›› 2019, Vol. 39 ›› Issue (10): 1142-.doi: 10.3969/j.issn.1674-8115.2019.10.007

• Original article (Basic research) • Previous Articles     Next Articles

Construction of scell lines expressing large conductance Ca2+-activated K+ channel α-subunit and molecular mechanism of potassium excretion in this channel

YING Si-qi, ZHANG Ya, GUO Qin, YANG Yang, LIU Shuang, ZHANG Chong   

  1. Department of Nephrology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
  • Online:2019-10-28 Published:2019-11-22
  • Supported by:
    National Natural Science Foundation of China,81770706,81570634

Abstract: Objective · To construct scell lines expressing the large conductance Ca2+-activated K+ channel (MaxiK or BK) α-subunit and to explore the mechanism of potassium excretion via BKα channel. Methods · The BKα plasmid with Myc tag was constructed and transfected into HEK293 cell lineslipofectamine 2000. The positive monoclonal cell lines were screenedG418, and the of BKα was detectedWestern blotting and the location of BKαimmunofluorescence. The scell lines expressing BKα protein was cultured on slides to form a single cell layer, which was perfused with different potassium ion concentrations of 5 mmol/L and 100 mmol/L, and the single channel patch clamp recorded the ion flux of BKα. 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 of BKα was detectedWestern blotting. Results · Scell lines expressing BKα channel were selected HEK293 cells after transfection and cellular immunofluorescence verified the of BKα channel and its on the cell membrane. The channel open frequency (NPo) of BKα increased rapidly when perfused with 100 mmol/L potassium. After being transfected with wild type or mutants of Kir4.1, the membrane of BKα in the scell lines showed significant difference among these groups (PConclusion · The HEK293 cell lines stably expressing BKα have been successfully constructed. BKα channel can be activatedhigh potassium solutions. The function of the BKα subunit can be related to Kir4.1 channel, which may be attributed to the depolarization of the cells transfectedKir4.1 mutants.

Key words: large conductance Ca2+-activated K+ channel, inwardly rectifying potassium channel Kir4.1, renal tubule, K+ excretion

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