Characteristics of Kcnn4 channels in the apical membranes of an intestinal epithelial cell line
Characteristics of Kcnn4 channels in the apical membranes of an intestinal epithelial cell line
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DOI：
10.1152/ajpgi.00558.2010
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发表时间：
2011-11-01
影响因子：
4.5
通讯作者：
Wonderlin, William F.
中科院分区：
文献类型：
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作者：
Basalingappa, Kanthesh M.;Rajendran, Vazhaikkurichi M.;Wonderlin, William F.
Basalingappa KM、Rajendran VM、Wonderlin WF。肠上皮细胞系顶膜 Kcnn4 通道的特征。 Am J Physiol Gastrointest Liver Physiol 301：G905-G911，2011。首次发表于 2011 年 8 月 25 日； doi: 10.1152/ajpgi.00558.2010.-上皮细胞顶膜和基底外侧膜中的中导 K(+) (Kcnn4) 通道在肠和结肠中激动剂诱导的液体分泌中发挥重要作用。基底外侧 Kcnn4 通道已使用膜片钳方法在原位得到了很好的表征，但对顶膜中 Kcnn4 通道的原位研究受到了一层阻止密封形成的粘液的阻碍。在本研究中，我们使用膜片钳方法来表征 IEC-18 细胞（一种源自大鼠小肠的细胞系）顶膜中的 Kcnn4 通道。在渗透性支持物上生长的单层 IEC-18 细胞不含粘液，紧密连接能够选择性地进入顶膜。在由内而外的斑块中，用移液器溶液中存在的 iberiotoxin（一种 Kcnma1/大电导，Ca(2+)-激活的 K(+) 通道阻滞剂）和 apamin（一种 Kcnn1-3/小电导，Ca(2+)-激活的 K(+) 通道阻滞剂）观察到的 Ca(2+) 依赖性 K(+) 通道表现出 31 pS 的单通道电导，具有内向整流。 TRAM-34（一种 Kcnn4 阻断剂）可逆地阻断电流，IC(50) 为 8.7 +/- 2.0 mu M。当将 Kcnn4 通道的肽抑制剂 Charybdotoxin 添加到移液管溶液中时，未观察到通道。 TRAM-34 对顶膜斑块中 Kcnn4 通道的抑制作用低于基底外侧膜斑块中的 Kcnn4 通道，这与 Kcnn4c 和 Kcnn4b 亚型分别在顶膜和基底外侧膜中的优先表达一致。 RT-PCR 和蛋白质印迹分析证实了 IEC-18 细胞中两种亚型的表达。这是肠上皮细胞顶膜 Kcnn4 通道的首次表征。
Basalingappa KM, Rajendran VM, Wonderlin WF. Characteristics of Kcnn4 channels in the apical membranes of an intestinal epithelial cell line. Am J Physiol Gastrointest Liver Physiol 301: G905-G911, 2011. First published August 25, 2011; doi: 10.1152/ajpgi.00558.2010.-Intermediate-conductance K(+) (Kcnn4) channels in the apical and basolateral membranes of epithelial cells play important roles in agonist-induced fluid secretion in intestine and colon. Basolateral Kcnn4 channels have been well characterized in situ using patch-clamp methods, but the investigation of Kcnn4 channels in apical membranes in situ has been hampered by a layer of mucus that prevents seal formation. In the present study, we used patch-clamp methods to characterize Kcnn4 channels in the apical membrane of IEC-18 cells, a cell line derived from rat small intestine. A monolayer of IEC-18 cells grown on a permeable support is devoid of mucus, and tight junctions enable selective access to the apical membrane. In inside-out patches, Ca(2+)-dependent K(+) channels observed with iberiotoxin (a Kcnma1/large-conductance, Ca(2+)-activated K(+) channel blocker) and apamin (a Kcnn1-3/small-conductance, Ca(2+)-activated K(+) channel blocker) present in the pipette solution exhibited a single-channel conductance of 31 pS with inward rectification. The currents were reversibly blocked by TRAM-34 (a Kcnn4 blocker) with an IC(50) of 8.7 +/- 2.0 mu M. The channels were not observed when charybdotoxin, a peptide inhibitor of Kcnn4 channels, was added to the pipette solution. TRAM-34 was less potent in inhibiting Kcnn4 channels in patches from apical membranes than in patches from basolateral membranes, which was consistent with a preferential expression of Kcnn4c and Kcnn4b isoforms in apical and basolateral membranes, respectively. The expression of both isoforms in IEC-18 cells was confirmed by RT-PCR and Western blot analyses. This is the first characterization of Kcnn4 channels in the apical membrane of intestinal epithelial cells.