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 ICl 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 ICl 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.
Highlights
Accessory cholera enterotoxin (Ace) of Vibrio cholerae has been shown to contribute to diarrhea
Earlier studies had shown that purified Ace from aceϩ V. cholerae strains is a Ca2ϩ-dependent agonist that acts at the apical membrane of polarized intestinal epithelial cells (T84 models) to stimulate anion secretion [6]
Evidence that Ace-stimulated ClϪ secretion is not due to a rise of [Ca2ϩ]i is based on the following: 1) application of Ace did not raise [Ca2ϩ]i in mouse ANO6 fused to mCherry-expressing HEK293 cells or in Caco-2 cells; 2) in the presence of intracellular calcium (100 nM or 1 M), Ace failed to display significant whole-cell currents from ANO1-expressing
Summary
Accessory cholera enterotoxin (Ace) of Vibrio cholerae has been shown to contribute to diarrhea. We had demonstrated earlier that the biologically active recombinant Ace, purified from a specialized E. coli M15 (pREP4) strain, induced a dose-dependent Isc increase across T84 cell monolayers along with ATP stimulation This Isc response was significantly inhibited by bumetanide, an inhibitor of the Na,K,2Cl (NKCC) cotransporter, indicating that this current is predominantly carried by chloride ion (ClϪ) [7]. The experiments conducted in our present study demonstrated for the first time that essentially ANO6 is able to produce ClϪ current by stimulatory effects of phospholipid phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), commonly known as PIP2, through RhoA activation by recombinant Ace. We have used a combination of electrophysiological, biochemical, molecular biology mutagenesis, and pharmacological approaches along with in vivo mouse ileal loop assay to demonstrate whether alterations in PIP2 levels by the action of Ace affect native ANO6 function in intestinal epithelial cells. Our data revealed that ANO6 and PIP2 are powerful new additions to the mechanism of secretory diarrhea and have considerable implications for diarrheal disease therapy
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