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Am J Physiol Cell Physiol (February 20, 2002). doi:10.1152/ajpcell.00587.2001
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Articles in PresS, published online ahead of print February 20, 2002
Am J Physiol Cell Physiol, 10.1152/ajpcell.00587.2001
Submitted on December 10, 2001
Accepted on February 14, 2002

An ancient pre-vertebrate Na+/nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti)

Sylvia Y Yao1, Amy M Ng1, Shaun K Loewen1, Carol E Cass2, Stephen A Baldwin3, and James D Young1*

1 Physiology, University of Alberta, Edmonton, AB, Canada
2 Oncology, University of Alberta, Edmonton, AB, Canada
3 School of Biochemistry and Molecular Biology, University of Leeds, Leeds, United Kingdom

* To whom correspondence should be addressed. E-mail: james.young{at}ualberta.ca.

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 pre-vertebrate, 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 Km values of 10 - 45 µM. 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 carboxyl-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 fulfil important physiological functions.




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