Cysteine Scanning Mutagenesis on Transmembrane Domain 1 of the Liver‐Specific Organic Anion Transporting Polypeptide 1B3

Abstract

The liver-specific organic anion transporting polypeptide 1B3 (OATP1B3) is a multispecific transporter that mediates the uptake of a broad range of drugs and other xenobiotics into hepatocytes. To identify regions in OATP1B3 important for substrate binding and/or translocation, we performed cysteine scanning mutagenesis. Twenty-two amino acids in transmembrane domain 1 (TM1) and extracellular loop 1 (EL1) were individually mutated to cysteines, and mutant proteins were transiently expressed in HEK293 cells. To determine the effect of each mutation on protein function, we measured uptake of estrone-3-sulfate and estradiol-17β-glucuronide. Transport was significantly reduced in several mutants after correcting for surface expression, indicating that this stretch of amino acids may be important for protein function. Functional cysteine mutants were then incubated with the cysteine-modifying reagents MTSES or MTSET prior to measuring uptake. MTS pre-incubation strongly inhibited the function of those mutants which could be labeled with biotinylated maleimide. Preincubating cells with estradiol-17β-glucuronide prior to labeling with biotinylated maleimide reduced the extent to which several mutants were labeled. These data suggest that TM1 and EL1 in OATP1B3 play a significant role in substrate binding and/or translocation. Supported by NIH grants RR021940, GM077336.