Anoctamin 6 Contributes to Cl- Secretion in Accessory Cholera Enterotoxin (Ace)-stimulated Diarrhea: AN ESSENTIAL...

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.