Pharmacological analysis and molecular cloning of the canine equilibrative nucleoside transporter 1.
We studied the binding of [3H]nitrobenzylthioinosine (NBMPR) and the uptake of [3H]formycin B by the es (equilibrative inhibitor-sensitive) nucleoside transporter of Madin Darby Canine Kidney (MDCK) cells. NBMPR inhibited [3H]formycin B uptake with a Ki of 2.7+/-0.6 nM, and [3H]NBMPR had a KD of 1.3+/-0.3 nM for binding to these cells; these values are significantly higher than those obtained in human and mouse cell models. In contrast, other recognized es inhibitors, such as dipyridamole, were significantly more effective as inhibitors of [3H]NBMPR binding and [3H]formycin B uptake by MDCK cells relative to that seen for human cells. We isolated a cDNA encoding the canine es nucleoside transporter (designated cENT1), and assessed its function by stable expression in nucleoside transport deficient PK15NTD cells. The PK15-cENT1 cells displayed inhibitor sensitivities that were comparable to those obtained for the endogenous es nucleoside transporter in MDCK cells. These data indicate that the dog es/ENT1 transporter has distinctive inhibitor binding characteristics, and that these characteristics are a function of the protein structure as opposed to the environment in which it is expressed.
Hammond
James R
JR
Department of Physiology and Pharmacology, M216 Medical Sciences Building, University of Western Ontario, London, Ontario, Canada N6A 5C1. jhammo@uwo.ca
Stolk
Meaghan
M
Archer
Richard G E
RG
McConnell
Kristy
K
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004109:Dilazep / Q000494:pharmacology; D004176:Dipyridamole / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D033721:Equilibrative Nucleoside Transporter 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005573:Formycins / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010879:Piperazines / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D011869:Radioligand Assay; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014316:Tritium
D002352:Carrier Proteins; D018076:DNA, Complementary; D033721:Equilibrative Nucleoside Transporter 1; D005573:Formycins; D010879:Piperazines; C072040:draflazine; D014316:Tritium; C034561:formycin B; D013868:Thioinosine; D004176:Dipyridamole; D004109:Dilazep; C001789:4-nitrobenzylthioinosine