The human equilibrative
nucleoside transporter, hENT1, which is sensitive to inhibition by nitrobenzylthioinosine (
NBMPR), is expressed in a wide variety of tissues. hENT1 is involved in the uptake of natural
nucleosides, including regulation of the physiological effects of extracellular
adenosine, and transports
nucleoside drugs used in the treatment of
cancer and
viral diseases. Structure-function studies have revealed that transmembrane domains (TMD) 3 through 6 of hENT1 may be involved in binding of
nucleosides. We have hypothesized that
amino acid residues within TMD 3-6, which are conserved across equilibrative transporter sequences from several species, may have a critical role in the binding and transport of
nucleosides. Therefore, we explored the role of point mutations of two conserved
glycine residues, at positions 179 and 184 located in transmembrane domain 5 (TMD 5), using a GFP-tagged hENT1 in a yeast
nucleoside transporter assay system. Mutations of
glycine 179 to
leucine,
cysteine, or
valine abolished transporter activity without affecting the targeting of the transporter to the plasma membrane, whereas more conservative mutations such as
glycine to
alanine or
serine preserved both targeting to the plasma membrane and transport activity. Similar point mutations at
glycine 184 resulted in poor targeting of hENT1 to the plasma membrane and little or no detectable functional activity.
Uridine transport by G179A mutant was significantly lower (p < 0.05) and less sensitive (p < 0.05) to inhibition by
NBMPR when compared to the wild-type transporter (IC(50) 7.7 +/- 0.8 nM versus 46 +/- 14.6 nM). Based on these data, we conclude that when hENT1 is expressed in yeast,
glycine 179 is critical not only to the ability of hENT1 to transport
uridine but also as a determinant of hENT1 sensitivity to
NBMPR. In contrast,
glycine 184 is likely important in targeting the transporter to the plasma membrane. This is the first identification and characterization of a critical
amino acid residue of hENT1 that is important in both
nucleoside transporter function and sensitivity to inhibition by
NBMPR.