Transmembrane domain 5 of the LdNT1.1 nucleoside transporter is an amphipathic helix that forms part of the nucleoside translocation pathway.

Transporters of the equilibrative nucleoside transporter (ENT) family promote the uptake of nucleosides, nucleobases, and a variety of therapeutic drugs in eukaryotes from protozoa to mammals. Despite its importance, the translocation pathway that mediates the internalization of these substrates has not been identified yet in any of the ENT carriers. Previous genetic studies on the LdNT1.1 nucleoside transporter from Leishmania donovani defined two amino acid residues in predicted transmembrane domains (TMD) 5 and 7 that may line this translocation pathway. The role of TMD5 in forming a portion of the aqueous channel was investigated using the substituted-cysteine accessibility method. A series of 22 cysteine substitution mutants spanning predicted TMD5 were created from a fully functional, cysteine-less, parental LdNT1.1. Cysteine replacement at six positions (M(176)C, T(186)C, S(187)C, Q(190)C, V(193)C, and K(194)C) produced permeases that were inhibited by incubation with sulfhydryl-specific methanethiosulfonate reagents, denoting their solvent accessibility to the translocation pathway. Adenosine was able to block this thiol modification, implying that access to the domain becomes restricted as a consequence of the substrate binding. Strikingly, the Q(190)C substitution interacted differentially with the substrates adenosine and uridine, suggesting that binding of adenosine but not uridine might directly occlude this position. When superimposed on a helical model, all six mutants clustered along one face of the amphipathic alpha-helix predicted for TMD5, strongly suggesting its involvement in the translocation pathway through LdNT1.1.

Functional expression and characterization of a purine nucleobase transporter gene from Leishmania major.

Leishmania major, like all the other kinetoplastid protozoa, are unable to synthesize purines and rely on purine nucleobase and nucleoside acquisition across the parasite plasma membrane by specific permeases. Although, several genes have been cloned that encode nucleoside transporters in Leishmania and Trypanosoma brucei, much less progress has been made on nucleobase transporters, especially at the molecular level. The studies reported here have cloned and expressed the first gene for a L. major nucleobase transporter, designated LmaNT3. The LmaNT3 permease shows 33% identity to L. donovani nucleoside transporter 1.1 (LdNT1.1) and is, thus, a member of the equilibrative nucleoside transporter (ENT) family. ENT family members identified to date are nucleoside transporters, some of which also transport one or several nucleobases. Functional expression studies in Xenopus laevis oocytes revealed that LmaNT3 mediates high levels of uptake of hypoxanthine, xanthine, adenine and guanine. Moreover, LmaNT3 is an high affinity transporter with K(m) values for hypoxanthine, xanthine, adenine and guanine of 16.5 +/- 1.5, 8.5 +/- 0.6, 8.5 +/- 1.1, and 8.8 +/- 4.0 microM, respectively. LmaNT3 is, thus, the first member of the ENT family identified in any organism that functions as a nucleobase rather than nucleoside or nucleoside/nucleobase transporter.