Single Amino Acid Mutations in the Na+/Taurocholate Cotransporting Polypeptide (NTCP) Result in Substrate‐Dependent Effects

Abstract

Nonsynonymous polymorphisms can significantly impact the function of a protein. However, so far, the outcome of such mutations at non‐conserved positions are difficult to predict. Previous work showed that some non‐conserved positions have an unexpected outcome when they are mutated: if multiple amino acids are substituted, the results show progressive alterations like a “rheostat”, rather than an “all‐or‐nothing” effect often seen for mutations at conserved positions (“toggle”). To begin to define rules for predicting mutation outcomes at rheostat positions, we used the Na+/Taurocholate Cotransporting Polypeptide (NTCP) as a model transmembrane transporter. NTCP is a sodium dependent bile acid transporter expressed at the basolateral membrane of human hepatocytes. NTCP is important for the enterohepatic circulation of bile acids and also mediates the transport of steroid hormones like estrone‐3‐sulfate (E3S) or drugs like rosuvastatin. We initially focused on the S267F polymorphism that decreased taurocholate transport, stimulated rosuvastatin uptake, and showed no effect on E3S transport. We replaced the serine with all other nineteen amino acids by site directed mutagenesis, transfected the cDNAs into human embryonic kidney cells (HEK293), and determined the uptake of tritiated substrates. In addition, we identified the conserved G102 and the non‐conserved Y146 positions using multiple sequences alignments and homology modeling based on the structures of two bacterial homologues. Both positions fall in mobile regions of NTCP. Interestingly, mutations at S267 resulted in a clear rheostat behavior for taurocholate uptake, with S267F showing only 15% of the function of wild‐type NTCP. Similarly, rheostat behavior was observed for E3S and rosuvastatin, but several mutants transported these substrates 2‐ to 10‐fold higher than wild‐type NTCP, demonstrating that the different amino acid replacements resulted in substrate‐dependent effects. As expected, the non‐conserved Y146 demonstrated rheostat behavior for all three substrates. However, the conserved G102 turned out to be a rheostat position for taurocholate transport but a toggle for the uptake of E3S and rosuvastatin. In summary, we have determined that rheostat positions exist in human NTCP. Furthermore, the functional consequences of single amino acid mutations at the three positions studied so far, varied depending on the substrate tested. Therefore, functional predictions of amino acid mutations seem to be more challenging and will require further experiments at additional positions to potentially define a unifying set of rules.Support or Funding InformationNIH R01 GM077336, P30 GM118247, W.M. Keck FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.