NUCLEOSIDE TRANSPORTERS: CNTs AND ENTs - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 183

in possessing a long hydrophilic N-terminal region that contains a dileucine motif
(DE)XXXL(LI) for endosomal/lysosomal targeting. 6 ENT3 transcripts and proteins
are widely distributed in human and rodent tissues. In humans the highest levels
of mRNA were found in the placenta, uterus, ovary, spleen, lymph node, and bone
marrow; the lowest levels were found in brain and heart. 6 In contrast to ENT1 and
ENT2, the endogenous as well as GFP-tagged hENT3 proteins were found to reside
predominantly intracellularly in HeLa cells and to colocalize partially with lysosomal
markers. Truncation of the N-terminal region or mutation of the dileucine motif to
alanine (hENT3AA) relocated hENT3 to the plasma membrane in HeLa cells and in
Xenopus oocytes. 6
Characterization of a cell surface-expressed mutant hENT3AA suggested that
hENT3 is a broadly selective and low-affinity nucleoside transporter that transports
purine and pyrimidine nucleosides as well as the nucleobase adenine. 6 hENT3AA-
mediated transport is Na + independent but stimulated strongly by low pH, with the
optimum pH being 5.5. 6 At pH 5.5, hENT3AA exhibits about 40- and 10-fold lower
affinities for adenosine and uridine than for hENT1. Nucleoside analogs, including
cladribine, cordycepin (3 -deoxyadenosine), tubercidin (7-deazaadenosine), fludara-
bine, zebularine, ddI, ddC, and AZT, are transported by hENT3AA. hENT3AA is
relatively insensitive to the classical nucleoside transport inhibitors, such as NBMPR,
dipyridamole, and dilazep.

ENT4 (SLC29A4) The fourth member of the human SLC29 family, hENT4, was first
cloned and characterized in our laboratory. 7 Gene orthologs of ENT4 are also found in
mouse and rat. 7 , 87 hENT4 is a 530-residue protein which displays about 86% identity
to the 528-residue mouse homolog mENT4. The mENT4 and hENT4 proteins share
low but significant sequence identity (about 18 to 20%) to ENT1-3 and possess a
similar 11 TM membrane topology. However, hENT4 has a longer hydrophilic N-
terminus and exhibits a low overall sequence homology to other ENTs. Phylogenetic
analysis suggests that the mammalian ENT4 lineage is evolutionarily distinct from the
ENT1/2/3 lineage(s). 87 The gene encoding hENT4 is located at chromosome 7p22.1.
Our Northern blot analysis showed that hENT4 mRNA is expressed preferentially
in the human brain but is also found in skeletal muscle, kidney, heart, and liver. 7 In
human and mouse brain, hENT4 transcripts are widely distributed in different regions
of the CNS (Wang et al., unpublished data). 7 YFP tagging of hENT4 in MDCK cells
demonstrated that this protein is localized primarily to the plasma membrane.
Although ENT4 was originally suggested to be a nucleoside transporter, 3
extensive functional analysis carried out in this laboratory showed that except
for a moderate activity toward adenosine, hENT4 minimally interacts with other
nucleosides, nucleobases, or their analogs. 7 Instead, it mediates Na + -independent,
electrogenic transport of monoamine neurotransmitters such as serotonin and
dopamine ( K m = M, respectively). 7 Functional analysis of the mouse

ENT4 ortholog also confirmed that the monoamines are the physiologic substrates
of ENT4 (Wang et al., unpublished data). Therefore, we previously proposed an
alternative functional name, plasma membrane monoamine transporter (PMAT), for
ENT4. Our recent analysis showed that hENT4 also transports cationic xenobiotics
such as MPP + and TEA, and shares striking functional similarities to a genetically

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