Date of Award

Spring 5-15-2015

Author's School

College of Arts & Sciences

Author's Department/Program

Biology

Degree Name

Bachelor of Arts

Abstract

Organic Cation Transporters (OCTs) are polyspecific, facilitative transporters that play major roles in metabolite and drug clearance. OCTs are promising drug targets and elucidating their mechanisms of substrate recognition is crucial for rational drug design. OCT-mediated transport of polyvalent cations remains unexplored. OCT-expressing Xenopus laevis oocytes were used to assess transport of polyamines, ubiquitous polyvalent cations of broad physiological import, but for which transport mechanisms are unknown. Dose-response analysis of radiolabelled substrate uptake revealed that polyamines are relatively low affinity, but high turnover substrates for OCTs compared to model substrate methyl-4-phenylpyridinium (MPP+). Polyamine analogs of varying hydrophobic character were screened for competition against MPP+, and hydrophobicity was demonstrated to be a principal requirement for polycationic substrate recognition, and OCT3 exhibits significantly higher hydrophobicity requirements than other isoforms. A hydrophobic cleft capable of accommodating a variety of structures has been identified by homology modelling of OCT1. In OCT3, replacement of a conserved residue within this pocket, D475, by charge reversal, neutralization, or replacement, abolishes MPP+ uptake, suggesting it to be obligatory for OCT3-mediated transport by stabilization of positive charges within the substrate binding pocket. Mutations at residues which line the binding pocket not conserved in OCT3 from OCT1 recapitulate the selectivity profile of OCT1. Interactions of polyamines and OCT1 blockers with wild-type OCT3 are weak, but are significantly potentiated in mutant OCT3. This suggests that substrate specificity in OCTs is determined at the putative hydrophobic cleft, and that residues identified above are key contributors to substrate affinity and/or sensitivity in OCTs.

Language

English (en)