The Research On Interaction Between Expression Of Acid-sensitive Ion Channel La And PolyQ-expanded Ataxin-3

Objective:In order to provide new clues to the pathogenesis and clinical treatment research on SCA3/MJD, this study is intended to clarify whether acid-sensitive ion channel1a and ataxin-3could influence the expression level of each other in SCA3/MJD cell model.Methodes:1.pEGFP-N1-ataxin-3-20Q and pEGFP-N1-ataxin-3-70Q were transfected into HEK293cells in order to construct the cell models expressing wide type or polyQ-expanded ataxin-3.2. The pH of the cell culture medium was titrated to DMEM medium(pH=7.4, abbreviated as DMEM medium), pH7.4buffer, pH6.0buffer, and then the expression level of ataxin-3was detected by Western blot in order to observe whether the pH level of the cell culture medium would affect the expression of ataxin-3.3. The ASIC1a inhibitor Amiloride hydrochloride (5-N, N-dimethyl amiloride, DMA) of four concentrations,0,50,100,150was put into the cell culture medium and then the expression level of ataxin-3was detected by Western blot in order to observe the impact of DMA on the expression level of ataxin-3.4. The vectors pEGFP-Nl-ataxin-3-20Q/70Q and pCMV-HA-ASIC1a were co-transfected into HEK293cells to construct the cell models expressing both wide type or polyQ-expanded ataxin-3and ASIC1a. In order to observe the influence of activated or inhibited ASIC1a on the expression level of ataxin-3, ASIC1a was activated by lowering the pH level of the cell culture medium, or inhibited by adding different concentrations of DMA to the cell culture medium, and then the expression level of ataxin-3was detected by Western blot.5. The vectors pEGFP-N1-ataxin-3-20Q/70Q and pCMV-HA-ASIC1a were co-transfected into HEK293cells. The expression of ASIC1a was detected by Western blot in order to observe whether polyQ-expanded ataxin-3can affect the expression level of ASIC1a or not.Results:1. There is no relationship between the expression of wild type ataxin-3and ASIC1a.2. The expression of ataxin-3-20Q or ataxin-3-70Q in cells expressing only ataxin-3protein (without ASIC1a expressing) showed no significant differences among groups of different pH values. Among different cell groups cultured in DMEM medium which was added different concentrations, both the expression level of ataxin-3-20Q and ataxin-3-70Q showed no significant differences.3. The expression level of ataxin-3-20Q in cells expressing both ataxin-3and ASIC1a were cultured in different pH mediums showed no significant differences among different groups, while the expression level of ataxin-3-70Q in group of pH6.0buffer was much higher than the other two groups(P<0.01). In the culture medium of pH6.0buffer, the expression level of ataxin-3-70Q showed no difference between group without DMA and50μM DMA group, while the expression level of ataxin-3-70Q showed significant difference between100μM DMA group or150μM DMA group and group without DMA(P<0.05and P<0.01)4. Cells expressing both ataxin-3and ASIC1a were cultured in the medium of DMEM, pH7.4buffer or pH6.0buffer. The expression level of ASIC1a in cells expressing ataxin-3-70Q was much higher than cells expressing ataxin-3-20Q (P＜0.05)Conclusions:1. There is no relationship between the expression of wild type ataxin-3and ASIC1a.2. Activated by acid environment, ASIC1a could significantly increase the expression level of polyQ-expanded ataxin-3.3. As one ASIC1a inhibitor, DMA could reverse the effects of activated ASIC1a on the expression level of polyQ-expanded ataxin-3.4. PolyQ-expanded ataxin-3could increase the expression level of ASIC1a.