Molecular cloning of a Na+-dependent nucleoside transporter from rabbit intestine.
OBJECTIVE
Substantial species differences in the transport kinetics of nucleosides and therapeutic nucleoside analogs have been observed in various experimental systems. To explain these differences at a molecular level, it is necessary to clone the relevant transporters and examine their functional characteristics in heterologous expression systems. The goal of the present study was to clone the nucleoside transporters present in rabbit, an important preclinical animal model, and to functionally characterize the clone(s).


METHODS
A Polymerase Chain Reaction (PCR)-based homology cloning approach in conjunction with Rapid Amplification of cDNA Ends (RACE) was used to isolate a full-length cDNA. Characterization of this transporter was accomplished through heterologous expression in Xenopus laevis oocytes.


RESULTS
A full-length cDNA encoding a purine-selective, Na+-dependent nucleoside transporter, rbSPNT1, was isolated from rabbit small intestine. The encoded protein is 658 amino acid residues in length. Hydropathy analysis suggests that rbSPNT1 has 11 to 14 transmembrane domains. In Xenopus laevis oocytes expressing rbSPNT1, the uptake of uridine and inosine was enhanced significantly; uridine transport was inhibited by purine, but not pyrimidine nucleosides. mRNA transcripts for rbSPNT1 were detected primarily in intestine, lung, and kidney and at lower levels in liver, brain, and heart.


CONCLUSIONS
A full-length functional nucleoside transporter was cloned. Sequence analysis and functional characterization suggest that rbSPNT1 is the rabbit homolog of the purine-selective nucleoside transporter, N1. The cloned rbSPNT1 can be used to understand the molecular mechanisms responsible for the observed species differences in the transport of nucleosides and therapeutic nucleoside analogs.
Gerstin
K M
KM
Department of Biopharmaceutical Sciences, University of California, San Francisco 94143-0446, USA. kmg@itsa.ucsf.edu
Dresser
M J
MJ
Wang
J
J
Giacomini
K M
KM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis*; D003001:Cloning, Molecular; D007421:Intestine, Small / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014981:Xenopus
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C065171:cif nucleoside transporter