Department of Gastroenterology, Hannover Medical School, 30625 Hannover, Germany.
Department of Pediatric Pneumonology, Allergology and Neonatology, Hannover Medical School, 30625 Hannover, Germany.
Int J Mol Sci. 2023 Sep 18;24(18):14214. doi: 10.3390/ijms241814214.
Studies in human colonic cell lines and murine intestine suggest the presence of a Ca-activated anion channel, presumably TMEM16a. Is there a potential for fluid secretion in patients with severe cystic fibrosis transmembrane conductance regulator () mutations by activating this alternative pathway? Two-dimensional nondifferentiated colonoid-myofibroblast cocultures resembling transit amplifying/progenitor (TA/PE) cells, as well as differentiated monolayer (DM) cultures resembling near-surface cells, were established from both healthy controls (HLs) and patients with severe functional defects in the gene (PwCF). F508del mutant and CFTR knockout (null) mice ileal and colonic mucosa was also studied. HL TA/PE monolayers displayed a robust short-circuit current response (ΔI) to UTP (100 µM), forskolin (Fsk, 10 µM) and carbachol (CCH, 100 µM), while ΔI was much smaller in differentiated monolayers. The selective TMEM16a inhibitor Ani9 (up to 30 µM) did not alter the response to luminal UTP, significantly decreased Fsk-induced ΔI, and significantly increased CCH-induced ΔI in HL TA/PE colonoid monolayers. The PwCF TA/PE and the PwCF differentiated monolayers displayed negligible agonist-induced ΔI, without a significant effect of Ani9. When TMEM16a was localized in intracellular structures, a staining in the apical membrane was not detected. TMEM16a is highly expressed in human colonoid monolayers resembling transit amplifying cells of the colonic cryptal neck zone, from both HL and PwCF. While it may play a role in modulating agonist-induced CFTR-mediated anion currents, it is not localized in the apical membrane, and it has no function as an apical anion channel in cystic fibrosis (CF) and healthy human colonic epithelium.
在人类结肠细胞系和鼠肠中进行的研究表明，存在一种钙激活的阴离子通道，推测为 TMEM16a。通过激活这种替代途径，是否有可能在严重囊性纤维化跨膜电导调节因子（CFTR）突变患者中引起液体分泌？从健康对照（HL）和严重 CFTR 基因功能缺陷患者（PwCF）中建立了二维未分化结肠类器官-肌成纤维细胞共培养物（类似于转运扩增/祖细胞（TA/PE）细胞）以及分化单层（DM）培养物（类似于近表面细胞）。还研究了 F508del 突变和 CFTR 敲除（null）小鼠的回肠和结肠黏膜。HL TA/PE 单层对 UTP（100 µM）、 forskolin（Fsk，10 µM）和卡巴胆碱（CCH，100 µM）表现出强大的短路电流反应（ΔI），而分化单层中的ΔI 要小得多。选择性 TMEM16a 抑制剂 Ani9（高达 30 µM）不会改变对腔 UTP 的反应，显著降低 Fsk 诱导的ΔI，并显著增加 HL TA/PE 结肠类器官单层中 CCH 诱导的ΔI。PwCF TA/PE 和 PwCF 分化单层显示出可忽略不计的激动剂诱导的ΔI，Ani9 没有明显作用。当 TMEM16a 定位于细胞内结构时，在顶膜中未检测到染色。TMEM16a 在 HL 和 PwCF 来源的类似于结肠隐窝颈部区域的 TA/PE 结肠类器官单层中高度表达。虽然它可能在调节激动剂诱导的 CFTR 介导的阴离子电流中发挥作用，但它不在顶膜中定位，并且在囊性纤维化（CF）和健康人结肠上皮中没有作为顶膜阴离子通道的功能。
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