NUCLEOSIDE TRANSPORTERS: CNTs AND ENTs - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 181

apparent affinity for purine nucleosides. 61 Using a similar approach, Loewen et al.
confirmed these findings in hCNT1 and hCNT2, and further identified Ser319/Gln320
and Ser353/Leu354 as essential determinants for substrate selectivity. 63 In addition,
mutation of a conserved residue Phe316 in TM7 of hCNT1 to tyrosine or alanine
increased transporter sensitivity to guanosine. 64 Together, these studies demonstrated
that TMs 7 and 8 are essential components of the substrate binding and transloca-
tion channel, and that substrate selectivity of the CNTs may be determined by a few
key amino acid residues located on TMs 7 and 8. Cysteine-accessibility analysis of
hCNT3 suggests that TMs 11 and 12 may also participate in forming the nucleoside
translocation pathway. 65 In contrast, TMs 1 to 3 appear not essential for CNT function.
When TMs 1 to 3 were truncated from rCNT1 or hCNT1, the resulting transporters
retained significant Na + -dependent transport activity. 15 NupC, a H + -coupled CNT
from E. coli , lacks TMs 1 to 3, further suggesting that TMs 1 to 3 may not be required
for CNT function. 66

8.3. EQUILIBRATIVE NUCLEOSIDE TRANSPORTERS (SLC29)

8.3.1. Family Members

ENT1 (SLC29A1) Purification and N-terminal sequencing of the prototype es trans-
porter from human erythrocytes led to the cloning of hENT1 in 1997. 67 Human ENT1
is a 456-residue glycoprotein with 11 predicted TMs (Figure 8.2B). The hENT1 gene
is localized to chromosome 6p21.1-21.2. ENT1 orthologs were cloned from rat and
mouse. 68-70 rENT1 (457 a.a.) and mENT1.1 (460 a.a.) are about 78 to 79% identical
to hENT1. 68-70 A splice variant, mENT1.2, has been reported for mouse. 70 Studies
at both the mRNA and protein levels have revealed that ENT1 is almost ubiqui-
tously distributed in human and rodent tissues, although its abundance varies among
tissues. 12 , 67 , 70 High expression of hENT1 protein or mRNA has been demonstrated
and confirmed in many tissues, including erythrocytes, placenta, brain, heart, liver,
lung, and colon. 12 , 71 However, striking intertissue and interindividual differences in
the expression levels of hENT1 were observed. 12 GFP- or YFP (yellow fluorescence
protein)-tagged hENT1 is localized predominantly on the basolateral membrane of
differentiated MDCK cells, whereas a small amount of hENT1 also appears on the
apical membrane. 17 , 72 Besides plasma membrane, Lai et al. showed that hENT1, but
not mENT1, is also present in mitochondria, which may contribute to enhanced mi-
tochondrial toxicity of certain antiviral nucleoside analogs. 73 A PEXN motif located
in the first extracellular loop of hENT1 was recently identified as an important signal
for mitochondrial targeting of hENT1. 74
Recombinant human, rat, and mouse ENT1 transporters transport both purine
and pyrimidine nucleosides and exhibit typical es -type activities. The K m values of
hENT1 toward endogenous nucleosides range from 50

M
for cytidine. 75 Transport is Na + independent and inhibited by nanomolar NBMPR.
hENT1 does not interact with nucleobases. 67 , 75 hENT1 interacts with many nucleo-
side analogs widely used in the treatment of cancer and viral infections. Cladribine,

μ M for adenosine to 680 μ