Na⁺-dependent purine nucleoside transporter from human kidney: cloning and functional characterization

Abstract

Many purine nucleosides and their analogs are actively transported in the kidney. Using homology cloning strategies and reverse transcriptase-polymerase chain reactions, we isolated a cDNA encoding a Na+-dependent nucleoside transporter, hSPNT1, from human kidney. Functional expression in Xenopus laevis oocytes identified hSPNT1 as a Na+-dependent nucleoside transporter that selectively transports purine nucleosides but also transports uridine. The Michaelis constant ( K m) of uridine (80 μM) in interacting with hSPNT1 was substantially higher than that of inosine (4.5 μM). hSPNT1 (658 amino acids) is 81% identical to the previously cloned rat Na+-nucleoside transporter, SPNT, but differs markedly from SPNT in terms of its primary structure in the NH2 terminus. In addition, an Alu repetitive element (∼282 bp) is present in the 3′-untranslated region of the hSPNT1 cDNA. Northern analysis revealed that multiple transcripts of hSPNT1 are widely distributed in human tissues including human kidney. In contrast, rat SPNT transcripts are absent in kidney and highly localized to liver and intestine. The hSPNT1 gene was localized to chromosome 15. This is the first demonstration of a purine nucleoside transporter in human kidney.

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; …”

“…CaCNT-producing oocytes transported all purine nucleosides tested (adenosine, inosine, guanosine). Although uridine was also a good CaCNT permeant, no significant transport was detected for other pyrimidine nucleosides (cytidine, thymidine), suggesting a cif -like transport profile similar to that of hCNT2 and rCNT2 (Che et al, 1995;Yao et al, 1996a;Wang et al, 1997;Ritzel et al, 1998). The purine nucleobase hypoxanthine was also not transported, suggesting that CaCNT functions exclusively as a nucleoside transport protein.…”

“…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).…”

Nucleoside anticancer drugs: the role of nucleoside transporters in resistance to cancer chemotherapy

“…hCNT1 is predicted to have 13 transmembrane domains. Human cDNAs encoding hCNT2 (also termed hSPNT1), which has cif activity, were isolated from the kidney and intestine (Wang et al, 1997;Ritzel et al, 1998) by hybridization/RT-PCR cloning. The human homologs were almost identical (658 amino acids), with the exception of a single amino-acid substitution at position 75, which is arginine in hCNT2 (Ritzel et al, 1998) and serine in hCNT2/hSPNT1 (Wang et al, 1997).…”

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].…”