Identification of Key Amino Acids that Impact Organic Solute Transporter α/β (OSTα/β)

Organic solute transporter α/β (OSTα/β) is a bidirectional bile acid transporter localized on the basolateral membrane of hepatic, intestinal, and renal epithelial cells. OST α / β plays a critical role in intestinal bile acid reabsorption and is upregulated in hepatic diseases characterized by elevated bile acids, whereas genetic variants in SLC51A / B have been associated with clinical cholestasis. OST α / β also transports and is inhibited by commonly used medications. However, there is currently no high-resolution structure of OST α / β , and structure-function data for OST α , the proposed substrate-binding subunit, are lacking. The present study addressed this knowledge gap and identified amino acids in OST α that are important for bile acid transport. This was accomplished using computational modeling and site-directed mutagenesis of the OST α subunit to generate OST α / β mutant cell lines. Out of the 10 OST α / β mutants investigated, four (S228K, T229S, Q269E, Q269K) exhibited decreased [ 3 H]-taurocholate (TCA) uptake (ratio of geometric means relative to OST α / β wild type (WT) of 0.76, 0.75, 0.79, and 0.13, respectively). Three OST α / β mutants (S228K, Q269K, E305A) had reduced [ 3 H]-TCA efflux % (ratio of geometric means relative to OST α / β WT of 0.86, 0.65, and 0.79, respectively). Additionally, several OST α / β mutants demonstrated altered expression and cellular localization when compared with OST α / β WT. In summary, we identified OST α residues (Ser228, Thr229, Gln269, Glu305) in predicted transmembrane domains that affect expression of OST α / β and may influence OST α / β -mediated bile acid transport. These data advance our understanding of OST α / β structure/function and can inform future studies designed to gain further insight into OST α / β structure or to identify additional OST α / β substrates and inhibitors. SIGNIFICANCE STATEMENT: OST α / β is a clinically important transporter involved in enterohepatic bile acid recycling with currently no high-resolution protein structure and limited structure-function data. This study identified four OST α amino acids (Ser228, Thr229, Gln269, Glu305) that affect expression of OST α / β and may influence OSTα/β-mediated bile acid transport. These data can be utilized to inform future investigation of OST α / β structure and refine molecular modeling approaches to facilitate the identification of substrates and/or inhibitors of OST α / β .