Cysteine Scanning Mutagenesis of Transmembrane Domain 10 in Organic Anion Transporting Polypeptide 1B1

نویسندگان

  • Shuichi Ohnishi
  • Amanda Hays
  • Bruno Hagenbuch
چکیده

Organic anion transporting polypeptide (OATP) 1B1 is an important drug transporter expressed in human hepatocytes. Previous studies have indicated that transmembrane (TM) domain 2, 6, 8, 9, and in particular 10 might be part of the substrate binding site/translocation pathway. To explore which amino acids in TM10 are important for substrate transport, we mutated 34 amino acids individually to cysteines, expressed them in HEK293 cells, and determined their surface expression. Transport activity of the two model substrates estrone-3-sulfate and estradiol-17β-glucuronide as well as of the drug substrate valsartan for selected mutants was measured. Except for F534C and F537C, all mutants were expressed at the plasma membrane of HEK293 cells. Mutants Q541C and A549C did not transport estradiol-17β-glucuronide and showed negligible estrone-3-sulfate transport. However, A549C showed normal valsartan transport. Pretreatment with the anionic and cell impermeable sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) affected the transport of each substrate differently. Pretreatment of L545C abolished estrone-3-sulfate uptake almost completely, while it stimulated estradiol-17β-glucuronide uptake. Further analyses revealed that mutant L545C in the absence of MTSES showed biphasic kinetics for estrone-3-sulfate that was converted to monophasic kinetics with a decreased apparent affinity, explaining the previously seen inhibition. In contrast, the apparent affinity for estradiol-17β-glucuronide was not changed by MTSES treatment, but the Vmax value was increased about 4-fold, explaining the previously seen stimulation. Maleimide labeling of L545C was affected by preincubation with estrone-3-sulfate but not with estradiol-17β-glucuronide. These results strongly suggest that L545C is part of the estrone-3-sulfate binding site/translocation pathway but is not directly involved in binding/translocation of estradiol-17β-glucuronide.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Frequencies of two functionally significant SNPs and their haplotypes of organic anion transporting polypeptide 1B1 SLCO1B1 gene in six ethnic groups of Pakistani population

Objective(s):Organic anion transporter polypeptide 1B1 (OATP1B1) encoded by solute carrier organic transporter 1B1 (SLCO1B1) gene; a transporter involved in the uptake of drugs and endogenous compounds is present in hepatocyte sinusoidal membrane. Aim of this study was to investigate the frequencies of functionally significant SNPs (388A>G and 521T>C) and their haplotypes in 6 ethnic groups of ...

متن کامل

The Signature Sequence Region of the Human Drug Transporter Organic Anion Transporting Polypeptide 1B1 Is Important for Protein Surface Expression

The organic anion transporting polypeptides (OATPs) encompass a family of membrane transport proteins responsible for the uptake of xenobiotic compounds. Human organic anion transporting polypeptide 1B1 (OATP1B1) mediates the uptake of clinically relevant compounds such as statins and chemotherapeutic agents into hepatocytes, playing an important role in drug delivery and detoxification. The OA...

متن کامل

Functional analysis of the extracellular cysteine residues in the human organic anion transporting polypeptide, OATP2B1.

Organic anion transporting polypeptide (OATP) superfamily member 2B1 (OATP2B1) mediates the uptake of steroid hormone precursors and selected drugs in the placenta, liver, mammary gland, brain, and intestine. This action is modulated by sulfhydryl reagents. Common to all OATPs is a large extracellular loop between transmembrane domains IX and X with 10 conserved cysteines. To elucidate the stru...

متن کامل

Conserved tryptophan residues within putative transmembrane domain 6 affect transport function of organic anion transporting polypeptide 1B1.

The organic anion-transporting polypeptides (OATPs, gene symbol SLCO) are a family of transporters that play important roles in the absorption, distribution, metabolism, and excretion of various drugs. Although substrate specificity of transporter proteins is under extensive study, the underlying mechanisms for substrate binding and/or recognition remain largely unknown. Transmembrane domain 6 ...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:

دوره 53  شماره 

صفحات  -

تاریخ انتشار 2014