Role of Extracellular Histidine Residues for the Function and pH Sensitivity of Human Organic Anion Transporting Polypeptide 1B3

Organic anion transporting polypeptide 1B3 (OATP1B3) is a liver-specific transporter that mediates uptake of various substances from blood into hepatocytes. The transport function of OATP1B3 was shown to be pH-sensitive. As the protonation state of extracellular histidine residues can be affected by the environmental pH, in the present study, the role of 7 extracellular histidine residues in the function and pH sensitivity of OATP1B3 has been examined. Our results showed that H115 had the most significant effect on the function of OATP1B3. The Cryo-EM structure of OATP1B3 indicated that H115 is involved in the binding and release of bicarbonate during a transport cycle. Functional studies on H115 mutants suggested that a hydrogen-bond forming group was preferred over a positively charged group at site 115, indicating that a hydrogen bond is optimum for bicarbonate's binding/release cycle. This may also explain why OATP1B3 showed lower transport function at pH 4.5 than at pH 7.4, as H115 is positively charged at pH 4.5 but neutral at pH 7.4. In addition, the H115A mutation largely compromised the pH sensitivity of OATP1B3, probably due to the loss of its protonation state switching capability. Taken together, H115 plays an important role in the function and pH sensitivity of OATP1B3.