NUCLEOSIDE TRANSPORTERS: CNTs AND ENTs - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 184

unrelated drug transporter family, organic cation transporters (OCTs) (reviewed in
Chapter 2). 8 Because ENT4 does not typically function as a nucleoside transporter,
we will not include it for further discussion.
8.3.2. Transport Mechanisms
ENT1 and ENT2 are facilitated carriers that transport substrates down their concen-
tration gradients. However, in many cell types, cellular uptake of nucleosides and
nucleoside analogs are tightly coupled to intracellular metabolism (e.g., phosphory-
lation). Rapid enzymatic conversion of nucleosides to metabolites (e.g., nucleotides)
can thus provide a metabolic “driving force” to promote cellular uptake. 1 Efflux oc-
curs only when intracellular concentrations of free nucleoside exceed those outside
the cell. The transport mechanism of the lysosome-localized ENT3 is not known
yet. ENT3 activity is strongly stimulated by proton, exhibiting maximal activity at
pH 5.5 and no activity at pH 8.0. It is unclear whether the pH dependence of ENT3
reflects a proton-nucleoside cotransport mechanism, or an evolutionary adaptation of
ENT3 to the acidic environment of lysosomes. Mammalian ENT homologs are also
found in fungi, protozoans, nematodes, insects, and plants, but not in bacteria. 87 Inter-
estingly, ENT members from Leishmania donovani function as electrogenic proton
cotransporters, 88 suggesting that ENT-type transporters are not always “equilibrative”
and can be electrogenic and concentrative in certain species.
Like the CNTs, the 3 -OH of the ribose moiety is critical for substrate interaction
with ENT1 and ENT2, whereas the 2 and 5 hydroxyl groups are less important. 58 , 85 , 89
Halogen modifications on most positions at the base are generally accepted. In gen-
eral, hENT2 is more tolerant than hENT1 to modification at the ribose ring and even
transports nucleobases that lack the ribose moiety. Analysis of a series of uridine
analogs with hENT1 and hENT2 suggests that the C(2 )-OH is a structural determi-
nant for uridine-hENT1 but not for uridine-hENT2 interactions. 89 Moreover, hENT2
displayed more tolerance than hENT1 to removal of C(5 )-OH. The changes in bind-
ing energies between transporter proteins and the various uridine analogs suggest that
hENT1 may form strong interactions with C(3 )-OH and moderate interactions with
C(2 )-OH and C(5 )-OH of uridine, whereas hENT2 may form strong interactions with
C(3 )-OH, weak interactions with C(5 )-OH, and no interaction with C(2 )-OH. 89
Glycosylation scanning and antibody studies have confirmed the originally pro-
posed 11 TM topology of hENT1 (Figure 8.2B). 90 hENT1 is N-glycosylated at a single
site and hENT2 at two sites in the large extracellular loop linking TMs 1 and 2. 90 Gly-
cosylation is not required for the transport activity of hENT1 but may slightly affect
the binding affinity to transport inhibitors such as NBMPR. 91 Glycosylation does not
change hENT2 function but is required for efficient targeting of hENT2 protein to
the plasma membrane. 92 Investigation of human and rat ENT1/2 has begun to iden-
tify functionally important domains of these transporters. The human and rat ENT1
transporters exhibit similar binding affinities toward NBMPR, but there is more than
a 50-fold difference between hENT1 and rENT1 in interacting with dipyridamole. 79
Using chimeric constructs of hENT1 and rENT1, Sundaram et al. first demonstrated
that TMs 1 to 6 of hENT1 are required for interaction with dipyridamole and dilazep,