| Objective.To construct stable cell lines expressing the Large Conductance Ca2+-Activated K+Channel（MaxiK or BK）α-subunit and to explore the mechanism of potassium excretion via BKαchannel.Methods.The BKαplasmid with Myc tag was constructed and transfected into HEK293 cell lines by lipofectamine 2000.The positive monoclonal cell lines were screened by G418,and the expression of BKαwas detected by Western blotting and the location of BKαby immunofluorescence.The stable cell lines expressing BKαprotein was cultured on slides to form a single cell layer,which was Perfused with different potassium ion concentrations of 5 mM and 100 mM,and the single channel patch clamp recorded the ion flux of BKα.Wild type and mutants of Kir4.1 were transfected into HEK293 cells stably expressing BKαby calcium phosphate transfection,and BKαion flux was recorded by single channel patch clamp.Wild type and mutants（G77R,G130R,C140R and R297C）of the inwardly rectifying potassium channel（Kir4.1）were transfected into HEK293 cells stably transfected with BKα,and then the membrane protein was extracted.The expression of BKαwas detected by Western blotting.Results.Stable cell lines expressing BKαchannel were selected from HEK293 cells after transfection and cellular immunofluorescence verified the expression of BKαchannel and its expression on the cell membrane.The channel open frequency（NPo）of BKαincreased rapidly when perfused with 100 mM potassium.After transfected with wild type or mutants of Kir4.1,the membrane expression of BKαin the stable cell lines showed significantly difference among these groups（p<0.05）.Conclusion.The HEK293 cell lines stably expressing BKαhave been successfully constructed;BKαis activited by high potassium;the Membrane expression of BKαchannel is related to the activity of Kir4.1channel,which may be attributed to the depolarization of the cells by transfected with Kir4.1 mutants. |
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