Biomedical Engineering Reference
In-Depth Information
5-fluoro-2
-deoxyuridine, zebularine, and gemcitabine) and purine (cladribine, clo-
farabine, and fludarabine) nucleoside analogs.
47
,
43
Low uptake is also observed for
AZT, ddC, and ddI.
47
Recent electrophysiology studies demonstrated further that rib-
avirin and 3-deazauridine are also hCNT3 substrates.
51
So far, specific high-affinity
inhibitors have not been described for hCNT3 and other CNTs.
8.2.2. Transport Mechanisms
Mammalian CNTs are Na
+
-coupled transporters which utilize the inwardly directed
Na
+
gradient established by Na
+
/K
+
-ATPase to translocate substrates into cells.
Early radio tracer uptake studies in cell and tissue preparations suggested that the
Na
+
/nucleoside coupling stoichiometry is 1 : 1 for
cit
(CNT1) and
cif
(CNT2); and 2 : 1
for
cib
(CNT3).
1
Recent electrophysiological studies have confirmed these coupling
stoichiometry for hCNT1 and hCNT3.
51-53
Electrophysiological analysis of hCNT1
also suggested an ordered simultaneous transport model in which Na
+
first binds to
the transporter in order to enhance subsequent binding of nucleoside.
52
Interestingly,
while ion coupling of hCNT1 and hCNT2 is Na
+
specific, hCNT3 exhibits unique
broad cation specificity and can use H
+
and Li
+
to substitute Na
+
for coupling.
53
Chimeric studies between hCNT1 and hCNT3 suggested that the site for Na
+
ion
interaction may reside in the C-terminal half.
53
CNT homologs have also been found
in bacteria, yeast, nematodes, insects, and hagfish.
4
,
54
CNTs isolated from
Escherichia
coli
,
Candida albicans
, and
Caenorhabditis elegans
use a proton gradient as the
driving force, whereas the hagfish hfCNT is coupled to Na
+
.
53
From the substrate point of view, CNT1-3 specifically transport nucleosides and
their analogs. CNT1-3 do not transport nucleobases (i.e., free purine or pyrimidine
bases) or nucleotides. A number of recent studies have examined the structure-activity
relationships of the CNTs.
19
,
55-58
Besides isoform-dependent specificity toward the
purine or pyrimidine moieties, several common features of the ribose structure are
important for CNT-substrate interaction. Generally speaking, the 3
-hydroxyl group
of the ribose ring is essential for substrate interaction with the CNTs, and modifications
at the C(3
) position are usually not tolerated. CNTs are less sensitive to modifications
at the 5
-hydroxyl group and generally tolerate changes at the 2
-hydroxyl group.
From the protein standpoint, mammalian CNTs are proposed to have 13 trans-
membranes (TMs) with an intracellular N-terminus and an extracellular C-terminus
based on antibody and glycosylation studies (Figure 8.2A).
15
hCNT1, hCNT2, and
hCNT3 all contain potential N-glycosylation sites and are likely to exist in the glyco-
sylated form.
5
,
50
Mutational analysis of the three putative N-linked glycosylation sites
in rCNT2 suggests that glycosylation does not affect transporter function or mem-
brane sorting.
59
Protein chimera and site-directed mutagenesis studies have been
used to examine the structure-function relationships of CNTs. Domain-swapping
studies in rCNT1 and rCNT2 first identified TMs 7 and 8 as the essential sites for
substrate recognition and discrimination.
60
Site-directed mutagenesis studies further
identified Ser318 on TM 7 of rCNT1 as essential for its pyrimidine selectivity.
61
Replacing Ser318 with glycine transformed rCNT2 into a broadly selective CNT3-
like transporter.
61
,
62
A Gln319Met mutation augmented this effect by enhancing the
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