Molecular cloning and functional expression of cDNAs encoding a human Na+-nucleoside cotransporter (hCNT1)

Abstract

We report identification of a new human nucleoside transporter protein by molecular cloning and functional expression of its cDNA. Previously, we used expression selection in Xenopus oocytes to isolate a cDNA from rat jejunal epithelium encoding the pyrimidine-selective Na+-dependent nucleoside transporter rCNT1 (Q.-Q. Huang, S. Y. M. Yao, M. W. L. Ritzel, A. R. P. Paterson, C. E. Cass, and J. D. Young. J. Biol. Chem. 269: 17757-17760, 1994). cDNAs for a human homologue of rCNT1, designated hCNT1, have been isolated from human kidney by hybridization cloning and reverse transcriptase polymerase chain reaction amplification strategies. hCNT1 was 83% identical to rCNT1 in amino acid sequence and exhibited the transport characteristics of an Na+-dependent nucleoside transporter with selectivity for pyrimidine nucleosides and adenosine when expressed in Xenopus oocytes. Deoxyadenosine, which undergoes net renal secretion, and guanosine were poor permeants. hCNT1 did, however, transport 3'-azido-3'-deoxythymidine. This is the first demonstration that members of the CNT family exist in human cells and provides evidence of their involvement in the renal transport of physiological nucleosides and nucleoside drugs. The hCNT1 gene was mapped to chromosome 15q25-26.

Functional characterization of a H+/nucleoside co‐transporter (CaCNT) from Candida albicans, a fungal member of the concentrative nucleoside transporter (CNT) family of membrane proteins

“…Kinetic parameters (apparent K m and V max ) derived from the CaCNT-mediated transport data, which were consistent with simple Michaelis-Menten kinetics, are presented in Table 1. Apparent K m values varied between 16 and 64 µM (adenosine < uridine < inosine, guanosine) and were in the same range as values obtained previously for recombinant mammalian CNT proteins (Huang et al, 1994;Che et al, 1995;Yao et al, 1996a;Ritzel et al, 1997;Wang et al, 1997; …”

“…Healthy stage VI Xenopus oocytes were injected (Inject + Matic System) with 20 nl CaCNT RNA transcript (1 ng/nl) or 20 nl water alone and incubated at 18 • C in MBM for 3 days with a daily change of medium before the assay of transport activity (Huang et al, 1994;Ritzel et al, 1997;Yao et al, 2000).…”

“…Human (h) and rat (r) ENT1 and ENT2 transport pyrimidine and purine nucleosides and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and vasoactive drugs, and by the ability of hENT2 and rENT2 to also transport nucleobases (Griffiths et al, 1997a(Griffiths et al, , 1997bYao et al, 1997;Crawford et al, 1998;Yao et al, 2002a). CNT1 and CNT2 both transport uridine and adenosine, but are otherwise selective for pyrimidine (hCNT1 and rCNT1) and purine (hCNT2 and rCNT2) nucleosides (Huang et al, 1994;Che et al, 1995, Yao et al, 1996aWang et al, 1997;Ritzel et al, 1997Ritzel et al, , 1998. hCNT3, its mouse (m) orthologue mCNT3 and a close relative from Eptatretus stouti, an ancient marine prevertebrate, transport both pyrimidine and purine nucleosides (Ritzel et al, 2001;Yao et al, 2002b).…”

“…CNT1/2/3 proteins transport a broad spectrum of antiviral and anticancer nucleoside analogues (Huang et al, 1995;Yao et al, 1996b;Ritzel et al, 1997Ritzel et al, , 1998Ritzel et al, , 2001Mackey et al, 1999;unpublished observation). hCNT1 and rCNT1, for instance, transport the antiviral drugs 3 -azido-3 -deoxythymidine (AZT) and 2 3 -dideoxycytidine (ddC), but not 2 3 -dideoxyinosine (ddI).…”

Kinetic study of anti-viral ribavirin uptake mediated by hCNT3 and hENT1 in Xenopus laevis oocytes

“…As the concentration of sodium ions is much lower inside the cell than outside, CNTs can function as active transporters according to the sodium concentration gradient. So far, three subtypes of CNTs have been identified in humans: hCNT1, which is pyrimidine nucleoside-selective; hCNT2, which is purine nucleoside-selective; and hCNT3, which is broadly selective for both pyrimidine and purine nucleosides [7,8,9,10]. Generally, CNTs have a higher affinity for nucleosides than that of equilibrative nucleoside transporters (ENTs) [5].…”