Browsing by Author "Suotunen, Pauliina"

The OATP1B3 belongs to the organic anion transporting polypeptides (OATPs) encoded by the SLCO (solute carrier organic anion) genes which belongs to the SLC (solute carriers) gene superfamily. It is an influx transporter which is primarily expressed on the basolateral membrane of the hepatocytes. It transports many endogenous substrates as well as clinically important drugs such as thyroid hormones and statins into hepatocytes and thus participates in the first step of hepatic metabolism. Single nucleotide polymorphisms (SNPs) of the SLCO1B3 gene can affect the pharmacokinetics and pharmacodynamics of its substrates. The aim of this study was to set up and optimize an in vitro method to study the function and expression of the OATP1B3 transporter and its genetic variants. SNPs 334T> G (Ser112Ala), 699G> A (Met233Ile) and 767G> C (Gly256Ala) and stop codon TAA were introduced into the SLCO1B3 gene by site-directed mutagenesis. Recombinant baculovirus vectors containing the genetic information of OATP1B3 and its variants were used to transiently transfect the HEK293 cells. After optimizing the substrate incubation time and concentration, as well as the viral load and selecting the fluorescent substrate (8-FcA), the uptake assay was used to determine the transport activity of the OATP1B3 variants in HEK293 cells. The transport activity of the artificial WTP variant was also investigated in this study. The transport activities of the Ser112Ala, Met233Ile and Gly256Ala variants did not change significantly from the wild type although the transport activity of the Met233Ile variant appears to be slightly impaired. In turn the WTP variant was unable to transport 8-FcA. Based on this study the function of OATP1B3 variants can be studied using recombinant baculovirus to transiently transfect the HEK293 cells. 8-FcA can be used as a probe substrate in these studies. The results of this study confirm previous knowledge of the functioning of Ser112Ala, Met233Ile and Gly256Ala variants. More studies are needed about the effects of these variants on the transport of OATP1B3 drug substrates. Also studies about the location, cell membrane and total cell expression of the WTP variant are needed to evaluate reliably the reasons of its inactivity.