The Research On The High Throughout Screening Model Of ASIC1a Inhibitors

Mechanisms controlling ischemic stroke are very complicated,but studies on ASICs indicated that they played important roles in ischemic stroke.Therefore,ASICs are popular research subjects now.ASICs are ligand-gated cation channels activated by extracellular acidification.Recent progress suggests that Ca²⁺ overlode mediated by acid-sensitive channel ASIC1a is a critical factor in ischemic stroke.Thus,discovery of specific and high-activity ASIC1a inhibitors will be a key for ischemic stroke therapy.ASIC1a has low expression in the membrane of neurons,axons,or dendritic spine,but high in the cytoplasm.In the present study,we created a recombinant plasmid of pcDNA3.1/Zeocin-mASIC1a-myc-His and transfected it into diffirent cell lines of COS-7, 293-T,293-A,SMMC-7221,MMT,FRT,or CHO cells in order to detect ASIC1a distribution. FRT and CHO cells were found to be the most suitable ones for High Thoughout Screeing (HTS).It was reported that coexpression of p11 and ASIC1a led an increase in the expression of ASIC1a at the cell membrane.Therefore,we created a recombinant plasmid of pBudce4.1-mP11-mASIC1a in order to construct stable cell lines expressing both p11 and ASIC1a.The expression were confirmed by RT-PCR and immunofluorescence techniques.In addition,the suitability of HTS methods for detecting ASIC1a inhibitors were discussed in this paper.To screen the ASIC1a inhibitors by HTS,a calcium-sensitive fluorescent protein(cameleon-2),a calcium-sensitive fluorescent dye Fura-2/AM and cell toxicity test(LDH release assay) were used to detect ASIC1a functional expression.The results suggested that LDH release assay was optimum but stability and accuracy of LDH release assay still need to be improved.As I stated above,this system gives us a new sight for looking for inhibitors and activators of small molecule ASIC1a with specific,high-activity and less side effects.Furthermore,it will be benefit for research on mechanisms controlling ischemic stroke-induced neuronal injury and therapy.