Equilibrative nucleoside transporters: mapping regions of interaction for the substrate analogue nitrobenzylthioinosine (NBMPR) using rat chimeric proteins.
The rat equilibrative nucleoside transporters rENT1 and rENT2 belong to a family of integral membrane proteins with 11 potential transmembrane segments (TMs) and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine (NBMPR). Structurally, the proteins have a large glycosylated extracellular loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. In the present study, we have generated chimeras between NBMPR-sensitive rENT1 and NBMPR-insensitive rENT2, using splice sites at rENT1 residues 99 (end of TM 2), 171 (between TMs 4 and 5), and 231 (end of TM 6) to identify structural domains of rENT1 responsible for transport inhibition by NBMPR. Transplanting the amino-terminal half of rENT2 into rENT1 rendered rENT1 NBMPR-insensitive. Domain swaps within the amino-terminal halves of rENT1 and rENT2 identified two contiguous regions, TMs 3-4 (rENT1 residues 100-171) and TMs 5-6 (rENT1 residues 172-231), as the major sites of NBMPR interaction. Since NBMPR is a nucleoside analogue and functions as a competitive inhibitor of zero-trans nucleoside influx, TMs 3-6 are likely to form parts of the substrate translocation channel.
Sundaram
M
M
Membrane Transport Research Group, Departments of Physiology and Oncology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Yao
S Y
SY
Ng
A M
AM
Cass
C E
CE
Baldwin
S A
SA
Young
J D
JD
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D033705:Equilibrative Nucleoside Transport Proteins; D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2; D009865:Oocytes; D010449:Peptide Mapping / Q000379:methods*; D011684:Purine Nucleosides / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism*; D014529:Uridine / Q000378:metabolism; D014981:Xenopus
D000345:Affinity Labels; D002352:Carrier Proteins; D033705:Equilibrative Nucleoside Transport Proteins; D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2; D011684:Purine Nucleosides; D011993:Recombinant Fusion Proteins; C438652:Slc29a1 protein, rat; C438655:Slc29a2 protein, rat; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D014529:Uridine