Effect of Mutations in the Substrate Binding Cavity of Human Organic Anion Transporters 1 and 3

Organic anion transporters (OAT) mediate the excretion of anionic xenobiotics and their metabolites in kidney and barrier tissues. Currently, an understanding of OAT structure, substrate interaction and translocation is limited to point mutations and transport kinetics. OAT structural models have been developed from the crystal structure of the glycerol‐3‐phosphate transporter from E.Coli, a protein that possesses sequence conservation and similar folding patterns with the OATs. Within this investigation, the structural models are employed to determine the substrate handling of two human OAT isoforms, hOAT1 and hOAT3, which possess sequence similarity (49%) but differ in substrate specificity. Docking of several OAT substrates (p‐aminohipurate, estrone sulfate, glutarate) indicates additional aromatic (hOAT1/hOAT3: Y230/Y218 and F438/F426) and basic (hOAT1/hOAT3: K431/K419) amino acids are involved in hydrogen bonding or pi‐pi stacking interactions. Mutating each residue to alanine decreased substrate docking affinities in the structural models. These results were validated in cell models expressing hOAT1 or hOAT3. Substrate uptake was reduced in all in vitro mutants when compared to wild‐type expression; however expression levels of some mutants were lower as shown by immunolocalization experiments. These preliminary data demonstrate that OAT structural models provide a mechanism to gain insight into substrate specificity. This research was supported by the Intramural Research Program of the NIH, NIEHS.