Effects And Mechanism Study Of ABCB1 Nucleotide Polymorphism On The Transportation Of Antiepileptic Drugs By P-glycoprotein

Objective:The aim of this study is to investigate the effect of single nucleotide polymorphisms on the transtortation of anti-epiletic drugs by P-gp and its underlying mechanism at cellular and molecular levels.Materials and methods:In this project,we established a highly sensitive cell model containing ABCB1 polymorphic loci,and studied the effects of SNP loci 1236,2677,3435 of ABCB1 on P-gp transporting antiepileptic drugs and their molecular mechanisms by various means.(1)The following plasmplasmids were constructed at 1236,2677 and 3435 loci by dot mutation at 1236,2677 and 3435 sites:ABCB1-1236/2677/2677/3435-C/G/C,ABCB1-1236/1236/2677/2677/2677/2677/3435-T/G/C,ABCB1-1236/1236/2677/2677/3435-C/T/C,ABCB1-1236/1236/266/26776/26776/2677/3477/3435-35-C/C/G/G/T,AB1-1236/26776 and 3477 and 3435-T/T/T/C,AB1-1236/2677/2677 and 3435-T/343435-C/T/T,ABCB1-1236/2677/3435-T/T/T.(2)The constructed plasmid was successfully transfected into LLC-PK1 cells to obtain LLC-PK1 cells stably expressing ABCB1 polymorphism loci and to screen out cell lines containing the same protein expression.(3)The LC-PK1 monolayer cell model containing ABCB1 polymorphism was validated to obtain well-functioning and stable expression cell lines.(4)Single-layer cell model was used to detect the effect of SNP loci on the transport of different antiepileptic drugs by P-gp.(5)Flow cytometry was used to detect the effect of SNP sites on P-gp transport fluorescent substrates.(6)ATPase assay was used to detect the effect of ABCB1 SNP on the ATPase activity of P-gp in order to explore its possible molecular mechanism.Results:Through stable transfection,We obtained cell lines with different site mutations by stable transfection.Among them,ABCB1-1236/2677/3435-C/G/C obtained 11 monoclones,ABCB1-1236/2677/343 5-T/G/C 13 monoclones,ABCB1-1236/2677/3435-C/T/C 13 monoclones,ABCB1-1236/2677/3435-C/G/T 12 monoclones,ABCB1-1236/2677/3435-C/G/T 10 monoclones,ABCB1-1236/2677/3435-T/C 10 monoclones and ABCB1-1236/263/2635-T/C.Twelve monoclones were obtained from 435-T/G/T,13 from ABCB1-1236/2677/3435-C/T/T,11 from ABCB1-1236/2677/3435-T/G/T and 11 from ABCB1-1236/2677/3435-T/T.Through screening,16 cell lines with the same relative expression levels of protein and mRNA were obtained.Sixteen cell lines with similar expression levels were screened out from eight plasmid-transfected cell clones,namely ABCB1-1236/2677/3435-C/G(1),ABCB1-1236/2677/3435-C/G(4),ABCB 1-1236/2677/343 5-C/G/C(8),ABCB 1-1236/2677/3435-C/G/C(11),CB1-1236/2677/3435-T/C(11),CB1-1236/2677/343 5-C/T/C(11),and ABCB1-3477/C/C/C(4)respectively.)、ABCB1-1236/2677/3435-C/G/T(6)、ABCB1-1236/2677/3435-C/G/T(9)、ABCB1-1236/2677/3435-T/T/C(9)、ABCB1-1236/2677/3435-T/G/T(1)、ABCB1-1236/2677/3435-T/G/T(8)、ABCB1-1236/2677/3435-C/T/T(3)、ABCB1-1236/2677/3435-C/T/T(4)、ABCB1-1236/2677/3435-C/T/T(7)、ABCB1-1236/2677/3435-T/T/T(3)、ABCB1-1236/2677/343 5-T/T/T(4),ABCB1-1236/2677/3435-T/T/T(8).(1)In the single-layer cell model test of ABCB1 polymorphism gene transporting antiepileptic drugs,the function of P-gp(Con.difference at 180 min/protein level/mRNA level)was evaluated by the concentration difference between basal and top layers of the drug at 180 min,due to the different expression levels of P-gp in different cell lines.After calibrating the P-gp protein and RNA levels of each cell line,there was no significant difference in the ability of transporting antiepileptic drugs with different mutation sites(p=0.02),indicating that the mutations at 1236,2677 and 3435 loci did not affect the transport function of P-gp.This result is close to the trend of Drug efflux assay.(2)Drug efflux assay was used to detect the effect of cloned cells with different mutation sites on the transport of P-gp fluorescent substrate Rhodamine 123.The fluorescence of P-gp transported Rhodamine 123 in different cell lines was divided by the relative expression of the mRNA in each cell line.There was no significant difference in the transportability of Rhodamine 123 between cells with different mutation sites(p=0.02)after corrected by the level of P-gp mRNA.The results showed that the mutations at 1236,2677 and 3435 loci did not lead to the enhancement of P-gp transport function.(3)After extracting membrane proteins from different cell lines,the expression levels of membrane proteins in all cell lines were detected by Western Blotting.The standard curve of inorganic phosphorus(Pi)and fluorescence intensity was established,and the release of inorganic Pi was analyzed according to the fluorescence intensity.ATPase releases inorganic phosphorus per unit time/relative membrane protein expression.Different antiepileptic drugs were added to the membrane proteins of different cell lines.The content of inorganic phosphorus released by ATPase in unit time was determined.The expression of inorganic phosphorus was corrected by the expression of each cell mesangial protein.The results showed that mutations at 1236,2677 and 3435 sites stimulated by antiepileptic drugs did not affect the activity of ATPase of P-gp.Conclusions:In this study,we established a single-layer cell transport model with different mutation sites to elucidate the effect of single nucleotide polymorphism on P-glycoprotein transporting antiepileptic drugs at the cellular level.It was found that 1236,2677 and 3435 single nucleotide polymorphism sites did not affect the transport function of P-gp.At the same time,we found that ABCB1 single nucleotide polymorphism did not affect the binding ability of P-gp to antiepileptic drugs through ATPase activity detection,and further explored the effect of SNP on the function of P-gp at the molecular level.Our study will provide direct evidence for determining the association between ABCB1 polymorphism and drug-resistant epilepsy.It is of great significance to reveal the pathogenesis of drug-resistant epilepsy,predict the efficacy of antiepileptic drugs,and design new antiepileptic drugs and P-gp inhibitors.