Comprehensive examination of charged intramembrane residues in a nucleoside transporter

Permeases of the equilibrative nucleoside transporter family mediate the uptake of nucleosides and/or nucleobases in a diverse array of eukaryotes and transport a host of drugs used for treatment of cancer, heart disease, AIDS, and parasitic infections. To identify residues that play central roles in transport function, we have systematically substituted by site-directed mutagenesis all the charged residues located within predicted transmembrane domains of the Leishmania donovani nucleoside transporter 1.1, LdNT1.1, which transports adenosine and the pyrimidine nucleosides. Substitution of three of these ten residues by uncharged amino acids resulted in loss of >95% transport activity, and we hence designated them "key" residues. These amino acids were Glu⁹⁴, Lys¹⁵³, and Arg⁴⁰⁴ located in transmembrane domains 2, 4, and 9, respectively. In addition, previous studies on the related LdNT2 inosine/guanosine transporter identified the highly conserved Asp³⁸⁹ and Arg³⁹³ (equivalent to Asp ³⁷⁴ and Arg³⁷⁸ in LdNT1.1) in transmembrane domain 8 as key residues. Among these residues, the mutants in Arg³⁹³ (LdNT2) and Arg⁴⁰⁴ were strongly impaired in trafficking to the plasma membrane, but the other mutants were expressed with high to moderate efficiency at the cell surface, indicating that their mutation impaired transport activity per se. A conservative K153R substitution exhibited a change in substrate specificity, acquiring the ability to transport inosine, a nucleoside that is not a substrate for the wild-type LdNT1.1 permease. These results imply that the Glu ⁹⁴, Lys¹⁵³, and Asp³⁷⁴ residues may play central roles in the mechanism of substrate translocation in LdNT1.1.