Conserved tryptophan residues within putative transmembrane domain 6 affect transport function of organic anion transporting polypeptide 1B1.

The organic anion-transporting polypeptides (OATPs, gene symbol SLCO) are a family of transporters that play important roles in the absorption, distribution, metabolism, and excretion of various drugs. Although substrate specificity of transporter proteins is under extensive study, the underlying mechanisms for substrate binding and/or recognition remain largely unknown. Transmembrane domain 6 (TM6) is a relatively conserved region within OATP family members, and several amino acid residues on its extracellular half are part of the OATP family signature sequence D-X-RW-(I,V)-GAWWX-G-(F,L)-L. In the present study, two adjacent tryptophan residues (Trp258 and Trp259) within TM6 were identified as critical amino acids for the transport function of OATP1B1. Kinetic studies showed that substitution of Trp258 with alanine resulted in monophasic kinetics for estrone-3-sulfate uptake, with a significantly higher Km value (Km = 12.0 ± 2.8 μM) than the high-affinity component of wild-type OATP1B1 (Km = 0.38 ± 0.06 μM). On the other hand, W259A retained the biphasic characteristic of the transporter. Km values of the high- and low-affinity components for estrone-3-sulfate of W259A are 1.93 ± 0.76 μM and 30.8 ± 4.4 μM, respectively. Further studies revealed that W258A retained transport function of another prototypic substrate, taurocholate, while W259A displayed a dramatically reduced uptake of the substrate and exhibited an 8-fold increase in the Km value compared with that of the wild-type and W258A. Our results suggest that Trp258 and Trp259 may play different roles in the uptake of different substrates by OATP1B1.