Effect Of Protein Palmitoylation In The Inhibition Of AMPA Receptor Transport By Aluminum

Objective:To study the effect of protein palmitoylation in the inhibition of?-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid?AMPA?receptor transport by aluminum.Methods:1.Rat adrenal pheochromocytoma cells?PC12 cells?were cultured and treated with different concentrations of Maltol aluminum?Al?mal?₃?for different times.CCK8 was used to detect the cell survival rate and determine the exposure time and dose.2.At the end of the exposure,membrane protein?M?and total protein?T?were extracted respectively.The expression levels of GluR1 and GluR2 of AMPA receptor subunits in membrane protein and total protein were detected by Western blot.The ratios of membrane protein to total protein?M/T?were used to measure the transport of AMPA receptor.3.The palmitoylation levels of GluR1 and GluR2 of AMPA receptor subunits were detected by Immunoprecipitation-Acyl-Biotin Exchange assay?IP-ABE?.4.Western-blot was used to detect the protein expression of zinc finger,DHHC-type containing 3?zDHHC3?and palmitoyl protein thioesterase 1?PPT1?.5.The gene level of miRNA-134 gene was detected by q-PCR.6.Statistical methods:One way ANOVA was used for the difference between groups;if the variance was the same,LSD method was used for comparison between the two groups;if the variance was not the same,Dunnet-t method was used for analysis;Pearson correlation analysis was used for correlation test.The test level is?=0.05 on both sides.Results:1.The results of CCK8 showed that the cell survival rate of PC12 cells treated with100,200,400?mol/L Al?mal?₃ for 24 hours was more than 80%,the exposure time was determined to be 24 hours,and the exposure concentration was control group?0?mol/L Al?mal?₃?,low dose group?100?mol/L Al?mal?₃?,medium dose group?200?mol/L Al?mal?₃?,high dose group?400?mol/L Al?mal?₃?.2.The ratio of membrane protein to total protein?M/T?showed that the M/T of GluR1 and GluR2 membrane protein to total protein of AMPA receptor subunits gradually decreased with the increase of Al?mal?₃ concentration,that is,the transport of AMPA receptor subunits GluR1 and GluR2 to the cell membrane decreased.The M/T of GluR1 in 200?mol/L Al?mal?₃ group was lower than that in the control group?P<0.05?,and in 400?mol/L Al?mal?₃ group was lower than that in the control group and100?mol/L Al?mal?₃ group?P<0.05?.The M/T of GluR2 in 100,200 and 400?mol/L Al?mal?₃ groups was lower than that in the control group.3.The results of IP-ABE showed that the palmitoylation levels of GluR1 and GluR2gradually decreased with the increase of Al?mal?₃ concentration.The palmitoylation level of GluR1 protein in 100,200 and 400?mol/L Al?mal?₃ groups was lower than that in the control group?P<0.05?.The palmitoylation level of GluR1 protein in 200 and 400?mol/L Al?mal?₃ group was lower than that in 100?mol/L Al?mal?₃ group?P<0.05?,and there was a difference between 400?mol/L Al?mal?₃ group and 200?mol/L Al?mal?₃ group?P<0.05?.The palmitoylation level of GluR2 protein in 100,200 and 400?mol/L Al?mal?₃groups was lower than that in the control group?P<0.05?.4.Western-blot showed that with the increase of Al?mal?₃ concentration,the expression level of zDHHC3 gradually decreased,and the expression level of PPT1gradually increased.Compared with the control group,the expression level of zDHHC3in 200 and 400?mol/L Al?mal?₃ group decreased?P<0.05?.Compared with the control group,the expression level of PPT1 in the three groups of 100,200 and 400?mol/L Al?mal?₃ increased?P<0.05?.The expression level of PPT1 in 400?mol/L Al?mal?₃ group was higher than that in 100?mol/L Al?mal?₃ group?P<0.05?.5.The results of q-PCR showed that the gene level of miRNA-134 gradually increased with the increase of Al?mal?₃ concentration.The miRNA-134 gene level of 100,200 and 400?mol/L Al?mal?₃ group was higher than that of the control group?P<0.05?;the miRNA-134 gene level of 200 and 400?mol/L Al?mal?₃ group was higher than that of100?mol/L Al?mal?₃ group?P<0.05?.6.Correlation analysis showed that there was a positive correlation between the palmitoylation and the transport of GluR1?P<0.05?,a negative correlation between expression of PPT1 and transport of GluR1 and GluR2?P<0.05?,and a negative correlation between the gene level of miRNA-134 and expression of zDHHC3?P<0.05?Conclusion:1.Aluminum can inhibit the transport of AMPA receptor,and the decrease of palmitoylation level of AMPA receptor may be one of its mechanisms.2.The lower espression of zDHHC3 and the higher expression of PPT1 may be one of the mechanism of aluminum inhibiting palmitoylation of AMPA receptor.3.The increase of miRNA-134 gene expression may be the upstream mechanism of the decrease of zDHHC3 expression induced by aluminum,but the specific regulatory mechanism needs to be further studied.