title: An ancient prevertebrate Na+-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti).
abstract: The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1, hCNT2, and hCNT3 are pyrimidine nucleoside-selective (system cit), purine nucleoside-selective (system cif), or broadly selective for both pyrimidine and purine nucleosides (system cib), respectively. All have orthologs in other mammalian species and belong to a gene family (CNT) that has members in insects, nematodes, pathogenic yeast, and bacteria. Here, we report the cDNA cloning and functional characterization of a CNT family member from an ancient marine prevertebrate, the Pacific hagfish (Eptatretus stouti). This Na+-nucleoside symporter, designated hfCNT, is the first transport protein to be characterized in detail in hagfish and is a 683-amino acid residue protein with 13 predicted transmembrane helical segments (TMs). hfCNT was 52, 50, and 57% identical in sequence to hCNT1, hCNT2, and hCNT3, respectively. Similarity to hCNT3 was particularly marked in the TM 4-13 region. When produced in Xenopus oocytes, hfCNT exhibited the transport properties of system cib, with uridine, thymidine, and inosine apparent K(m) values of 10-45 microM. The antiviral nucleoside drugs 3'-azido-3'-deoxythymidine, 2',3'-dideoxycytidine, and 2',3'-dideoxyinosine were also transported. Simultaneous measurement of uridine-evoked currents and radiolabeled uridine uptake under voltage-clamp conditions gave a Na+-to-uridine coupling ratio of 2:1 (cf. 2:1 for hCNT3 and 1:1 for hCNT1/2). The apparent K50 value for Na+ activation was >100 mM. A 50:50 chimera between hfCNT and hCNT1 (TMs 7-13 of hfCNT replaced by those of hCNT1) exhibited hCNT1-like cation interactions, establishing that the structural determinants of cation stoichiometry and binding affinity were located within the carboxy-terminal half of the protein. The high degree of sequence similarity between hfCNT and hCNT3 may indicate functional constraints on the primary structure of the transporter and suggests that cib-type CNTs fulfill important physiological functions.
authors.affiliation: Department of Physiology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Sylvia Y
authors.initials: SY
authors.lastname: Yao
authors.forename: Amy M
authors.initials: AM
authors.lastname: Ng
authors.forename: Shaun K
authors.initials: SK
authors.lastname: Loewen
authors.forename: Carol E
authors.initials: CE
authors.lastname: Cass
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Baldwin
authors.forename: James D
authors.initials: JD
authors.lastname: Young
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D019380:Anti-HIV Agents / Q000493:pharmacokinetics; D002678:Chimera; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D016049:Didanosine / Q000493:pharmacokinetics; D006196:Hagfishes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009865:Oocytes; D020033:Protein Isoforms / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus; D016047:Zalcitabine / Q000493:pharmacokinetics; D015215:Zidovudine / Q000493:pharmacokinetics
chemical_list: D019380:Anti-HIV Agents; D018076:DNA, Complementary; D020033:Protein Isoforms; D011994:Recombinant Proteins; D027981:Symporters; C073034:sodium-nucleoside cotransporter; D015215:Zidovudine; D016047:Zalcitabine; D016049:Didanosine