Functional Characterization of Position 271 in NTCP, a Predicted Rheostat Location

Background Human Na + /taurocholate cotransporting polypeptide (NTCP) is a sodium dependent bile acid transporter expressed at the basolateral membrane of human hepatocytes. It is important for the enterohepatic circulation of bile acids, such as taurocholate (TCA), but it also transports other compounds including estrone‐3‐sulfate (E3S) and rosuvastatin. We previously identified a rheostat position at a naturally occurring polymorphism, S267F. That is, when other amino acids were substituted into this position, progressive changes in function were observed. Aim The purpose of the current study is to determine whether position N271, a predicted rheostat, is indeed another rheostat position in NTCP. Methods The crystal structures of two bacterial NTCP homologs were used to model the structure of human NTCP in the inward‐open and outward‐open conformations. Mutations were then introduced into the structural models and the energetic effect on protein stability was evaluated using the Rosetta software. Based on these calculations we predicted that N271 is a rheostat. Mutants for position N271 were generated using site‐directed mutagenesis and the resulting proteins were transiently expressed in HEK293 cells. The functions of the mutants were characterized by measuring uptake of radiolabeled model substrates: [ 3 H]‐taurocholate, [ 3 H]‐estrone‐3‐sulfate, and [ 3 H]‐rosuvastatin. Plasma membrane expression levels were determined by surface biotinylation assays. Results Initial transport studies using nanomolar concentrations of TCA, E3S, and rosuvastatin indicated a variety of intermediate functional outcomes for the mutants. Furthermore, this alteration in function was substrate dependent, which is consistent with our findings for S267. Some mutants, such as N271A, transport substrates similar to wild‐type NTCP, while others, like N271K, show variation depending on the substrate. N271P consistently transports all substrates minimally. In addition, there is no correlation between the initial uptake results and the Rosetta energies. Summary and Conclusions Initial transport studies demonstrate that position N271 is a rheostat position, as predicted by the alteration in Rosetta energy scores. However, there was no correlation between the calculated energy scores and the functional results. This suggests that functional outcomes of specific amino acid replacements at position N271 may arise from more complex conformational changes than were captured by the two modeled states. Additional experiments should help to determine to what extent the observed change in function is due to variations in the substrate affinity, the turnover number or the transport capacity of the mutants. Support or Funding Information P30 GM118247, GM077336, and W. M. Keck Foundation