Browsing by Subject "ABCG2"

The ABCG2-protein is an ATP-dependent half transporter. It is found on apical membranes in intestine, liver, kidney, blood-brain barrier and placenta where it regulates absorption, distribution and elimination of many drugs, but also natural compounds and endogenous metabolites. Natural variation found on the ABCG2-gene can alter protein expression and transport activity. The altered function has been linked to pharmacokinetic changes and developing of diseases like gout. Studying natural ABCG2-variants and their effect gathers knowledge not only on their effect on pharmacokinetics but also on the ABCG2- transporters’ mechanism of function. The aim of this study was to combine an activating (I456V or H457R) and an inactivating (Q141K, F431L or T542A) non-synonymous single nucleotide variant in the same gene to study their combined effect on the ABCG2-transporter expression and active transport. Mutations were incorporated into the ABCG2- gene by site directed mutagenesis and the protein was expressed on HEK293-cells. The transport activity for Lucifer-Yellow and estrone sulfate was measured using HEK293-ABCG2-vesicles produced from cell membranes. The protein expression was measured with Western blot and mass spectrometry proteomics. Based on this study, different mutations together can alter each other’s effects, but the combined result is not always equal to the sum of variations. T542A-mutation did not show significant increase on the protein expression on any of the T542A-combinations, even though it has had such an effect in earlier studies. I456V, earlier expressed like wild type ABCG2, seemed to increase protein expression in all combinations. Q141K, F431L and T542A -mutations had lowering not expression dependent effect on the transport activity. F431L-mutation being so dominant that either of the two activating mutations could not restore the active transport in combinations. As seen before, H457R-variant seemed to cause a significant substrate specific activating effect on transport activity also in this study when combined with other mutations. However, H457R had a strong lowering effect on the protein expression and two of the combinations did not produce enough protein for active transport. As seen in this study, the ABCG2-doublemutations can cause altered ABCG2-function and lead to pharmacokinetic changes. These types of in vitro studies are important in studying these less common genetic variants which in lack of study subjects can be hard to study on clinical trials.