Abstract
Organic cation transporter 1 (OCT1, SLC22A1) is localized in the sinusoidal membrane of human hepatocytes and mediates hepatic uptake of weakly basic or cationic drugs and endogenous compounds. Common amino acid substitutions in OCT1 were associated with altered pharmacokinetics and efficacy of drugs like sumatriptan and fenoterol. Recently, the common splice variant rs35854239 has also been suggested to affect OCT1 function. rs35854239 represents an 8 bp duplication of the donor splice site at the exon 7-intron 7 junction. Here we quantified the extent to which this duplication affects OCT1 splicing and, as a consequence, the expression and the function of OCT1. We used pyrosequencing and deep RNA-sequencing to quantify the effect of rs35854239 on splicing after minigene expression of this variant in HepG2 and Huh7 cells and directly in human liver samples. Further, we analyzed the effects of rs35854239 on OCT1 mRNA expression in total, localization and activity of the resulting OCT1 protein, and on the pharmacokinetics of sumatriptan and fenoterol. The 8 bp duplication caused alternative splicing in 38% (deep RNA-sequencing) to 52% (pyrosequencing) of the minigene transcripts when analyzed in HepG2 and Huh7 cells. The alternatively spliced transcript encodes for a truncated protein that after transient transfection in HEK293 cells was not localized in the plasma membrane and was not able to transport the OCT1 model substrate ASP+. In human liver, however, the alternatively spliced OCT1 transcript was detectable only at very low levels (0.3% in heterozygous and 0.6% in homozygous carriers of the 8 bp duplication, deep RNA-sequencing). The 8 bp duplication was associated with a significant reduction of OCT1 expression in the human liver, but explained only 9% of the general variability in OCT1 expression and was not associated with significant changes in the pharmacokinetics of sumatriptan and fenoterol. Therefore, the rs35854239 variant only partially changes splicing, causing moderate changes in OCT1 expression and may be of only limited therapeutic relevance.
Highlights
OCT1 (SLC22A1) is by far the most strongly expressed transporter of organic cations in the sinusoidal membrane of the human liver (Nies et al, 2009; Wang et al, 2015; Drozdzik et al, 2019)
Morphine, sumatriptan or fenoterol and endogenous compounds like thiamine belong to substrates transported by OCT1 (Wang et al, 2002; Tzvetkov et al, 2013; Chen et al, 2014; Matthaei et al, 2016; Tzvetkov et al, 2018)
OCT1 is encoded by the SLC22A1 gene, which is located on the long arm of human chromosome 6 (6q26) and contains 11 exons and 10 introns (Koehler et al, 1997; Zhang et al, 1997)
Summary
OCT1 (SLC22A1) is by far the most strongly expressed transporter of organic cations in the sinusoidal membrane of the human liver (Nies et al, 2009; Wang et al, 2015; Drozdzik et al, 2019). Morphine, sumatriptan or fenoterol and endogenous compounds like thiamine belong to substrates transported by OCT1 (Wang et al, 2002; Tzvetkov et al, 2013; Chen et al, 2014; Matthaei et al, 2016; Tzvetkov et al, 2018). A loss of OCT1 function was shown to increase plasma concentrations of several drugs including sumatriptan and fenoterol (Kerb et al, 2002; Shu et al, 2007; Tzvetkov et al, 2013; Arimany-Nardi et al, 2016; Matthaei et al, 2016; Tzvetkov et al, 2018). The resulting OCT1 protein has 554 amino acids and is composed of 12 transmembrane helices (TMHs) with intracellularly localized N- and C-termini
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