Grad Paper Feature of the Month:
Cysteine residues in the TM9-TM11 region of the human equilibrative nucleoside transporter
subtype 1 play an important role in inhibitor binding and translocation function
Jamie S. Park and James R. Hammond
Human equilibrative nucleoside transporter 1 (hENT1) is the main mediator of endogenous nucleoside and cytotoxic nucleoside analogue flux across cell membranes. Inhibitor and substrate interactions with equilibrative nucleoside transporter 1 (ENT1; SLC29A1) are known to be affected by cysteine-modifying reagents. By using site-directed mutagenesis, we were able to determine the role of cysteine residues involved in hENT1 function. These studies established that Cys378 is an extracellular-located residue modified by MTSET to inhibit the binding of NBMPR to intact cells. Mutation of Cys414 led to an enhancement of the ability of MTSET to inhibit NBMPR binding and this enhancement was eliminated by the co-mutation of Cys378, highlighting a potential structural linkage between TM9, TM10 and intracellular loop 5. Mutation of Cys416 led to the loss of the ability of the charged sulfhydryl reagents to inhibit NBMPR binding in isolated membranes, and also led to the loss of transport function. This finding further supports a conformational linkage between the fifth intracellular loop and the NBMPR binding domain, and implicates this region in the translocation function of hENT1.