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 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 K 50 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.