Mutation in Transmembrane Domain 8 of Human Urate Transporter 1 Disrupts Uric Acid Recognition and Transport

[Image: see text] Human urate transporter 1 (hURAT1) is the most pivotal therapeutic target for hyperuricemia. Due to a lack of crystal structure information, the atomic structure of URAT1 is not clearly understood. In this study, a multiple sequence alignment was performed, and K393, a positively charged residue in transmembrane domain (TMD) 8, was observed to be highly conserved in organic anion transporters (OATs). K393 was substituted with a positively, negatively, and neutrally charged amino acid via site-directed mutagenesis and then used to transfect HEK293 cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) analyses indicated that mutants of K393 showed mRNA and protein expression levels similar to those in the WT group. The nonpositively charged mutants K393A, K393D, and K393E eliminated 70–80% of (14)C-uric acid transport capacity, while the K393H mutant showed slight and the K393R mutant showed no reduced transport capacity compared with the WT group. Binding assays indicated that K393A, K393D, and K393E conferred lowered uric acid binding affinity. As indicated by the K(m) and V(max) values obtained from saturation kinetic experiments, K393A, K393D, and K393E showed increased K(m) values, but K393R and K393H showed K(m) values similar to those in the WT group. K393 also contributed to a high affinity for benzbromarone (BM) interaction. The inhibitory effects of BM were partly abolished in K393 mutants, with increased IC(50) values compared with the WT group. BM also exhibited weaker inhibitory effects on (14)C-uric acid binding in K393R and K393H mutants. In an outward homology model of URAT1, K393 was located in the inner part of the transport tunnel, and further molecular docking analysis indicated that uric acid and BM showed possible hydrogen bonds with K393. Mutants K393R and K393H showed possible interactions with uric acid, and positive charges confer high affinity for uric acid as revealed by their surface electrostatic potential. In conclusion, our data provide evidence that K393 is an important residue for the recognition of uric acid or inhibitors by URAT1.