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J. Biol. Chem., Vol. 277, Issue 1, 395-401, January 4, 2002
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From the a Canadian Institutes of Health Research
Group in the Molecular Biology of Membrane Proteins and
b Membrane Transport Research Group, Departments of
h Biochemistry, c Oncology, and e Physiology,
University of Alberta and the i Cross Cancer Institute,
Edmonton, Alberta T6G 1Z2, Canada and the f School of
Biochemistry and Molecular Biology, University of Leeds,
Leeds LS2 9JT, United Kingdom
Received for publication, June 8, 2001, and in revised form, October 26, 2001
Human equilibrative nucleoside transporters
(hENT) 1 and 2 differ in that hENT1 is inhibited by nanomolar
concentrations of dipyridamole and dilazep, whereas hENT2 is 2 and 3 orders of magnitude less sensitive, respectively. When a yeast
expression plasmid containing the hENT1 cDNA was randomly mutated
and screened by phenotypic complementation in Saccharomyces
cerevisiae to identify mutants with reduced sensitivity to
dilazep, clones with a point mutation that converted Met33
to Ile (hENT1-M33I) were obtained. Characterization of the mutant protein in S. cerevisiae and Xenopus laevis
oocytes revealed that the mutant had less than one-tenth the
sensitivity to dilazep and dipyridamole than wild type hENT1, with no
change in nitrobenzylmercaptopurine ribonucleoside (NBMPR) sensitivity
or apparent uridine affinity. To determine whether the reciprocal
mutation in hENT2 (Ile33 to Met) also altered sensitivity
to dilazep and dipyridamole, hENT2-I33M was created by site-directed
mutagenesis. Although the resulting mutant (hENT2-I33M) displayed
>10-fold higher dilazep and dipyridamole sensitivity and >8-fold
higher uridine affinity compared with wild type hENT2, it retained
insensitivity to NBMPR. These data established that mutation of residue
33 (Met versus Ile) of hENT1 and hENT2 altered the dilazep
and dipyridamole sensitivities in both proteins, suggesting that a
common region of inhibitor interaction has been identified.
Cellular uptake and release of nucleosides and nucleoside analog
drugs is mediated by integral membrane nucleoside transporter proteins
(1-4). These proteins are involved in salvage of extracellular nucleosides for nucleotide biosynthesis in mammalian cells, especially those that lack de novo synthesis pathways such as
enterocytes and hemopoietic cells. They are critical for the cellular
uptake of cytotoxic nucleoside analogs used in the treatment of human hematologic malignancies, solid tumors, and viral diseases (5, 6).
Nucleoside transporters also affect the cell surface concentration of
adenosine, which is a signaling molecule that binds to G
protein-coupled cell surface adenosine receptors, affecting
physiological processes such as coronary vasodilation, renal
vasoconstriction, neuromodulation, platelet aggregation, and lipolysis
(7, 8).
Mammalian nucleoside transporters are classified into two structurally
and functionally distinct families: the concentrative nucleoside
transporters (CNTs)1 and the
equilibrative nucleoside transporters (ENTs). CNTs mediate Na+-dependent transport against the nucleoside
concentration gradient and are found primarily in specialized cells
such as intestinal and renal epithelia. Three CNT isoforms, a
pyrimidine-nucleoside preferring (CNT1), a purine-nucleoside and
uridine preferring (CNT2), and a broadly selective (CNT3) protein, have
been identified by molecular cloning from mammalian tissues (9-14).
Mammalian ENTs are responsible for facilitated diffusion of nucleosides across cell membranes and have a broad tissue distribution. Two ENT
isoforms have been identified by molecular cloning and functional expression from mammalian tissues and mediate nucleoside transport processes that are functionally distinguished by their differential sensitivity to inhibition by NBMPR (1-4). NBMPR-sensitive nucleoside transport processes that bind NBMPR with high affinity,
(Kd = 0.1-1 nM), have been assigned the
functional designation es (equilibrative
sensitive) and are mediated by ENT1 proteins.
NBMPR-insensitive nucleoside transport processes are resistant to
inhibition by micromolar concentrations of NBMPR, are functionally
designated as ei (equilibrative
insensitive), and are mediated by ENT2 proteins. ENTs are
pharmacological targets for the coronary vasodilators dilazep,
dipyridamole, and draflazine, which have been shown to inhibit
transport and NBMPR binding (3, 15-17). Adenosine interacts with G
protein-coupled cell surface receptors of endothelial and smooth muscle
cells to induce vasodilation. Transporter-mediated adenosine uptake is
the major means by which this interaction is terminated, a mechanism
that is blocked by coronary vasodilator binding to the human ENT
isoforms hENT1 and hENT2 (7).
hENT2 shares 50% amino acid identity with hENT1 and is 2 and 3 orders
of magnitude less sensitive, respectively, to inhibition by
dipyridamole and dilazep than hENT1, whereas both rat isoforms (rENT1
and rENT2) are completely insensitive to these inhibitors (18, 19).
Human and rat ENT1 and ENT2 proteins share a common membrane
architecture, recently confirmed by hydropathy analysis and
glycosylation-scanning mutagenesis (20), with 11 transmembrane (TM)
segments, a large glycosylated loop between TM segments 1 and 2, and a
large intracellular loop between TM segments 6 and 7. In a previous
study, chimeric recombinant proteins were created between hENT1 and
rENT1 to identify the structural domains of hENT1 that are responsible
for interaction with dilazep and dipyridamole (21). The inhibitor
sensitivities of the chimeras suggested that TM segments 3-6 contain
the major site(s) of interaction with secondary contributions from TM
segments 1-2, providing the first insight into the regions of hENT1
that are important for interaction with dilazep and dipyridamole. The
individual amino acid residues responsible for interaction with dilazep
and dipyridamole have not yet been identified.
The goal of the current study was to identify amino acid residues
involved in dilazep and dipyridamole interaction by using a phenotypic
complementation assay to screen a library of randomly mutated yeast
expression plasmids containing the hENT1 cDNA (pYPhENT1) for
functional thymidine transport-competent mutants with reduced sensitivity to dilazep. The complementation assay is based on the
ability of recombinant hENT1 produced in Saccharomyces
cerevisiae to import thymidine under conditions of dTMP
starvation, thereby allowing growth, which is inhibited by the addition
of dilazep to the assay medium (22-24). hENT1 cDNAs were isolated
from the resulting mutant clones and sequenced, revealing a mutation in codon 33 that converted Met33 to Ile (M33I). When mutant
and wild type recombinant hENT1 were produced in S. cerevisiae and Xenopus laevis oocytes to quantitate dilazep and dipyridamole sensitivity, a significant decrease in sensitivity was observed for the mutated protein. The corresponding residue in hENT2 (Ile33) was therefore converted to a Met
by site-directed mutagenesis, and the sensitivity of the resulting
mutant to dilazep and dipyridamole was assessed. The results suggested
that residue 33 in the first TM segment (Met versus Ile)
contributes importantly to the ability of dilazep and dipyridamole to
interact with hENT1 and hENT2.
Strains and Media--
KY114 (MAT Plasmid Construction and Site-directed Mutagenesis--
For
S. cerevisiae expression, the hENT1 open reading frame was
amplified by PCR methodology using the primers (restriction sites underlined) 5'-XbaIes (5'-CCC TCT
AGA ATG ACA ACC AGT CAC CAG CCT C-3') and
3'-KpnIes (5'-CCC GGT ACC TCA CAC AAT
TGC CCG GAA CAG G-3') and inserted into the yeast expression
vector pYPGE15 to generate pYPhENT1. For X. laevis oocytes
expression, the hENT1-M33I cDNA was cloned into pBluescript II KS
(+) (Stratagene) to generate pKS (+)-hENT1-M33I as previously described
for the generation of pKS (+)-hENT1 (21, 28).
The hENT2 open reading frame was amplified by PCR using the
primers (restriction sites underlined) 5'-XbaIei
(5'-CCC TCT AGA ATG GCC CGA GGA GAC GCC-3') and
3'-KpnIei (5'-CCC GGT ACC TCA GAG CAG
CGC CTT GAA G-3') and inserted into pYPGE15 to generate pYPhENT2.
The hENT2 point mutant resulting in the I33M change in amino acid
sequence was generated using megaprimer PCR methodology (29). All
reactions were performed using Pwo polymerase (Roche Molecular
Biochemicals), and the resulting PCR products were verified by DNA
sequencing using an ABI PRISM 310 sequence detection system (PerkinElmer Life Sciences).
Random Mutagenesis of pYPhENT1--
Double-stranded plasmid DNA
(10 µg) was precipitated with ethanol/sodium acetate and resuspended
in 500 µl of freshly prepared hydroxylamine solution (90 mg of NaOH,
350 mg of hydroxylamine HCl, pH ~6.5, in 5 ml of H2O).
The DNA was incubated for 16 h at 37 °C, and the reactions were
terminated by the addition of 15 µl of 4 M NaCl, 50 µl
of 1 mg/ml bovine serum albumin followed by precipitation with 1 ml of
95% ethanol. The DNA was resuspended in 100 µl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and precipitated again with 15 µl of 4.0 M NaCl, 250 µl of 95% ethanol.
The resuspension-precipitation procedure was repeated three times in
total with a final resuspension in 20 µl of TE buffer.
Phenotypic Complementation and Screening of Mutants--
The
complementation assay was based on the ability of recombinant hENT1
produced in yeast to salvage exogenously supplied thymidine under
conditions of dTMP starvation (23). In brief, KTK cells transformed
with pYPhENT1 using a lithium acetate procedure (27) were plated
directly onto CMM/GLU plates containing methotrexate (MTX) at 50 µg/ml and sulfanilamide (SAA) at 6 mg/ml (CMM/GLU/MTX/SAA). Colonies
formed with an efficiency of ~105 transformants/µg of
DNA after incubation at 30 °C for 3.5 days in the presence of 10 µM thymidine, and complementation was prevented when 10 µM dilazep was also present. Hydroxylamine-treated
pYPhENT1 (20 µg) was transformed into KTK cells, which were then
plated onto CMM/GLU/MTX/SAA with 10 µM thymidine and 10 µM dilazep and incubated at 30 °C for 3.5 days.
Colonies with apparent resistance to dilazep inhibition of
complementation were isolated, grown in 5 ml of liquid CMM/GLU for 2 days, and restreaked onto CMM/GLU/MTX/SAA plates with 10 µM thymidine and 10 µM dilazep. The mutant
hENT1 cDNAs were amplified from the yeast colonies by PCR,
subcloned back into nonmutated pYPGE15, and sequenced.
Uridine Transport in S. cerevisiae--
The plasmids pYPhENT1,
pYPhENT1-M33I, pYPhENT2, and pYPhENT2-I33M were transformed into
fui1::TRP1 yeast, a strain that lacks the endogenous uridine
permease FUI1 (25). The transport of [3H]uridine (Moravek
Biochemicals, Brea, CA) by logarithmically proliferating yeast was
measured as described previously using the "oil stop" method (30,
31) with the following modifications. Yeast were grown in CMM/GLU to an
A600 of 0.7-1.5, washed once with fresh medium,
and resuspended to an A600 of 2.0 in fresh medium. All transport assays were performed at room temperature and pH
7.0. 1-ml portions of yeast culture were distributed into 15-ml plastic
centrifuge tubes to which 5-10-µl portions of stock dilazep,
dipyridamole, or NBMPR (Sigma) solution or solvent alone (H2O, ethanol, or dimethyl sulfoxide) were added to achieve
the desired final concentration. To allow for steady-state
equilibration, the yeast were incubated in the presence of inhibitor
for 30 min before addition of radiolabeled permeant (32-35). Transport
reactions were initiated by the rapid addition of a small volume of
[3H]uridine to a final concentration of 2 µM. Transport reactions were terminated at graded time
intervals by pipetting triplicate 200-µl portions of yeast suspension
into 1.5-ml microcentrifuge tubes containing 200 µl of transport oil;
the tubes were immediately centrifuged at 12,000 × g
for 2 min. The supernatants were removed by aspiration, the resulting
pellets were solubilized with 5% Triton X-100 for 24 h, and the
radioactive content was determined by liquid scintillation counting.
Functional Expression of hENT1 and hENT1-M33I in X. laevis
Oocytes--
In vitro synthesized transcripts were prepared
from pKS(+)-hENT1 and pKS(+)-hENT1-M33I (SP6 MEGAscript Kit Ambion,
Austin, TX) in water and injected into isolated mature stage VI oocytes from X. laevis as described previously (14). Mock-injected
oocytes were injected with water alone. Transport assays were performed as described previously (21, 28) on groups of 10 oocytes at 20 °C
using [14C]uridine (Amersham Life Biosciences) (1 µCi/ml) in 200 µl of transport buffer containing 100 mM
NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 7.5. The initial rates of uridine uptake (10 µM) were
determined using incubation periods of 5 min.
Uridine Transport by Recombinant hENT1 and hENT2 in
Yeast--
Time courses for influx of [3H]uridine were
measured into fui1::TRP1, a uridine transport-defective
strain of yeast (25), that contained pYPhENT1 or pYPhENT2 to determine
incubation times that provided significant signal-to-noise ratios while
also maintaining the initial rates of uptake (Fig.
1). The time course for nonmediated uridine influx was obtained by assessing uridine uptake into
pYPGE15-containing yeast and yielded a rate of 0.11 ± 0.01 pmol/mg protein/s. Time courses for uridine uptake into pYPhENT1- and
pYPhENT2-containing yeast for the first 10 s (Fig. 1,
inset) gave rates of 1.03 ± 0.40 and 1.63 ± 0.45 pmol/mg protein/s, respectively. Uptake time courses over 40 min were
linear for both pYPhENT1- and pYPhENT2-containing yeast and yielded
rates, respectively, of 0.93 ± 0.02 and 1.4 ± 0.02 pmol/mg
protein/s. Uptake rates over the first 10 s were not different
from the rates calculated from 40-min time courses, indicating that
initial rates representing uridine transport were maintained over long
periods of time. The extended linear time courses were likely due to
efficient substrate "trapping" by conversion of uridine to UMP by
uridine kinase, thereby minimizing backflow of
[3H]uridine from the small intracellular compartment to
the much larger extracellular volume. Uridine transport rates were
determined for all subsequent experiments using incubation times of 10 or 20 min.
Random Mutagenesis and Screening--
MTX and SAA prevent the
conversion of dUMP to dTMP by yeast thymidylate synthase and thus cause
depletion of intracellular dTMP pools and inhibition of growth (22).
KTK yeast producing recombinant hENT1 and H. simplex
thymidine kinase can salvage thymidine via transporter-mediated uptake
when low concentrations (e.g. 10 µM) are
present in the growth medium, thereby allowing yeast to circumvent
MTX/SAA-imposed growth arrest. Because thymidine salvage can be blocked
by the inclusion of 10 µM dilazep in the complementation
growth medium (23), this inhibition of thymidine rescue was used to
screen a hENT1 random mutant library for functional proteins with
reduced affinity for dilazep. pYPhENT1 was treated in vitro
with the mutagen hydroxylamine, transformed into KTK yeast, and
screened for dilazep resistance. Dilazep-resistant yeast colonies were
isolated, and the hENT1 cDNA was amplified and subcloned into
nonmutated pYPGE15. Twenty-one resistant mutant cDNA clones were
sequenced and shown to be identical, with a point mutation in codon 33 that converted Met to Ile.
A Comparison of Sequences of Inhibitor-sensitive and -insensitive
Mammalian ENTs--
Recombinant human and mouse ENT1 proteins are
highly sensitive to transport inhibition by dipyridamole, whereas
recombinant human and mouse ENT2 proteins are much less sensitive (18,
36). For example, the reported IC50 values for mENT1 and
mENT2 produced in X. laevis oocytes were 75 and 2204 nM, respectively, which corresponds to a 29.4-fold
difference (36). A transport-deficient cultured cell line stably
transfected with recombinant hENT1 or hENT2 exhibited a 70-fold
difference between the two proteins in sensitivity to dipyridamole with
IC50 values of 5 and 356 nM, respectively (19).
The rat ENT isoforms (rENT1 and rENT2) are completely insensitive to
dipyridamole and dilazep transport inhibition when produced in X. laevis oocytes (18).
Multiple sequence alignment of the predicted amino acid sequences for
the human, mouse, and rat ENT1 and ENT2 proteins revealed that the
identity of the amino acid at residue 33 was consistent with the
dilazep and dipyridamole sensitivity of the recombinant transporters
(Fig. 2). Residue 33 is a Met in human
and mouse ENT1, the most inhibitor-sensitive transporters, whereas it
is an Ile in rat ENT1 and human, mouse, and rat ENT2 proteins, all of
which exhibit transport activity that is insensitive to inhibition by
dilazep and dipyridamole (18, 28, 36, 37). The predicted topology model
for hENT1 suggests that position 33 is the last residue in the first TM
segment and may therefore be solvent-accessible and/or in the plane of
the extracellular bilayer/solvent interface (20, 28).
Effect of Met-Ile Interconversion at Residue 33 of hENT1 and hENT2
on Uridine Transport Inhibition by Dilazep, Dipyridamole, and
NBMPR--
Uridine transport was measured in fui1::TRP1
yeast containing pYPhENT1 or pYPhENT1-M33I in the presence or absence
of a single high concentration of dilazep, dipyridamole, or NBMPR (Fig.
3A). hENT1-mediated uridine
transport was inhibited
Although hENT2 can be inhibited by high concentrations of dilazep and
dipyridamole, it is 2 and 3 orders of magnitude less sensitive,
respectively, to these compounds than hENT1 (19). To investigate the
role of residue 33 in inhibitor sensitivity of hENT2, Ile33
was converted to Met using site-directed mutagenesis, and the effects
of dilazep, dipyridamole and NBMPR on uridine transport were determined
in fui1::TRP1 yeast containing either pYPhENT2 or
pYPhENT2-I33M (Fig. 3B). Dilazep (10 µM)
and dipyridamole (1 µM) had no effect on hENT2-mediated
uridine transport, whereas both strongly inhibited hENT2-I33M-mediated
transport. In contrast, uridine transport in yeast with either mutant
or wild type hENT2 remained insensitive to NBMPR, a result that was
consistent with the lack of an effect of the opposite conversion on
NBMPR sensitivity of hENT1. These data, together with the data from
Fig. 3A, indicated that residue 33 plays a key role in
dilazep and dipyridamole inhibition of transport of both hENT1 and
hENT2 and is not involved in NBMPR inhibition of transport.
Kinetic Properties of Uridine Transport for hENT1, hENT1-M33I,
hENT2, and hENT2-I33M--
The effect of mutating residue 33 (Met
versus Ile) of hENT1 and hENT2 on the kinetics of uridine
transport was assessed by determining the concentration dependence of
initial rates of uridine uptake (Table
I). hENT1 and hENT1-M33I showed similar
kinetic parameters for uridine transport with Km
values of 110 ± 12 and 110 ± 28 µM,
respectively, and Vmax values of 5893 ± 1399 and 5215 ± 562 pmol/mg protein/min, respectively, suggesting that uridine interaction with hENT1 was unaffected by the mutation. In
contrast, Km values were 729 ± 53 and
87.2 ± 13.8 µM, respectively, for hENT2 and
hENT2-I33M, indicating an 8.4-fold increase in the apparent affinity
for uridine. Vmax values of 8370 ± 1091 and 6555 ± 1616 pmol/mg protein/min were obtained, respectively, for wild type and mutant hENT2. The
Vmax values for the mutant and wild type hENT1
and hENT2 proteins were not significantly different (p > 0.05) based on an unpaired two-tailed t test, suggesting
that expression of the recombinant proteins in yeast was not affected
by mutation of residue 33. The
Vmax:Km ratios for mutant and
wild type hENT1 were similar (47 and 53 pmol/mg
protein/min/µM, respectively), whereas the ratios for mutant hENT2 were much larger than those for wild type hENT2 (75 and 12 pmol/mg protein/min/µM, respectively).
Concentration-Effect Relationships for Dilazep, Dipyridamole, and
NBMPR--
The relative changes in inhibitor sensitivities of mutant
and wild type hENT1 and hENT2 were determined by assessing the
concentration dependence of uridine transport inhibition for the
recombinant proteins produced in fui1::TRP1 yeast. The yeast
were incubated with graded concentrations of inhibitors and then
assayed for [3H]uridine transport (Fig.
4). The Hill coefficients determined from
these relationships were not significantly different from unity based
on a t test against the theoretical value of 1.00 resulting
in p > 0.05, which was consistent with (i) the
presence of a single class of binding sites and (ii) the findings of
previous studies (21, 23).
The IC50 values obtained from the data of Fig. 4 and the
kinetic constants of Table I were used to compute apparent
Ki values, assuming that dilazep, dipyridamole, and
NBMPR inhibit uridine transport in a reversible and strictly
competitive manner at the concentration equal to the IC50
value (Table II) (17, 33, 38-40). The
transport of uridine by wild type hENT1 was potently inhibited by
dilazep (Ki, 18.7 ± 2.0 nM),
whereas hENT1-M33I-mediated transport was an order of magnitude less
sensitive to dilazep inhibition (Ki, 195 ± 51 nM). In contrast, hENT2-I33M was 46-fold more sensitive to
dilazep inhibition than wild type hENT2 with Ki
values of 2.91 ± 0.79 and 134 ± 40 µM, respectively. Thus, the mutations at residue 33 decreased the differences in dilazep sensitivity between hENT1 and hENT2. The mutant
proteins displayed a 15-fold difference (hENT1-M33I > hENT2-I33M), whereas the wild type proteins displayed a 7000-fold
difference (hENT1 > hENT2) in sensitivity to inhibition by
dilazep.
For both hENT1 and hENT2, the relative differences between the mutant
and wild type proteins in sensitivity to dipyridamole were similar to
those observed for dilazep (Table II). Ki values of
47.9 ± 8.9 and 528 ± 165 nM were obtained for
dipyridamole inhibition of transport for wild type and mutant hENT1,
respectively, translating into an 11-fold decrease in sensitivity. The
dipyridamole sensitivities of hENT2 (Ki, 6230 ± 900 nM) and hENT2-I33M (Ki, 461 ± 74 nM) differed by 13.5-fold. Wild type hENT2 was
128-fold less sensitive to dipyridamole than hENT1, which is consistent
with the results of previous studies (19), whereas the mutant proteins
displayed approximately equal sensitivities to dipyridamole.
The results of Fig. 3 suggested that mutant and wild type hENT1 were
highly sensitive to NBMPR because complete inhibition of transport was
observed for both at 0.1 µM NBMPR. In the experiments of
Table II, Ki values of 5.83 ± 1.08 and
3.34 ± 0.97 nM were obtained for hENT1 and
hENT1-M33I, respectively, demonstrating that both were potently
inhibited by NBMPR, with no statistically significant difference in
Ki values. The NBMPR sensitivities of hENT2 and
hENT2-I33M were not determined because the experiments of Fig.
3B had established that neither protein was inhibited by
NBMPR.
In a previous study (21), recombinant chimeric proteins were
constructed by domain substitutions between hENT1, which is sensitive
to inhibition by dilazep and dipyridamole, and its rat isoform, rENT1,
which is insensitive to both compounds and functionally characterized
in X. laevis oocytes. The results suggested that TM segments
1-6 of hENT1 are required for interaction with dilazep and
dipyridamole, with TM segments 3-6 being the major site of interaction
and TM segments 1-2 making a secondary contribution. Because residue
33 is predicted to be the last residue in TM segment 1, recombinant
hENT1-M33I was produced in X. laevis oocytes (Fig. 5) to assess the functional
characteristics of the mutated protein in the same recombinant
expression system as the chimera study. When oocytes producing mutant
and wild type hENT1 were assayed for uridine uptake in the presence of
graded concentrations of dipyridamole, IC50 values were
3640 ± 1410 and 300 ± 79 nM, respectively, corresponding to a 12.1-fold lower sensitivity for the mutant protein.
This relative decrease in sensitivity was similar to that observed when
the recombinant proteins were produced in yeast.
The results of molecular cloning and functional expression studies
on recombinant ENTs are consistent with the findings of studies on
es- and ei-type transport processes in cultured
cell lines and erythrocytes. The human and mouse es-type
transporters, which correspond to the hENT1 and mENT1 proteins, are
highly sensitive to dilazep and dipyridamole (3, 16, 41, 42). In
contrast, rat es and human, mouse, and rat ei
transporters are relatively insensitive to transport inhibition by
dilazep and dipyridamole, and these observed effects have been
correlated with the transport-inhibition phenotypes of recombinant
rENT1, hENT2, mENT2, and rENT2 (3, 41, 42). The current study provides
evidence that mutation of residue 33 of the hENT1 and hENT2 proteins
affects interaction with dilazep and dipyridamole significantly. The
identity of this residue (Met versus Ile) corresponds with
the relative dilazep and dipyridamole sensitivities of the known
mammalian ENTs, being a Met in human and mouse ENT1 and an Ile in rat
ENT1 and human, mouse, and rat ENT2 proteins (Fig. 2) (18, 19, 21, 28, 36, 37).
Mutation of Met33 to Ile in hENT1 decreased the sensitivity
of uridine transport to inhibition by dilazep and dipyridamole (as seen
by the >10-fold increase in Ki values) but did not alter the affinity for uridine (similar Km values)
or the sensitivity to inhibition of uridine transport by NBMPR (similar Ki values). In contrast, the sensitivity of hENT2 to dilazep and dipyridamole was increased >10-fold when Ile33
was converted to Met, the affinity for uridine was increased 8.4-fold,
and NBMPR sensitivity was not affected. These results, which implicated
residue 33 in uridine interaction with hENT2 but not hENT1, suggested a
difference in the permeant binding pockets of the two proteins. hENT1
and hENT2 are known to have different permeant binding properties
because hENT2 is capable of transporting nucleobases and antiviral
dideoxynucleoside analogs, whereas hENT1 is not (43, 44).
The apparent Km value for uridine transport obtained
for recombinant hENT1 in yeast (Table I) was 110 ± 12 µM, whereas values of 200-260 µM have been
obtained for recombinant hENT1 in other expression systems (cultured
cells, X. laevis oocytes) and for the native protein in
human erythrocytes (19, 28, 45). The basis for this discrepancy is
uncertain but may have been due to the human protein being inserted
into the yeast plasma membrane environment and/or an altered state of
glycosylation, resulting in subtle changes in the conformation of the
uridine-binding pocket.
Previous work in which chimeric recombinant proteins were created by
substituting domains between inhibitor-sensitive hENT1 and
inhibitor-insensitive rENT1 suggested that the region including residues 100-231 (which includes TM segments 3-6) is the major site
of interaction with dilazep and dipyridamole and that residues 1-99
(TM segments 1-2) play a secondary role (21). TM segments 3-6 were
also implicated in the interaction of rENT1 with NBMPR (46). The
chimera studies demonstrated that the N-terminal half of hENT1 is
critical for interaction with the inhibitors. In this work, when
recombinant hENT1-M33I was characterized in the same expression system
(X. laevis oocytes) as was utilized in the chimera study,
the relative effect of the mutation on dipyridamole sensitivity was
comparable with that observed in yeast. These oocyte results confirmed
participation of Met33, which is predicted to be the last
residue in TM segment 1, in binding of dilazep and dipyridamole. That
the M33I mutation reduced but did not abolish inhibitor sensitivity in
hENT1 (compared with rENT1 and rENT2, which are totally resistant to
inhibition) suggests that binding of dipyridamole and dilazep is likely
to be complex, involving contributions from several amino acid residues
from different regions of hENT1.
The results of equilibrium binding studies in cells with the
es transport process, for which ENT1 proteins are believed
to be responsible, have led to the conclusion that dilazep and
dipyridamole are competitive inhibitors for a single or overlapping
exofacial NBMPR and permeant binding site (17, 34, 39, 40, 47). However, results from other studies have suggested that dilazep and
dipyridamole display characteristics of allosteric ligands when present
at high concentrations (33, 35, 39, 48). A unifying model that has been
suggested for permeant and inhibitor binding to hENT1 describes two
binding sites in which permeants, NBMPR, and other inhibitors such as
dilazep and dipyridamole compete for a single high affinity site, which
is subject to allosteric modulation by a distinct broad specificity low
affinity site that binds nucleosides, nucleobases, and inhibitors when
present at very high concentrations (3). The contribution of the
potential allosteric binding site of hENT1 was likely to be negligible
in the experiments of the current study because the Hill coefficients indicated the presence of a single class of binding sites. These results suggested that mutation of residue 33 affected dilazep and
dipyridamole binding to the competitive binding site.
The current study established that residue 33 of hENT1 and hENT2 is
important for dilazep and dipyridamole interaction. It is not clear
whether residue 33 of hENT1 and hENT2 is directly involved in permeant
or inhibitor binding or whether the effects observed when it was
mutated were due to changes in the tertiary structure of these
proteins. The alternatives are difficult to resolve in the absence of
detailed structural data. Future studies include using different random
mutagenesis and screening approaches to identify other residues that
may be important for interaction with nucleoside transport inhibitors.
*
This work was supported by a Canadian Cancer Society grant
from the National Cancer Institute of Canada (to C. E. C.),
grants from the Canadian Institutes of Health Research (to C. E. C. and J. D. Y.) and the Wellcome Trust and Medical
Research Council (to S. A. B.).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.
d
Supported by studentships from the Alberta Heritage
Foundation for Medical Research and the Endowed Ph.D. Studentship in Oncology.
g
Heritage Scientist of the Alberta Heritage Foundation for
Medical Research.
j
Canada Research Chair in Oncology. To whom correspondence
should be addressed: Dept. of Oncology, Cross Cancer Inst., 11560 University Ave., Edmonton, AB T6G 1Z2, Canada. Tel.:
780-432-8320; Fax: 780-432-8425; E-mail:
carol.cass@cancerboard.ab.ca.
Published, JBC Papers in Press, October 31, 2001, DOI 10.1074/jbc.M105324200
The abbreviations used are:
CNT, concentrative
nucleoside transporter;
ENT, equilibrative nucleoside transporter;
NBMPR, nitrobenzylmercaptopurine ribonucleoside
(6-[(4-nitrobenzyl)thiol]-9-
Mutation of Residue 33 of Human Equilibrative Nucleoside
Transporters 1 and 2 Alters Sensitivity to Inhibition of Transport
by Dilazep and Dipyridamole*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, gal, ura3-52, trp1,
lys2, ade2, hisd2000) was the parental yeast strain used to
generate KTK, which produces recombinant Herpes simplex
thymidine kinase (22), and fui1::TRP1, which contains a
disruption in the gene encoding the endogenous uridine permease (FUI1)
(25). Other strains were generated by transformation of the
yeast/Escherichia coli shuttle vector pYPGE15 (26) into KTK
and fui1::TRP1 using a standard lithium acetate method (27).
cDNA inserts were under the transcriptional control of the
constitutive PGK promoter. Yeast strains were maintained in
complete minimal medium (CMM) containing 0.67% yeast nitrogen base
(Difco, Detroit, MI), amino acids (as required to maintain auxotrophic
selection), and 2% glucose (CMM/GLU). Agar plates contained CMM with
various supplements and 2% agar (Difco). Plasmids were propagated in
E. coli strain TOP10F' (Invitrogen) and maintained in Luria
broth with ampicillin (100 µg/ml).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 1.
Time courses of [3H]uridine
uptake for recombinant hENT1 and hENT2 produced in S. cerevisiae. Yeast cells containing pYPhENT1
(circles), pYPhENT2 (squares), or pYPGE15
(triangles) were incubated with 2 µM
[3H]uridine for the indicated time periods. The
inset shows the time courses for the first 10 s of
[3H]uridine influx. Each point represents mean uridine
uptake (± S.E., n = 3); S.E. values are not presented
where the size of the point is larger than the S.E.
View larger version (34K):
[in a new window]
Fig. 2.
Multiple sequence alignment of predicted
amino acid sequences of the ENT1 and ENT2 proteins in transmembrane
segment 1 (TM1) of humans, mice, and rats. The position of the
M33I mutation is indicated by the arrow. The
GenBankTM accession numbers are AAC51103 (hENT1) (28),
AAF78452 (mENT1) (36), AAB88049 (rENT1) (18), AAC39526 (hENT2) (37),
AAF78477 (mENT2) (36), and AAB88050 (rENT2) (18). Multiple sequence
alignment was performed with DNAMAN version 4.03 software using the
BLOSUM 62 substitution matrix.
80% by 0.1 µM dilazep and 0.3 µM dipyridamole, whereas hENT1-M33I was capable of
transport at 60% of the maximal rate in the presence of both inhibitors. These results suggested that hENT1-M33I was substantially less sensitive to dilazep and dipyridamole than wild type hENT1. In
contrast, uridine transport was completely inhibited by 0.1 µM NBMPR in yeast with either recombinant protein,
suggesting that residue 33 was not involved in the binding of
NBMPR.
View larger version (11K):
[in a new window]
Fig. 3.
Inhibition of uridine transport mediated by
recombinant hENT1, hENT1-M33I, hENT2, and hENT2-I33M by dilazep,
dipyridamole, and NBMPR. Yeast cells containing pYPhENT1
(A, solid bars), pYPhENT1-M33I (A,
open bars), pYPhENT2 (B, solid bars),
or pYPhENT2-I33M (B, open bars) were incubated
for 20 min in the presence of 2 µM
[3H]uridine with or without the indicated concentration
of inhibitor. Uridine transport rates (means ± S.E.,
n = 3) in the presence of inhibitor are represented as
percentages of the rates observed in the absence of inhibitor
(control), which were 48.1 ± 4.7, 40.6 ± 1.5, 27.4 ± 0.2, and 100.2 ± 0.7 pmol/mg protein/min,
respectively, for hENT1, hENT1-M33I, hENT2, and hENT2-I33M. Three
separate experiments gave qualitatively similar results.
Kinetic properties of uridine transport for hENT1, hENT1-M33I, hENT2,
and hENT2-I33M
View larger version (16K):
[in a new window]
Fig. 4.
The concentration dependence of transport
inhibition of recombinant hENT1, hENT1-M33I, hENT2, and hENT2-I33M by
dilazep, dipyridamole, and NBMPR. Yeast cells containing pYPhENT1
(closed circles), pYPhENT1-M33I (open circles),
pYPhENT2 (closed squares), or pYPhENT2-I33M (open
squares) were incubated for 20 min in the presence of 2 µM [3H]uridine with or without graded
concentrations of dilazep (A), dipyridamole (B),
or NBMPR (C). Uridine transport rates (mean ± S.E.,
n = 3) in the presence of inhibitor are represented as
percentages of the rates observed in the absence of inhibitor
(control), and S.E. values are not presented where the size
of the point is larger than the S.E. Mean values (± S.E.) for control
uridine transport rates were 78.4 ± 5.5, 70.1 ± 2.6, 31.8 ± 0.7, and 143.6 ± 2.2 pmol/mg protein/min for hENT1,
hENT1-M33I, hENT2, and hENT2-I33M, respectively. Three separate
experiments gave similar results. IC50 values and Hill
coefficients were determined using GraphPad Prism version 3.0 software
by nonlinear regression analysis. The Ki values
given in Table II were calculated using the equation of Cheng and
Prusoff (38) with the experimentally determined IC50 values
for each inhibitor and the uridine Km values for
each recombinant protein (Table I).
Ki values for dilazep, dipyridamole, and NBMPR inhibition of
uridine transport for hENT1, hENT1-M33I, hENT2, and hENT2-I33M
View larger version (16K):
[in a new window]
Fig. 5.
The concentration dependence of inhibition of
recombinant hENT1 and hENT1-M33I by dipyridamole in X. laevis oocytes. Initial rates of
[14C]uridine uptake were determined in the presence of
graded concentrations of dipyridamole and were corrected for endogenous
uridine transport activity by subtracting uptake values obtained in
water-injected oocytes. The oocytes were pretreated with dipyridamole
for 1 h to allow for complete binding site equilibration. Uridine
transport rates (mean ± S.E., n = 10-12) in the
presence of inhibitor are represented as percentages of the rates
observed in the absence of inhibitor (control), and the S.E.
values are not presented where the size of the point is larger than the
S.E. Mean values (± S.E.) for control uridine transport rates were
2.13 ± 0.10 and 2.15 ± 0.11 pmol/oocyte/5 min,
respectively, for hENT1 and hENT1-M33I. IC50 values were
determined using GraphPad Prism version 3.0 software by nonlinear
regression analysis and were 300 ± 79 and 3640 ± 1400, respectively, for hENT1 and hENT1-M33I.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
ABBREVIATIONS
-D-ribofuranosyl purine);
TM, transmembrane;
h, human;
m, mouse;
r, rat;
CMM, complete minimal
medium;
GLU, glucose;
MTX, methotrexate;
PGK, phosphoglycerate kinase;
SAA, sulfanilamide.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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J. Zhang, F. Visser, M. F. Vickers, T. Lang, M. J. Robins, L. P.C. Nielsen, I. Nowak, S. A. Baldwin, J. D. Young, and C. E. Cass Uridine Binding Motifs of Human Concentrative Nucleoside Transporters 1 and 3 Produced in Saccharomyces cerevisiae Mol. Pharmacol., December 1, 2003; 64(6): 1512 - 1520. [Abstract] [Full Text] [PDF]
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G. Li, K. Liu, S. A. Baldwin, and D. Wang Equilibrative Nucleoside Transporters of Arabidopsis thaliana: cDNA CLONING, EXPRESSION PATTERN, AND ANALYSIS OF TRANSPORT ACTIVITIES J. Biol. Chem., September 12, 2003; 278(37): 35732 - 35742. [Abstract] [Full Text] [PDF]
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S. Arastu-Kapur, E. Ford, B. Ullman, and N. S. Carter Functional Analysis of an Inosine-Guanosine Transporter from Leishmania donovani: THE ROLE OF CONSERVED RESIDUES, ASPARTATE 389 AND ARGININE 393 J. Biol. Chem., August 29, 2003; 278(35): 33327 - 33333. [Abstract] [Full Text] [PDF]
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 Y. Acimovic and I. R. Coe Molecular Evolution of the Equilibrative Nucleoside Transporter Family: Identification of Novel Family Members in Prokaryotes and Eukaryotes Mol. Biol. Evol., December 1, 2002; 19(12): 2199 - 2210. [Abstract] [Full Text] [PDF]
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 N. Sankar, J. Machado, P. Abdulla, A. J. Hilliker, and I. R. Coe Comparative genomic analysis of equilibrative nucleoside transporters suggests conserved protein structure despite limited sequence identity Nucleic Acids Res., October 15, 2002; 30(20): 4339 - 4350. [Abstract] [Full Text] [PDF]
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S. Y. M. Yao, A. M. L. Ng, M. F. Vickers, M. Sundaram, C. E. Cass, S. A. Baldwin, and J. D. Young Functional and Molecular Characterization of Nucleobase Transport by Recombinant Human and Rat Equilibrative Nucleoside Transporters 1 and 2. CHIMERIC CONSTRUCTS REVEAL A ROLE FOR THE ENT2 HELIX 5-6 REGION IN NUCLEOBASE TRANSLOCATION J. Biol. Chem., July 5, 2002; 277(28): 24938 - 24948. [Abstract] [Full Text] [PDF]
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