title: 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.
abstract: 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 IClof 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 IClwas 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+]irise 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 PIP2directly to ANO6 in HEK293 cells indicate likely PIP2regulation of ANO6. The biophysical and pharmacological properties of Ace stimulated Cl-current along with intestinal fluid accumulation, and binding of PIP2to 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-PIP2signaling.
authors: From the Molecular Pathophysiology Division, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme-XM, Beliaghata, Kolkata 700010, India.
authors: Joydeep
authors: J
authors: Aoun
authors: the Department of Physiology, Kansai Medical University, 5-1, Shimmachi 2, Hirakata, 573 1010 Osaka, Japan.
authors: Mikio
authors: M
authors: Hayashi
authors: From the Molecular Pathophysiology Division, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme-XM, Beliaghata, Kolkata 700010, India.
authors: Irshad Ali
authors: IA
authors: Sheikh
authors: From the Molecular Pathophysiology Division, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme-XM, Beliaghata, Kolkata 700010, India.
authors: Paramita
authors: P
authors: Sarkar
authors: From the Molecular Pathophysiology Division, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme-XM, Beliaghata, Kolkata 700010, India.
authors: Tultul
authors: T
authors: Saha
authors: From the Molecular Pathophysiology Division, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme-XM, Beliaghata, Kolkata 700010, India.
authors: Priyanka
authors: P
authors: Ghosh
authors: From the Molecular Pathophysiology Division, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme-XM, Beliaghata, Kolkata 700010, India.
authors: Rajsekhar
authors: R
authors: Bhowmick
authors: the Department of Biotechnology, College of Science and Technology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, India, and.
authors: Dipanjan
authors: D
authors: Ghosh
authors: the Department of Biochemistry, Bose Institute, P-1/12 CIT Road, Scheme-VIIM, Kolkata 700054, India.
authors: Tanaya
authors: T
authors: Chatterjee
authors: the Department of Biochemistry, Bose Institute, P-1/12 CIT Road, Scheme-VIIM, Kolkata 700054, India.
authors: Pinak
authors: P
authors: Chakrabarti
authors: From the Molecular Pathophysiology Division, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme-XM, Beliaghata, Kolkata 700010, India.
authors: Manoj K
authors: MK
authors: Chakrabarti
authors: From the Molecular Pathophysiology Division, National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme-XM, Beliaghata, Kolkata 700010, India, kmh_niced@yahoo.co.in.
authors: Kazi Mirajul
authors: KM
authors: Hoque
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000075368:Anoctamins; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D018938:Caco-2 Cells; D002118:Calcium / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D002771:Cholera / Q000139:chemically induced / Q000378:metabolism*; D002772:Cholera Toxin / Q000633:toxicity*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors; D057809:HEK293 Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018408:Patch-Clamp Techniques; D049294:Phospholipid Transfer Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000187:drug effects; D014734:Vibrio cholerae / Q000472:pathogenicity; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: C081530:Ace toxin; C555078:ANO6 protein, human; D000075368:Anoctamins; C505032:CFTR protein, human; D002712:Chlorides; D049294:Phospholipid Transfer Proteins; C066737:RHOA protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D002772:Cholera Toxin; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D020742:rhoA GTP-Binding Protein; D002118:Calcium
keywords: epithelial cell; epithelial cell adhesion molecule (EpCAM); intestinal epithelium; ion channel; membrane; pathogenesis; phosphatidylinositol; signaling