上海交通大学学报（医学版）

上海交通大学学报（医学版） ›› 2019, Vol. 39 ›› Issue (10): 1142-.doi: 10.3969/j.issn.1674-8115.2019.10.007

• 论著·基础研究 • 上一篇    下一篇

稳定表达大电导钙激活钾通道α亚基的细胞株构建及其排钾的分子机制研究

应思琦，张 娅，郭 琴，杨 阳，刘 爽，张 翀   

  1. 上海交通大学医学院附属新华医院肾脏科，上海200092
  • 出版日期:2019-10-28 发布日期:2019-11-22
  • 通讯作者: 张 翀，电子信箱：zhangchong@xinhuamed.com.cn。
  • 作者简介:应思琦（1993—），女，硕士生；电子信箱：zjsyysysq@sjtu.edu.cn。
  • 基金资助:
    国家自然科学基金面上项目（81770706，81570634）

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

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摘要： 目的·构建稳定表达大电导钙激活钾通道(large conductance Ca2+-activated K+ channel， MaxiK或BK）α亚基的HEK293细胞株，探讨BKα通道的排钾机制。方法·构建带Myc标签的BKα质粒，采用脂质体转染方法将BKα质粒转染至HEK293细胞系中，用药物G418筛选阳性单克隆细胞株，分别以Western blotting和细胞免疫荧光法检测BKα蛋白表达及定位。将稳定表达BKα蛋白的HEK293细胞系培养于玻片，形成单细胞层，分别给予5 mmol/L和100 mmol/L钾离子浓度的浴液， 膜片钳单通道记录BKα的离子流。内向整流性钾通道Kir4.1的野生型和突变型（G77R、G130R、C140R和R297C）分别转染稳定转染BKα的HEK293细胞后提取膜蛋白，Western blotting检测BKα的表达情况。结果·转染后的HEK293细胞中挑选表达BKα通道的细胞株，细胞免疫荧光验证了BKα通道表达及其在细胞膜上表达。在给予100 mmol/L钾离子浓度浴液的情况下，BKα的通道开放频率（NPo）快速增加。Kir4.1的野生型和突变型分别转染稳定转染BKα的HEK293细胞后提取膜蛋白，Western blotting检测发现转染Kir4.1突变体质粒的HEK293细胞的BKα表达较野生型增加（P0.05）。结论·成功地建立了稳定表达BKα的HEK293细胞株，BKα通道可以被高钾溶液激活；BKα通道的膜表达水平与Kir4.1通道功能状态有关，可能是由于突变的Kir4.1通道导致细胞去极化，从而激活BKα。

关键词: 大电导钙激活钾通道, 内向整流通道Kir4.1, 肾小管, 钾离子排泄

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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