Aoun Joydeep, Hayashi Mikio, Sheikh Irshad Ali, Sarkar Paramita, Saha Tultul, Ghosh Priyanka, Bhowmick Rajsekhar, Ghosh Dipanjan, Chatterjee Tanaya, Chakrabarti Pinak, Chakrabarti Manoj K, Hoque Kazi Mirajul
From the Molecular Pathophysiology Division, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme-XM, Beliaghata, Kolkata 700010, India.
the Department of Physiology, Kansai Medical University, 5-1, Shimmachi 2, Hirakata, 573 1010 Osaka, Japan.
J Biol Chem. 2016 Dec 23;291(52):26816-26836. doi: 10.1074/jbc.M116.719823. Epub 2016 Oct 31.
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 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-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 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 [Ca] rise in Caco-2 cells, but cellular abundance of phosphatidylinositol 4,5-bisphosphate (PIP) increased. Identification of the PIP-binding motif at the N-terminal sequence among human and mouse ANO6 variants along with binding of PIP directly to ANO6 in HEK293 cells indicate likely PIP regulation of ANO6. The biophysical and pharmacological properties of Ace stimulated Cl current along with intestinal fluid accumulation, and binding of PIP to the proximal KR motif of channel proteins, whose mutagenesis correlates with altered binding of PIP, 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-PIP signaling.
霍乱弧菌的辅助霍乱肠毒素（Ace）已被证明与腹泻有关。然而，Ace激活的氯离子通道的信号传导机制和具体类型仍不清楚。我们在此表明，重组Ace蛋白诱导顶端质膜电流，该电流被经典的钙激活氯离子通道（CaCC）阻滞剂抑制。令人惊讶的是，Ace引发的电流上升既不受ANO1（TMEM16A）特异性抑制剂T16A-AO1（TAO1）的影响，也不受囊性纤维化跨膜电导调节因子（CFTR）阻滞剂CFTR inh-172的影响。Ace刺激了Caco-2细胞中的全细胞电流。然而，顶端电流通过敲低ANO6（TMEM16F）而减弱。在Caco-2细胞中重新表达ANO6可恢复这种受损的表型。在瞬时表达小鼠ANO1-mCherry或ANO6-GFP的HEK293细胞中对ANO电流进行的全细胞膜片钳记录证实Ace诱导了氯离子分泌。应用Ace产生了ANO6电流，但没有产生ANO1电流。Ace无法在Caco-2细胞中诱导钙离子升高，但磷脂酰肌醇4,5-二磷酸（PIP）的细胞丰度增加。在人和小鼠ANO6变体的N端序列中鉴定出PIP结合基序，以及PIP在HEK293细胞中直接与ANO6结合，表明ANO6可能受PIP调节。Ace刺激的氯离子电流的生物物理和药理学特性以及肠液积聚，以及PIP与通道蛋白近端KR基序的结合，其诱变与PIP结合改变相关，与ANO6刺激相当。我们得出结论，ANO6主要在肠道上皮细胞中表达，在那里它通过RhoA-ROCK-PIP信号的钙非依赖性机制，由Ace刺激导致分泌性腹泻。
