Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Pharmacology and Toxicology

Supervisor

Dr. James R. Hammond

Abstract

Human equilibrative nucleoside transporter 1 is the main mediator of bi-directional nucleoside flux and is found ubiquitously. Inhibitor and substrate interactions with ENT1 are known to be affected by cysteine-modifying reagents. Our aim was to investigate the importance of cysteine residues in hENT1 function and identify which residues were sensitive to thiol modification for further application of cysteine scanning mutagenesis on extracellular loop 5. Transporter function was assessed by the binding of [3H]NBMPR and the cellular uptake of [3H]2-chloroadenosine. Treatment of hENT1 with the neutral sulfhydryl-modifier methyl methanethiosulfonate (MMTS) enhanced [3H]NBMPR binding but decreased [3H]2-chloroadenosine uptake. The membrane impermeable positively charged reagent [2-(Trimethylammonium)ethyl] methane-thiosulfonate (MTSET) but not the negatively charged reagent sodium-(2-sulfonatoethyl)-methanethiosulfonate (MTSES), inhibited [3H]NBMPR binding and enhanced [3H]2-chloroadenosine uptake. Furthermore, all three sulfhydryl modifiers decreased [3H]NBMPR binding when allowed cytoplasmic access. Site-directed mutagenesis on Cys222 eliminated the effect of MMTS on NBMPR binding. Mutation of Cys378 abolished the effect of MTSET on NMBPR binding and indicated that Cys378 is an extracellular-located residue. Mutation of Cys414 led to an enhancement of the ability of MTSET to inhibit NBMPR binding and this effect was eliminated by co-mutation of Cys378. Mutation of Cys416 abolished the effect of charged sulfhydryl reagents to inhibit NBMPR binding in isolated membranes,

and also eliminated transport function supporting a conformational linkage between the fifth intracellular loop and the NBMPR binding domain, and implicates this region in the translocation function of hENT1. To further confirm the importance of this region, extracellular loop 5 (EL5) was examined by cysteine scanning mutagenesis as residues in EL5 were individually mutated to cysteines. Mutation of N379, F390, E391, H392, and D393 to cysteine abolished uptake of [3H]2-chloroadenosine indicating their role in the transport mechanism of hENT1. Treatment of EL5 mutants with MTSET inhibited NBMPR binding in all but the V389C mutant. Co-incubation of NBMPR with MTSET was able to protect N379C from thiol modification while co-incubation of adenosine with MTSET protected R384C, Y385C, and L386C from MTSET effects. Our results indicate that adenosine may bind in close vicinity or in direct contact to these residues to prevent MTSET to attain access.