Identification of Structural and Molecular Features Involved in the Transport of 3′-Deoxy-Nucleoside Analogs by Human Equilibrative Nucleoside Transporter 3

Combination antiretroviral drug treatments depend on 3'-deoxy-nucleoside analogs such as 3'-azido-3'-deoxythymidine (AZT) and 2'3'-dideoxyinosine (DDI). Despite being effective in inhibiting human immunodeficiency virus replication, these drugs produce a range of toxicities, including myopathy, pancreatitis, neuropathy, and lactic acidosis, that are generally considered as sequelae to mitochondrial damage. Although cell surface-localized nucleoside transporters, such as human equilibrative nucleoside transporter 2 (hENT2) and human concentrative nucleoside transporter 1 (hCNT1), are known to increase the carrier-mediated uptake of 3'-deoxy-nucleoside analogs into cells, another ubiquitously expressed intracellular nucleoside transporter (namely, hENT3) has been implicated in the mitochondrial transport of 3'-deoxy-nucleoside analogs. Using site-directed mutagenesis, generation of chimeric hENTs, and 3H-permeant flux measurements in mutant/chimeric RNA-injected Xenopus oocytes, here we identified the molecular determinants of hENT3 that dictate membrane translocation of 3'-deoxy-nucleoside analogs. Our findings demonstrated that whereas hENT1 had no significant transport activity toward 3'-deoxy-nucleoside analogs, hENT3 was capable of transporting 3'-deoxy-nucleoside analogs similar to hENT2. Transport analyses of hENT3-hENT1 chimeric constructs demonstrated that the N-terminal half of hENT3 is primarily responsible for the hENT3-3'-deoxy-nucleoside analog interaction. In addition, mutagenic studies identified that 225D and 231L in the N-terminal half of hENT3 partially contribute to the ability of hENT3 to transport AZT and DDI. The identification of the transporter segment and amino acid residues that are important in hENT3 transport of 3'-deoxy-nucleoside analogs may present a possible mechanism for overcoming the adverse toxicities associated with 3'-deoxy-nucleoside analog treatment and may guide rational development of novel nucleoside analogs.