Functional Characterization Reveals the Significance of Rare Coding Variations in Human Organic Anion Transporting Polypeptide 2B1 (SLCO2B1)

Abstract

The organic anion transporting polypeptide 2B1 (OATP2B1), which is encoded by the SLCO2B1 gene, plays important roles in the absorption and disposition of its substrate drugs. Nonsynonymous variations of SLCO2B1 change its amino acid sequence and may alter its function. However, so far, very few genetic variants of SLCO2B1 have been functionally characterized.In the present study, first of all, 14 nonsynonymous single nucleotide variants (SNVs) of SLCO2B1 have been identified from the dbSNP database. Then, human embryonic kidney (HEK293) cells were employed as the expression system and functional studies were carried out for these 14 SNVs using substrates 4′,5′-dibromofluorescein (DBF), estrone-3-sulfate (E3S), atorvastatin, and rosuvastatin. Our results showed that four nonsynonymous rare variants, namely, SLCO2B1 c.332G > A (p.R111Q), c.1184C > A (p.P395H), c.1624G > A (p.V542M), and c.1998C > A (p.F666L), have great effect on the function of OATP2B1. Surface biotinylation and immunoblot analysis indicated that the variant c.1184C > A (p.P395H) almost completely disrupted OATP2B1's expression on the plasma membrane. According to the three-dimensional structural model of OATP2B1 we developed, these four mutated residues are not located at the substrate binding region of OATP2B1. Their significant effect on the function of OATP2B1 could probably be attributed to jeopardizing OATP2B1's surface expression as exemplified by c.1184C > A (p.P395H), altering the transporter's overall structure and affecting its interactions with other proteins or the lipid bilayer. Taken together, our results demonstrated that rare coding variants could have a great impact on the function and expression of OATP2B1.

Organic Anion Transporting Polypeptide 2B1 (OATP2B1) Genetic Variants: In Vitro Functional Characterization and Association With Circulating Concentrations of Endogenous Substrates

“…Such a result would be in keeping with its low CADD/REVEL/MetaLR scores. However, our findings for the OATP2B1 c.935G>A variant contrast with others that find a reduction in transport function for some substrates ( Yang et al, 2011 ; Nies et al, 2013 ; Yang et al, 2020 ).…”

“…However, we found that the OATP2B1 c.1457C>T variant had reduced transport function in vitro which was similar to other studies (Nozawa et al, 2002;Nies et al, 2013). But in contrast, two other studies found increased activity of OATP2B1 c.1457C>T (Ho et al, 2006;Yang et al, 2020). Lastly, we found that the most common OATP2B1 variant, namely c.935G>A, had rather benign functional consequences for substrates, except for a very slight reduction in rosuvastatin transport activity.…”

“…HEK293 cells are routinely used in drug transporter studies as they are efficiently transfected and express relatively low basal expression of drug transporters ( Ahlin et al, 2009 ). Previous studies which examined the transport function of OATP2B1 genetic variants have used the HEK293 cell line for transporter overexpression ( Nozawa et al, 2002 ; Nies et al, 2013 ; Yang et al, 2020 ). HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Scientific, Grand Island, NY, United States) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 2 mM L-glutamine (Invitrogen), at 37°C, 5% CO 2 .…”

“…In vitro studies have similarly provided heterogeneous results for the transport activity of OATP2B1 genetic variants. The OATP2B1 c.935G>A variant has mostly been associated with reduced transport activity, but its functional impact appears to be highly substrate-and experimental model-dependent (Nozawa et al, 2002;Ho et al, 2006;Yang et al, 2011;Nies et al, 2013;Yang et al, 2020). With the OATP2B1 c.1457C>T variant, in vitro studies are also conflicting with some reporting reduced transport activity (Nozawa et al, 2002;Nies et al, 2013), while for others, there was enhanced function (Ho et al, 2006;Yang et al, 2020), again with substrate-dependent effects.…”

“…The OATP2B1 c.935G>A variant has mostly been associated with reduced transport activity, but its functional impact appears to be highly substrate-and experimental model-dependent (Nozawa et al, 2002;Ho et al, 2006;Yang et al, 2011;Nies et al, 2013;Yang et al, 2020). With the OATP2B1 c.1457C>T variant, in vitro studies are also conflicting with some reporting reduced transport activity (Nozawa et al, 2002;Nies et al, 2013), while for others, there was enhanced function (Ho et al, 2006;Yang et al, 2020), again with substrate-dependent effects. Taken together, because of all the divergent and inconsistent findings from clinical and biochemical studies, the potential impacts of SLCO2B1 genetic variation to transporter activity remains to be understood.…”