The Effect of Single Nucleotide Variations in the Transmembrane Domain of OATP1B1 on in vitro Functionality

Abstract

Purpose Organic Anion Transporting Polypeptide 1B1 (OATP1B1) mediates hepatic influx and clearance of many drugs, including statins. The SLCO1B1 gene is highly polymorphic and its function-impairing variants can predispose patients to adverse effects. The effects of rare genetic variants of SLCO1B1 are mainly unexplored. We examined the impact of eight naturally occurring rare variants and the well-known SLCO1B1 c.521C > T (V174A) variant on in vitro transport activity, cellular localization and abundance. Methods Transport of rosuvastatin and 2,7-dichlorofluorescein (DCF) in OATP1B1 expressing HEK293 cells was measured to assess changes in activity of the variants. Immunofluorescence and confocal microscopy determined the cellular localization of OATP1B1 and LC–MS/MS based quantitative targeted absolute proteomics analysis quantified the amount of OATP1B1 in crude membrane fractions. Results All studied variants, with the exception of P336R, reduced protein abundance to varying degree. V174A reduced protein abundance the most, over 90% compared to wild type. Transport function was lost in G76E, V174A, L193R and R580Q variants. R181C decreased activity significantly, while T345M and L543W retained most of wild type OATP1B1 activity. P336R showed increased activity and H575L decreased the transport of DCF significantly, but not of rosuvastatin. Decreased activity was interrelated with lower absolute protein abundance in the studied variants. Conclusions Transmembrane helices 2, 4 and 11 appear to be crucial for proper membrane localization and function of OATP1B1. Four of the studied variants were identified as loss-of-function variants and as such could make the individual harboring these variants susceptible to altered pharmacokinetics and adverse effects of substrate drugs.

Functional characterization of six SLCO1B1 (OATP1B1) variants observed in Finnish individuals with a psychotic disorder

“…The current gene-based prescribing guideline from the Clinical Pharmacogenetics Implementation Consortium (CPIC) states that individuals with decreased and poor function phenotypes of OATP1B1 should limit statin doses and those with poor function phenotypes should avoid simvastatin altogether 6 . Based on our in vitro studies, c.629G>T (p.G210G>V) could be categorized as a poor function phenotype, since the in vitro activity and abundance is comparable to the SLCO1B1 c.521T>C genotype 1,14 . However, homozygous carriers of a rare variant like c.629G>T (p.G210G>V) are very uncommon.…”

“…Plasmids carrying the SLCO1B1 variants were created as described by Kiander et al 14 . The reference SLCO1B1 gene used was Genebank™ accession number AJ132573.1 and the mutagenesis primers are described in Supplementary Table 1.…”

“…After denaturation and break-down of the tertiary structure of the proteins, the crude membrane preparations were digested first with 1/100 LysC endopeptidase and after that with 1/100 TPCK-treated trypsin. Previously used isotope-labeled peptide mixture (3 fmol/µg protein) served as an internal standard 14 . Quantification was conducted with 6495 QQQ MS with a 1290 HPLC system and AdvanceBio peptide Map Column, 2.7 µm, 2.1 x 250 mm (Agilent Technologies, Santa Clara, CA, USA) as described previously 14,30 .…”

“…Previously used isotope-labeled peptide mixture (3 fmol/µg protein) served as an internal standard 14 . Quantification was conducted with 6495 QQQ MS with a 1290 HPLC system and AdvanceBio peptide Map Column, 2.7 µm, 2.1 x 250 mm (Agilent Technologies, Santa Clara, CA, USA) as described previously 14,30 . The SNVs did not alter the amino acids in the analyzed peptide sequences.…”

“…Pharmacogenetic research has suffered from small sample sizes, and finding a patient group of sufficient size is often challenging when exploring rare genetic variants 12 . Rare variants of SLCO1B1 are mainly unreported although their effect on drug safety can be as consequential as the effect of common variants 13,14 . Interpretation of genetic variants from vast amounts of whole exome sequencing data remains a challenge and computational tools are needed to predict the functional impact of pharmacogenetic variants before the identified variants can be explored at a biological level 15,16,17 .…”