Fine tuning of rabbit equilibrative nucleoside transporter activity by an alternatively spliced variant.
The full-length cDNA encoding an equilibrative nucleoside transporter (rbENT2) and its novel C-terminal variant, rbENT2A, were isolated from rabbit trachea. Rabbit ENT2 protein consists of 456 amino acid residues; rbENT2A is shorter by 41 residues. Both rbENT2 and rbENT2A transcripts are found in rabbit tissues including intestine, kidney cortex, kidney, and trachea, at varying levels of expression. When transfected in a heterologous expression system-Madin Darby canine kidney (MDCK) epithelial cell line-both rbENT2 and rbENT2A were expressed. rbENT2 had a molecular mass of 49 kDa; rbENT2A had a molecular mass of 44 kDa. Clones of both transporters yielded functional proteins that were capable of mediating uridine uptake and efflux without the needing to be coupled to a secondary ion (e.g. Na(+)). Remarkably, rbENT2A displayed a higher affinity (K(m) = 41 microM) and a lower capacity (V(max) = 0.6 nmol/mg protein/5 min) towards substrates than rbENT2 (K(m) = 272.8 microM, V(max) = 1.26 nmol/mg protein/5 min). Pharmacological profiles showed that nitro-benzyl-mercapto-purine-ribose (NBMPR) potently inhibited (3)H-uridine uptake mediated by rbENT2A, but not uptake mediated by rbENT2. The constitutive splicing, broad expression, markedly different kinetics, and distinct pharmacological characteristics of rbENT2A appear to act in conjunction with the wild type, rbENT2, to fine-tune basolateral nucleoside transport function in rabbit trachea.
Wu
Sharon K
SK
Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, 90089-9121, USA.
Ann
David K
DK
Kim
Kwang-Jin
KJ
Lee
Vincent H L
VH
D017398:Alternative Splicing / Q000235:genetics / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004285:Dogs; D016503:Drug Delivery Systems / Q000379:methods; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D033722:Equilibrative-Nucleoside Transporter 2 / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D014644:Genetic Variation / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008297:Male; D008954:Models, Biological; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013868:Thioinosine / Q000031:analogs & derivatives / Q000494:pharmacology; D014018:Tissue Distribution; D014132:Trachea / Q000166:cytology / Q000187:drug effects; D014316:Tritium / Q000737:chemistry; D014529:Uridine / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000493:pharmacokinetics
D033722:Equilibrative-Nucleoside Transporter 2; D012333:RNA, Messenger; C438654:SLC29A2 protein, human; D014316:Tritium; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D014529:Uridine