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.

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

“…Prokaryotic CNTs lack the first three TMs of their eukaryotic counterparts, and functional expression of N-terminally truncated human and rat CNT1 in Xenopus oocytes has established that these three TMs are not required for Na ϩ -dependent uridine transport activity (18). Consistent with this finding, chimeric studies involving hCNT1 and hfCNT (14) and hCNT1 and hCNT3 (19) have demonstrated that residues involved in Na ϩ -and H ϩ -coupling reside in the C-terminal half of the protein. Present in this region of the transporter, but of unknown function, is a highly conserved (G/A)XKX 3 NEFVA(Y/M/F) motif common to all eukaryote and prokaryote CNTs.…”

“…Together, hCNT1-3 account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized functionally include hfCNT, a second member of the CNT3 subfamily from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (14), CeCNT3 from Caenorhabditis elegans (15), CaCNT from Candida albicans (16), and the bacterial nucleoside transporter NupC from Escherichia coli (17). hfCNT is Na ϩ -but not H ϩ -coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H ϩ -coupled.…”

“…hfCNT is Na ϩ -but not H ϩ -coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H ϩ -coupled. Na ϩ : nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT3 (11,14). H ϩ :nucleoside coupling ratios for hCNT3 and CaCNT are 1:1 (11,16).…”

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

“…AY235425) ( Figure 1A), contained 13 predicted transmembrane helices (TMs) and had a putative molecular weight of 67.7 kDa. CaCNT was 33% identical (44% similar) to hCNT1, 34% identical (46% similar) to hCNT2, 38% identical (49% similar) to hCNT3, and 26% identical (37% similar) to the bacterial H + /nucleoside transporter NupC, the latter protein having only 10 predicted TMs (corresponding to TMs 4-13 of the other CNTs) (Hamilton et al, 2001;Yao et al, 2002a). The 13-TM membrane architecture of CaCNT is also predicted for C. elegans CeCNT3 (Hamilton et al, 2001) and suggests that this membrane topology may be common to all eukaryotic CNTs.…”

“…Two ENT and three CNT functional isoforms have been identified. 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.…”