Investigation Of The Genetic Polymorphisms Of Human OATP2B1

Organic anion transporting polypeptides(OATPs)belong to the superfamily of solute carriers(SLC),which are important membrane transporters in humans.OATPs are a family of multiple-specific transporters,mediating sodium-and ATP-independent membrane transport of numerous endogenous substances such as steroid hormones and bile acids and xenobiotics such as numerous drugs.Single nucleotide polymorphisms(SNPs)are variations in DNA sequences caused by mutations in a single nucleotide at the genomic level.They are the most common type of heritable variation in humans.OATP2B1 is highly expressed in the liver and intestine,which are important organs for drug absorption and metabolism.OATP2B1 gene mutation could lead to the alteration of its expression and function,which could in turn affect the intestinal absorption and hepatic uptake and thus the pharmacokinetic behavior of its substrate drugs.So far,relative few studies have been reported on the genetic polymorphisms of OATP2B1.Therefore,in the present study we selected OATP2B1 as our research object and investigated effect of genetic polymorphisms on the expression and function of OATP2B1.By searching the genetic databases,we selected 14 single nucleotide polymorphisms with altered amino acid sequences of OATP2B1,which were c.43C>T,c.332G>A,c.601G>A,c.853G>A,c.917G>A,c.935G>A,c.1184C>A,c.1457C>T(*3),c.1468A>G,c.1526G>A,c.1624G>A,c.1638C>A,c.1998C>A,and c.2077C>G.Using OATP2B1*1(WT)as a template(reference sequence),we successfully constructed these 14 mutants by site-directed mutagenesis,and investigated the effect of these polymorphisms on the expression and function of OATP2B1.The results showed that compared with wild type,c.332G>A,c.1184C>A,and c.1624G>A showed the greatest reduction of the transport functions for estrone-3-sulfate(E3S)and 4',5'-dibromofluorescein(DBF),while c.1184C>A,c.1624G>A,and c.1998C>A decreased the transport function for atorvastatin and rosuvastatin the most.Cell surface biotinylation and Western blot experiments showed that c.1184C>A and c.1624G>A had significant effect on protein surface expression and thus the decrease in their transport activity was mainly caused by the decrease in expression.c.332G>A and c.1998C>A had a slight effect on protein expression,and the decrease in their transport activity was related to the decrease in both protein expression and function.In addition,we selected mutants that had great effect on OATP2B1's function,namely c.332G>A,c.1624C>A,and c.1998C>A,for kinetic studies.In conclusion,our results indicated that genetic polymorphisms of OATP2B1 had significant effects on its expression and function,which in turn could affect the pharmacokinetics of its substrate drugs.