Identification of novel functional organic anion-transporting polypeptide 1B3 polymorphisms and assessment of substrate specificity

Ute I. Schwarz, Henriette E Meyer Zu Schwabedissen, Rommel G. Tirona, Atsuko Suzuki, Brenda F. Leake, Younes Mokrab, Kenji Mizuguchi, Richard H. Ho, Richard B. Kim

Research output: Contribution to journalArticle

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Abstract

Objective: The uptake carrier organic anion-transporting polypeptide 1B3 (OATP1B3, gene SLCO1B3) is involved in the hepatic clearance of xenobiotics including statins, taxanes, and mycophenolic acid. We thought to assess the SLCO1B3 coding region for yet unidentified polymorphisms and to analyze their functional relevance. Methods: We used DNA of ethnically diverse individuals for polymerase chain reaction, and determined polymorphisms by sequencing or temperature-dependent capillary electrophoresis. We then created variant OATP1B3 expression plasmids by site-directed mutagenesis, which were transiently expressed and functionally characterized in human cervical carcinoma (HeLa) cells using radiolabeled substrates. RESULTS: We identified six nonsynonymous polymorphisms including novel variants such as 439A>G (Thr147Ala), 767G>C (Gly256Ala), 1559A>C (His520Pro), and 1679T>C (Val560Ala). Allelic frequencies occurred to be ethnicity-dependent, with the latter observed only in African-Americans (3.6%). After expression in HeLa cells, His520Pro, Val560Ala, and Met233Ile or Met233Ile-Ser112Ala haplotype variants showed decreased uptake activity compared with wild type for cholecystokinin-8 and rosuvastatin, but not for atorvastatin. Kinetic cholecystokinin-8 analysis showed reduced Vmax without altering Km. His520Pro and Val560Ala exhibited decreased total and plasma membrane protein expressions. Val560 mapped onto a structural model of OATP1B3 showed that this is a key region for substrate-transporter interaction. His520 resides in a predicted extracellular region thought to be critical to the pH-dependent component of OATP1B3 activity. Loss of activity at pH 7.4 and 8.0 relative to pH 6.5 was significantly greater for the Pro520 variant. Conclusion: OATP1B3 polymorphisms that result in altered expression, substrate specificity, and pH-dependent activity may be of potential relevance to hepatic clearance of substrate drugs in vivo.

Original languageEnglish
Pages (from-to)103-114
Number of pages12
JournalPharmacogenetics and Genomics
Volume21
Issue number3
DOIs
Publication statusPublished - Mar 2011
Externally publishedYes

Fingerprint

Substrate Specificity
Anions
Peptides
HeLa Cells
Mycophenolic Acid
Hydroxymethylglutaryl-CoA Reductase Inhibitors
Taxoids
Liver
Structural Models
Capillary Electrophoresis
Xenobiotics
Site-Directed Mutagenesis
African Americans
Haplotypes
Blood Proteins
Membrane Proteins
Plasmids
Cell Membrane
Carcinoma
Polymerase Chain Reaction

Keywords

  • drug disposition
  • drug transporter
  • organic anion-transporting polypeptide 1B3 polymorphisms

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Molecular Medicine
  • Genetics(clinical)

Cite this

Identification of novel functional organic anion-transporting polypeptide 1B3 polymorphisms and assessment of substrate specificity. / Schwarz, Ute I.; Schwabedissen, Henriette E Meyer Zu; Tirona, Rommel G.; Suzuki, Atsuko; Leake, Brenda F.; Mokrab, Younes; Mizuguchi, Kenji; Ho, Richard H.; Kim, Richard B.

In: Pharmacogenetics and Genomics, Vol. 21, No. 3, 03.2011, p. 103-114.

Research output: Contribution to journalArticle

Schwarz, Ute I. ; Schwabedissen, Henriette E Meyer Zu ; Tirona, Rommel G. ; Suzuki, Atsuko ; Leake, Brenda F. ; Mokrab, Younes ; Mizuguchi, Kenji ; Ho, Richard H. ; Kim, Richard B. / Identification of novel functional organic anion-transporting polypeptide 1B3 polymorphisms and assessment of substrate specificity. In: Pharmacogenetics and Genomics. 2011 ; Vol. 21, No. 3. pp. 103-114.
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AU - Schwarz, Ute I.

AU - Schwabedissen, Henriette E Meyer Zu

AU - Tirona, Rommel G.

AU - Suzuki, Atsuko

AU - Leake, Brenda F.

AU - Mokrab, Younes

AU - Mizuguchi, Kenji

AU - Ho, Richard H.

AU - Kim, Richard B.

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N2 - Objective: The uptake carrier organic anion-transporting polypeptide 1B3 (OATP1B3, gene SLCO1B3) is involved in the hepatic clearance of xenobiotics including statins, taxanes, and mycophenolic acid. We thought to assess the SLCO1B3 coding region for yet unidentified polymorphisms and to analyze their functional relevance. Methods: We used DNA of ethnically diverse individuals for polymerase chain reaction, and determined polymorphisms by sequencing or temperature-dependent capillary electrophoresis. We then created variant OATP1B3 expression plasmids by site-directed mutagenesis, which were transiently expressed and functionally characterized in human cervical carcinoma (HeLa) cells using radiolabeled substrates. RESULTS: We identified six nonsynonymous polymorphisms including novel variants such as 439A>G (Thr147Ala), 767G>C (Gly256Ala), 1559A>C (His520Pro), and 1679T>C (Val560Ala). Allelic frequencies occurred to be ethnicity-dependent, with the latter observed only in African-Americans (3.6%). After expression in HeLa cells, His520Pro, Val560Ala, and Met233Ile or Met233Ile-Ser112Ala haplotype variants showed decreased uptake activity compared with wild type for cholecystokinin-8 and rosuvastatin, but not for atorvastatin. Kinetic cholecystokinin-8 analysis showed reduced Vmax without altering Km. His520Pro and Val560Ala exhibited decreased total and plasma membrane protein expressions. Val560 mapped onto a structural model of OATP1B3 showed that this is a key region for substrate-transporter interaction. His520 resides in a predicted extracellular region thought to be critical to the pH-dependent component of OATP1B3 activity. Loss of activity at pH 7.4 and 8.0 relative to pH 6.5 was significantly greater for the Pro520 variant. Conclusion: OATP1B3 polymorphisms that result in altered expression, substrate specificity, and pH-dependent activity may be of potential relevance to hepatic clearance of substrate drugs in vivo.

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