapeutic nucleoside analogs such as 2-chlorodeoxyadenosine,
azidothymidine, and 2',3'-dideoxycytidine used in the treatment of
cancer and viral infections (4-6). Na+-nucleoside
transporters exhibit distinct transport selectivity for purine and
pyrimidine nucleosides and have been classified into several subtypes
based on their substrate selectivity (1-3). The N1 (or cif) system is
selective for purine nucleosides, the N2 (or cit) system is selective
for pyrimidine nucleosides, and the N3 (or cib) system is broadly
selective (or nonselective), transporting both purine and pyrimidine
nucleosides. Uridine, a pyrimidine nucleoside, and adenosine, a purine
nucleoside, are ubiquitously transported by all three systems. Recently
the N1 and N2 transporters were cloned from rat and human (5, 7-9). Although the cloned N1 and N2 transporters have distinct substrate selectivity for pyrimidine and purine nucleosides, they share a high
sequence homology (60-70%) and a similar predicted membrane topology
(14 putative transmembrane domains). The broadly selective transporter,
N3, was characterized in a number of tissues and cells (10-12), but
the molecular identity of this transporter is currently unknown.
To determine the structural basis for substrate recognition and
discrimination in the Na+-nucleoside transporters, we
previously took advantage of the high sequence similarity and yet
distinct substrate selectivity of the cloned N1 and N2 transporters. By
constructing and analyzing a series of chimeric rat N1/N2 transporters,
we demonstrated that TMs 8 and 9 are the major sites for substrate
binding and discrimination (13). Interestingly, when TM8 of N2 was
replaced by that of N1, the resulting transporter, chimera T8, lost
pyrimidine selectivity and became a broadly selective (or nonselective)
transporter that accepts both purine and pyrimidine nucleosides as
substrates (13). Sequence alignment revealed that 5 amino acid residues
differ between rat N2 and N1 in the TM8 region (Fig. 1), suggesting
that simultaneous replacement of the five divergent residues in TM8 of
N2 with the corresponding residues in N1 would cause N2 to lose its
selectivity for pyrimidine nucleosides. It is possible that individual
residues in TM8 of N2 may gate the substrate permeation pathway, which
would only allow pyrimidine nucleosides and the common substrate,
adenosine, to pass through. Substitution of these residues removes the
gating validity, resulting in nonselective transporters
(e.g. chimera T8), which also allow the passage of purine nucleosides.
In this study, we focused on determining the specific residues
responsible for maintaining the substrate selectivity of N2. By
site-directed mutagenesis, the five residues in N2 were systematically mutated to the corresponding residues in N1. The substrate selectivity of each mutant was subsequently evaluated in the Xenopus
laevis oocyte expression system. The data suggest that a single
residue, serine 318, is responsible for maintaining the pyrimidine
selectivity of N2. An adjacent residue, glutamine 319, was found to be
important in influencing the apparent affinity for nucleosides.
 |
EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis and Sequence Analysis--
The
cDNAs of wild-type rat N1 (SPNT) and N2 (CNT1) transporters were
obtained by reverse transcriptase polymerase chain reaction (13). The
mutagenic oligonucleotides were synthesized in the Biochemical Resource
Center at the University of California, San Francisco. The Stratagene
ChameleonTM and QuickChangeTM site-directed mutagenesis kits
(Stratagene) were used to construct mutant cDNAs following the
manufacturer's protocols. Mutants with single amino acid substitutions
(S304T, T306A, S310A, S318G, and Q319M) were prepared using the
cDNA of wild-type rat N2 as template. The double mutant S318G/Q319M
was constructed by introducing a second mutation (Q to M) at position
319 of mutant S318G. The mutant T8.G318S was construct by changing
glycine 318 to serine in a previously described chimeric transporter T8
(13). The sequence of each mutant was confirmed by direct DNA
sequencing using an automated DNA sequencer (Applied Biosystems, Model
373A). Genetics Computer Group software package (Wisconsin Package,
Version 9) was used for sequence alignment and helical wheel analysis.
Transport Assays in X. laevis Oocytes--
cRNA of each mutant
was synthesized and injected into defolliculated oocytes as described
previously (5). Uptake was measured on groups of 10 oocytes 48-56 h
post-injection at 25 °C in 150 µl of transport buffer (2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4)
containing either 100 mM NaCl or 100 mM choline chloride and the respective 3H-labeled nucleoside (Moravek
Biochemicals). The kinetic parameters (apparent Km
and Vmax values) were determined by nonlinear least squares fits of substrate/velocity profiles to the
Michaelis-Menten equation using Kaleidagraph (Version 3.0, Synergy
Software). Because of the intrinsic variability in the expression level
of the transporters between batches of oocytes, the data are generally
expressed as the mean ±S.E. from experiments performed in the same
batch of oocytes. However, experiments were repeated at least twice in separate batches of oocytes.
 |
RESULTS |
Studies on chimeric transporters showed that replacing TM8 of N2
with that of N1 generated a chimera, T8, which lost the pyrimidine selectivity of N2 (Fig. 2, A-B). To identify the specific
residues responsible for maintaining the substrate selectivity of N2,
we individually changed the five divergent residues (Fig.
1) in TM8 of N2 to the corresponding
residues in N1. A double mutant (S318G/Q319M) and a reverse mutant
(T8.G318S) were also constructed. Mutants were first screened with the
common substrate, [3H]uridine, for activity. Significant
Na+-dependent uptake of uridine (10-70-fold
increase) was observed for all mutants (data not shown), suggesting
that all mutants were expressed and functional.

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Fig. 1.
Predicted secondary structures of chimera T8
and wild-type N1 and N2 transporters (A), primary sequences
of N1 and N2 transporters in the TM8 region (B), and amino
acid substitutions in mutants and in chimera T8 (C).
Panel A, wild-type N1 (659 amino acids), wild-type N2 (648 amino acids). Chimera T8 contains amino acid residues 1-300 of N2,
297-330 of N1, and 335-648 of N2. Panel B, the amino acid
sequences of N2 and N1 in TM8 region. The five residues that differ
between N2 and N1 in TM8 are shown in bold. Panel
C, systematic substitutions of the divergent residues in mutants.
The numbers refer to the sequence of rat N2. Chimera T8 has
all five divergent residues in the TM8 of N2 replaced by the
corresponding residues of N1.
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Substrate Selectivity of Mutants--
The substrate selectivity of
each mutant was examined in uptake experiments using
[3H]inosine as the model purine nucleoside and
[3H]thymidine as the model pyrimidine nucleoside. The
uptake of inosine (10 µM) and thymidine (10 µM) by N2, chimera T8, mutants S304T, T306A, S310A,
S318G, Q319M, and S318G/Q319M is shown in Fig.
2. Compared with water-injected oocytes,
significant Na+-dependent thymidine uptake
(13-133-fold increase) was observed in oocytes expressing mutants
S304T, T306A, S310A, and Q319M (Fig. 2, C-E and
2G). In contrast, there was no significant inosine uptake by
these mutants (Fig. 2. C-E and 2G), suggesting
that mutants S304T, T306A, S310A, and Q319M maintained the pyrimidine selectivity of N2. Therefore, single substitutions of these residues in
N2 with the corresponding residues in N1 did not affect the substrate
selectivity of N2.

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Fig. 2.
Uptake of 3H-labeled inosine and
thymidine by wild-type N2 (A), Chimera T8 (B),
and the mutants of N2 (C-H). X. laevis
oocytes were injected with H2O or 20 ng of cRNA of
wild-type N2, mutants of N2, or chimera T8 transporter. Uptake was
measured on groups of 10 oocytes at 25 °C in transport buffer in the
presence of Na+ (solid bars) or the absence of
Na+ (blank bars), and the respective
3H-labeled nucleoside (10 µM). Each value
represents the mean ±S.E. (n = 8-10).
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In contrast, in addition to thymidine, the mutant S318G also transports
inosine (Fig. 2F). In oocytes expressing mutant S318G, there
was a 41-fold increase in Na+-dependent inosine
uptake (3.27 ± 0.58 pmol/oocyte/30 min for S318G cRNA-injected
oocytes versus 0.08 ± 0.01 pmol/oocyte/30 min for
water-injected oocytes). These data suggest that changing serine 318 in
N2 to its equivalent residue in N1 (glycine) causes N2 to accept
inosine as a substrate. However, compared with the transport rate of
(10 µM) thymidine (14.90 ± 2.17 pmol/oocyte/30 min), S318G transports inosine (10 µM) at a rate 4.6-fold
lower (Fig. 2F). The data shown in Fig. 2F were
from one representative experiment in which the same batch of oocytes
was used. The experiment was performed several times. Although the rate
of nucleoside uptake varied among experiments (ranging from 0.71 to 3.3 for inosine and from 4.30 to 15.10 for thymidine, pmol/oocyte/30 min)
because of the intrinsic variability in the expression level between
batches of oocytes, significant Na+-dependent
inosine uptake (16- to 41-fold over water-injected oocytes) was
observed in S318G cRNA-injected oocytes in all experiments. Within a
single experiment, the thymidine uptake was consistently 4-6-fold
higher than the inosine uptake. These data suggest that although mutant
S318G accepts purine nucleosides as substrates, it still kinetically
favors the transport of pyrimidine nucleosides at the tested
concentration (10 µM).
Mutant Q319M maintained the substrate selectivity of wild-type N2;
however it transports thymidine with a significantly decreased rate
(Fig. 2A and 2G). Because glutamine 319 is also
adjacent to serine 318, we suspect that this residue may play a role in substrate binding. Therefore we introduced a second Q to M mutation at
position 319 of mutant S318G. This double mutant, S318G/Q319M, transports 10 µM inosine and thymidine at a comparable
but slow rate (1.07 ± 0.20 pmol/oocyte/30 min for inosine and
1.25 ± 0.20 pmol/oocyte/30 min for thymidine), generating an
uptake pattern similar to that of chimera T8 (Fig. 2, B and
H). These data indicate that changing glutamine 319 to
methionine in mutant S318G caused S318G to transport purine and
pyrimidine nucleosides without much kinetic bias.
The data presented in Fig. 2 suggested that serine 318 is important for
maintaining the pyrimidine selectivity of N2. Changing this residue in
N2 to glycine causes N2 to lose its substrate selectivity. To
investigate whether a reverse mutation can re-establish the pyrimidine
selectivity in chimera T8, we changed the glycine 318 in chimera T8
back to the serine residue in N2. Interestingly, this mutant (termed
T8.G318S) restored the pyrimidine selectivity of N2, transporting
thymidine but not inosine (Fig. 3). These data strongly suggest that serine 318 is essential for maintaining the
pyrimidine selectivity of the N2 transporter.

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Fig. 3.
Uptake of 3H-labeled inosine and
thymidine by mutant T8.G318S. X. laevis oocytes were
injected with H2O or 20 ng of cRNA of T8.G318S. Uptake was
measured on groups of 10 oocytes at 25 °C in transport buffer in the
presence of Na+ (solid bars) or the absence of
Na+ (blank bars), and the respective
3H-labeled nucleoside (10 µM). Each value
represents the mean ±S.E. (n = 8-10).
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Transport Kinetics of Mutants S318G and S318G/Q319M--
To
investigate why mutant S318G transports thymidine more favorably than
inosine at 10 µM (Fig. 2F) and how
substitution of Q319M in mutant S318G neutralized this imbalance (Fig.
2H), we examined the kinetics of thymidine and inosine
uptake mediated by mutant S318G and mutant S318G/Q319M (Fig.
4). Uptake of both nucleosides via mutant
S318G was saturable (Fig. 4A). The apparent Km of inosine was 273 ± 62 µM,
whereas that of thymidine was 27.5 ± 4.3 µM. The
Vmax of inosine was 28.8 ± 2.5 pmol/oocyte/30 min, whereas that of thymidine was 24.6 ± 0.9 pmol/oocyte/30 min (Fig. 4A). These data suggest that mutant
S318G has a much lower (~10-fold) apparent affinity for inosine than
for thymidine, whereas the apparent maximal rate of transport,
Vmax, for inosine is close to that for
thymidine. Therefore at low substrate concentrations (e.g.
10 µM), mutant S318G will favor the transport of
thymidine over that of inosine. The uptake of inosine and thymidine
mediated by mutant S318G/Q319M was also saturable (Fig. 4B).
For this mutant, the Km of inosine was 54.8 ± 14.7 µM, and the Km of thymidine was
79.3 ± 11.9 µM. The Vmax of
inosine was 10.8 ± 0.7 pmol/oocyte/30 min and that of thymidine
was 12.3 ± 0.5 pmol/oocyte/30 min (Fig. 4B). These
data suggest that mutant S318G/Q319M has similar apparent
Km and Vmax values for
inosine and thymidine, transporting these two compounds without much
kinetic bias. Compared with the mutant S318G (Fig. 4A), the
apparent affinity for inosine in the double mutant is greatly enhanced
(Km = 54.8 versus Km = 273 µM, p < 0.01), whereas the apparent affinity for thymidine is significantly decreased
(Km = 79.3 versus Km = 24.6 µM, p < 0.01).

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Fig. 4.
Michaelis-Menten studies of thymidine and
inosine uptake mediated by mutant S318G (A) and mutant
S318G/Q319M (B). Oocytes were injected with 20 ng of cRNA of
mutant S318G or S318G/Q319M. The initial velocities (30 min) of
[3H]thymidine uptake (solid circles) or
[3H]inosine uptake (squares) were determined
in Na+-buffer containing the respective nucleoside at
concentrations ranging from 1 to 1000 µM. Each
point represents the mean ±S.E. (n = 8-10). Apparent Km and Vmax
values were determined by fitting the data to the Michaelis-Menten
equations using a nonlinear fitting routine of Kaleidagraph.
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Helical Wheel Analysis of TM8--
To analyze the position of
serine 318 and glutamine 319 on TM8, helical wheels at a fixed angel of
100° are drawn for TM8 of N2 (Fig. 5).
The five substitutions in N1 are indicated by arrows.
Distinct amphipathic patterns (one side of the helix being hydrophobic,
and the other side hydrophilic) are observed, suggesting that one side
of TM8 may face an aqueous pore (e.g. the substrate binding
pore), whereas the other side may face the hydrophobic lipid. The
residue serine 318 is located in the center of hydrophilic side,
suggesting its side chain may directly interact with the substrates.
The residue glutamine 319 is located near the boundary of the
amphipathic interface. Its side chain may interact directly with the
substrates or may contribute indirectly to the conformation of the
substrate recognition sites.

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Fig. 5.
Helical wheel analysis of the TM8 of N2.
The transmembrane domain is assumed to be a standard -helix (3.6 residues/helical turn). Each residue in TM8 of N2 is plotted every
100° (360°/3.6) around the center of a circle. The
figures show the projection of the positions of the residues
on a plane perpendicular to the helical axis. Hydrophobic residues
(residues with positive hydrophobicity) are squared and
shown in blue, whereas hydrophilic residues (residues with
negative hydrophobicity) are shown in red. The positive and
negative of hydrophobicity are assigned according to the consensus
scale of Eisenberg et al. (20). The substitutions of the
five divergent residues in N1 are indicated by arrows. The
straight line indicates an arbitrary boundary between the
hydrophilic and the hydrophobic region.
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|
 |
DISCUSSION |
Our previous studies showed that replacing TM8 of N2 with that of
N1 caused N2 to lose its substrate selectivity (13). In this study, we
focused on determining the specific residues responsible for
maintaining the substrate selectivity of N2. By site-directed mutagenesis, the five divergent residues in N2 were systematically mutated to the corresponding residues in N1. Replacing serine 318 in N2
to its equivalent residue, glycine, resulted in a mutant (S318G), which
lost the pyrimidine selectivity of N2 and began to accept purine
nucleosides as substrates (Fig. 2F). In contrast, replacing
the other four residues with their equivalents did not alter the
selectivity of N2 (Fig. 2). Importantly, when the glycine residue in
the broadly selective chimera T8 was changed back to serine, the
resulting transporter (T8.G318S) regained pyrimidine selectivity (Fig.
3). These data strongly suggest that serine 318 is essential for
conserving the pyrimidine selectivity of wild-type N2.
Kinetic studies revealed that mutant S318G has a much lower
(~10-fold) apparent affinity for inosine than for thymidine (Fig. 4A), suggesting that although the ability of a mutant to
accept purine nucleosides is determined by whether a serine or a
glycine residue is present at position 318, other residues may
contribute to the kinetic differences in the transport of purine and
pyrimidine nucleosides by mutant S318G. Indeed, a second Q to M
mutation at position 319 following the S318G substitution resulted in a mutant (S318G/Q319M), which has comparable apparent
Km values for inosine and thymidine (Fig.
4B). These data suggest that although a single substitution
of glutamine 319 with methionine would not affect the pyrimidine
selectivity of N2 (Fig. 2G), changing it after the serine to glycine
substitution at position 318 would greatly enhance the apparent
affinity of mutant S318G toward purine nucleosides. In transport
kinetic analysis, the apparent affinity (i.e. apparent
Km) reflects not only substrate affinity for the
binding site but is also influenced by rate constants of substrate
translocation and dissociation, which occur subsequent to recognition
(14). Therefore, the observed changes in apparent affinity introduced
by the Q to M mutation in the S318G mutant may reflect changes in any
of these processes.
Studies of a number of membrane transporters suggest that the substrate
permeation pathway in a transporter is a channel-like structure formed
by several transmembrane helices (15-17). Charged and polar residues,
often found on one side of these helices, play critical roles in
interacting with the substrates (15, 18-20). Helical wheel analysis of
TM8 revealed a distinct amphipathic pattern (Fig. 5), suggesting that
one side of TM8 may face an aqueous channel for substrate permeation.
Serine 318 is located in the center of the hydrophilic side (Fig. 5),
suggesting that its side chain may protrude into the channel and act as
a gating residue through specific interactions with the substrates.
Substitution of a serine residue with a smaller residue, glycine, will
result in a loss of a methylene and a hydroxyl group on the side chain. These changes may cause a gain in the size of the substrate permeation channel and a loss of some specific chemical interactions
(e.g. a hydrogen bond), allowing the resulting transporter
S318G to tolerate the bulkier purine nucleosides as substrates. On the helical wheel diagram, the residue glutamine 319 is located near the
boundary of the amphipathic interface (Fig. 5). Its side chain may
interact directly with the substrates or may affect the apparent affinity by indirect influence of the conformation of the substrate recognition site in the channel. Substitution of this residue with a
methionine in mutant S318G may induce changes that make the transporter
interact with purine nucleosides with an increased apparent affinity.
However, it should be noted that the proposed topology of N2 consisting
of 14 transmembrane domains with an internal C and N termini needs
experimental validation. In addition, with the crystal structure of N2
unknown, the possibility that serine 318 may influence the selectivity
of N2 through indirect interactions with other sites in the protein
cannot be excluded.
In summary, we identified a single residue, serine 318, that is
primarily responsible for determining the substrate selectivity of the
N2 Na+-nucleoside transporter. The data suggest that serine
318 may be located in the nucleoside permeation pathway and act as a
gating residue that is important for the pyrimidine selectivity of N2. An adjacent residue, glutamine 319, was found to be important in
influencing the apparent affinity of the transporter for purine nucleosides. These studies provide important information about the
molecular mechanisms that govern the functional characteristics of
Na+-nucleoside transporters. Furthermore, the finding that
a few residues along the solute permeation pathway are responsible for the substrate selectivity and affinity of N2 may reflect a common molecular mechanism for substrate discrimination in some membrane transporters.
 |
FOOTNOTES |
*
This work is supported by National Institutes of Health
Grant GM42230.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.
To whom correspondence should be addressed: Dept. of
Biopharmaceutical Sciences and Cellular and Molecular Pharmacology,
University of California, S-926, 513 Parnassus Ave., San Francisco, CA
94143-0446. Tel.: 415-476-1936; Fax: 415-476-0688; E-mail:
kmg{at}itsa.ucsf.edu.
 |
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