Anoctamin 6 Contributes to Cl- Secretion in Accessory Cholera Enterotoxin (Ace)-stimulated Diarrhea AN ESSENTIAL ROLE FOR PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIP2) SIGNALING IN CHOLERA
Anoctamin 6 Contributes to Cl- Secretion in Accessory Cholera Enterotoxin (Ace)-stimulated Diarrhea AN ESSENTIAL ROLE FOR PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE (PIP2) SIGNALING IN CHOLERA
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DOI：
10.1074/jbc.m116.719823
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发表时间：
2016-12-23
影响因子：
4.8
通讯作者：
Hoque, Kazi Mirajul
中科院分区：
文献类型：
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作者：
Aoun, Joydeep;Hayashi, Mikio;Hoque, Kazi Mirajul
霍乱弧菌的副霍乱肠毒素（Ace）已被证明有助于腹泻。然而，Ace激活的Cl-通道的信号转导机制和具体类型尚不清楚.我们在这里已经表明，重组ACE蛋白诱导I-Cl顶端质膜，这是由经典的CaCC阻滞剂抑制。令人惊讶的是，ACE引起的电流升高既不受ANO 1（TMEM 16 A）特异性抑制剂T16 A（（inh））-A01（TAO 1）的影响，也不受囊性纤维化跨膜传导调节因子（CFTR）阻断剂CFTR inh-172的影响。Ace刺激Caco-2细胞中的全细胞电流。然而，顶端I-Cl通过敲低ANO 6（TMEM 16 F）而减弱。这种受损的表型通过在Caco-2细胞中重新表达ANO 6而恢复。瞬时表达小鼠ANO 1-mCherry或ANO 6-GFP的HEK 293细胞中ANO电流的全细胞膜片钳记录证实了Ace诱导Cl-分泌。Ace的应用产生ANO 6，但不产生ANO 1电流。Ace不能诱导Caco-2细胞中[Ca 2 +]（i）升高，但磷脂酰肌醇4，5-二磷酸（PIP 2）的细胞丰度增加。在人和小鼠ANO 6变体中N-末端序列处的PIP 2结合基序的鉴定以及PIP 2与HEK 293细胞中ANO 6的直接结合沿着表明ANO 6的可能的PIP 2调节。Ace刺激的Cl-电流沿着肠液积聚的生物物理和药理学性质以及PIP 2与通道蛋白的近端KR基序的结合（其诱变与PIP 2的改变的结合相关）与ANO 6刺激相当。我们的结论是，ANO 6主要表达在肠上皮细胞，在那里它有助于分泌性腹泻的ACE刺激的钙非依赖性机制的RhoA-ROCK-PIP 2信号。
Accessory cholera enterotoxin (Ace) of Vibrio cholerae has been shown to contribute to diarrhea. However, the signaling mechanism and specific type of Cl- channel activated by Ace are still unknown. We have shown here that the recombinant Ace protein induced I-Cl of apical plasma membrane, which was inhibited by classical CaCC blockers. Surprisingly, an Ace-elicited rise of current was neither affected by ANO1 (TMEM16A)specific inhibitor T16A((inh))-AO1(TAO1) nor by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker, CFTR inh-172. Ace stimulated whole-cell current in Caco-2 cells. However, the apical I-Cl was attenuated by knockdown of ANO6 (TMEM16F). This impaired phenotype was restored by re-expression of ANO6 in Caco-2 cells. Whole-cell patch clamp recordings of ANO currents in HEK293 cells transiently expressing mouse ANO1-mCherry or ANO6-GFP confirmed that Ace induced Cl- secretion. Application of Ace produced ANO6 but not the ANO1 currents. Ace was not able to induce a [Ca2+](i) rise in Caco-2 cells, but cellular abundance of phosphatidylinositol 4,5-bisphosphate (PIP2) increased. Identification of the PIP2-binding motif at the N-terminal sequence among human and mouse ANO6 variants along with binding of PIP2 directly to ANO6 in HEK293 cells indicate likely PIP2 regulation of ANO6. The biophysical and pharmacological properties of Ace stimulated Cl- current along with intestinal fluid accumulation, and binding of PIP2 to the proximal KR motif of channel proteins, whose mutagenesis correlates with altered binding of PIP2, is comparable with ANO6 stimulation. We conclude that ANO6 is predominantly expressed in intestinal epithelia, where it contributes secretory diarrhea by Ace stimulation in a calcium-independent mechanism of RhoA-ROCK-PIP2 signaling.