The Effect Of Two-Pore Domain Acid-Sensitive Potassium Channel TASK3 In Neuronal Injury Under High Extracellar Potassium Stress And The Construction And Expression Of One-Time Display TASK3 In Cells

In the early stage of acute hypoxia ischemia,the membrane channel function and membrane potential of neuronal changes,accompanying the efflux of K+and in tracellular Na+and Ca2+increase,and eventually leads to cell death.Acid-sensitive Potassium Channel-3(TASK3)belongs to the surperfamily of the two-pore K+ channels.At present,the research on the function of TASK3 is the most widely and most controversial.TASK3 channels have been proposed to mediate neuronal apopto sis.TASK3 channels functional status determines the resting potential of neuronal c ell,and controlsthedischarge frequency of neurons.However,there are few reports a bout the physiological function of TASK3,which is the way to exert its physiologi cal function in neurons hypoxia injury.The aim of this study was to investigate th e effect of TASK3 channel activity on the activity of neurons induced by high pota ssium,and to investigate the mechanism of TASK3 channel activity in the regulatio n of pathway activity in the case of high potassium injury.And this study focused o n constructing prokaryotic expression vector of TASK3,the expression of the recombinatio n protein TASK3-TAT.With the ability of Tat transduction peptide,The transient expr ession of TASK3 protein was constructed,which provides new technical means and scientific ideas for the further reserach on the function of TASK3 channel on neuro protection under ischemic injury.PART I Effect of TASK3 on SH-SY5 Y under high concentration potassium injuryObjectives To explore the effect of TASK3 channel on neuroprotection under high extracellular K+.Methods SH-SY5 Y cell was incubated in medium with different concentration K+for 6h,then the cell morphological changes were observed by inverted microscope and the viability of cells was detected by CCK-8.Cells were kept in a depolarizing medium with different concentration of TASK3 channel modulators,then the effect of TASK3 channel on cell proliferation was observed by CCK-8.The expression of pro-apotosis protein was confirmed by RT-PCR and Western Blotting.Results After cells were treated in a medium with extracellular high K+,the effect of extracellular potassium on cell vitality was in a dose-dependent and time-dependent manner.Adding different concentration of spermine TASK3 inhibitors(50?M,100 ?M,500 ?M,1000 ?M)can significantly decrease the SH SY5 Y cell vitality.And adding different concentration of TASK3 agonist isoflurane(0.4 m M,0.8 m M and 1.6 m M,8m M)can significantly enhance the cell vitality.Bax was down-regulated and Bcl-2/Bax was up-regulated in a depolarizing medium with isoflurane-TASK3 agonist,while Bax was up-regulated and Bcl-2/Bax was down-regulated in a depolarizing medium with spermine-TASK3 inhibitor.Conclusions The neuroprotection effect of TASK3 channel was achieved by regulating to the expression of pro-apoptosis protein under high concentration potassium injury.PART II Construction and identification of a recombinant protein with TASK3Objectives This study focused on constructing prokaryotic expression vector of TASK3 gene,the expression of the recombination protein TASK3,and transfection the protein into SH-SY5 Y cell line,aimed to achieve the transient expression of the recombination protein TASK3 in SH-SY5 Y cell line.Methods The TASK3-GFP-Tat gene was inserted into plasmid pET30.The expression of fusion protein was confirmed by Western Blot.Results Successfully constructed the prokaryotic expression recombination p ET30-TASK3-GFP-Tat.Here utilizing Tat fused to TASK3 to generate the fusion protein TASK3-GFP-Tat that was able to across cytomembrane.Conclusions The fusion protein TASK3 containing the Tat protein transduction domain could be efficiently transfect into SH-SY5 Y neuron cell successfully,which can be used for further research on biological functions of TASK3 subunits.