Whether zinc (Zn2+) regulates barrier functions by modulating tight-junction (TJ) proteins when pathogens such as Shigella alter epithelial permeability is still unresolved. We investigated the potential benefits of Zn2+ in restoring impaired barrier function in vivo in Shigella-infected mouse tissue and in vitro in T84 cell monolayers. Basolateral Shigella infection triggered a time-dependent decrease in transepithelial resistance followed by an increase in paracellular permeability of FITC-labeled dextran and altered ion selectivity. This led to ion and water loss into the intestinal lumen. Immunofluorescence studies revealed redistribution of claudin-2 and -4 to an intracellular location and accumulation of these proteins in the cytoplasm following infection. Zn2+ ameliorated this perturbed barrier by redistribution of claudin-2 and -4 back to the plasma membrane and by modulating the phosphorylation state of TJ proteins t hough extracellular signal-regulated kinase (ERK)1/2 dependency. Zn2+ prevents elevation of IL-6 and IL-8. Mice challenged with Shigella showed that oral Zn2+supplementation diminished diverse pathophysiological symptoms of shigellosis. Claudin-2 and -4 were susceptible to Shigella infection, resulting in altered barrier function and increased levels of IL-6 and IL-8. Zn2+ supplementation ameliorated this barrier dysfunction, and the inflammatory response involving ERK-mediated change of phosphorylation status for claudin-2 and -4. Thus, Zn2+ may have potential therapeutic value in inflammatory diarrhea and shigellosis. NEW & NOTEWORTHY Our study addresses whether Zn2+ could be an alternative strategy to reduce Shigella-induced inflammatory response and epithelial barrier dysfunction. We have defined a mechanism in terms of intracellular signaling pathways and tight-junction protein expression by Zn2+. Claudin-2 and -4 are susceptible to Shigella infection, whereas in the presence of Zn2+ they are resistant to infection-related barrier dysfunction involving ERK-mediated change of phosphorylation status of claudins.

This study explored the mechanism by which Ca2+-activated Cl− channels (CaCCs) encoded by the Tmem16a gene are regulated by calmodulin-dependent protein kinase II (CaMKII) and protein phosphatases 1 (PP1) and 2A (PP2A). Ca2+-activated Cl− currents ( IClCa) were recorded from HEK-293 cells expressing mouse TMEM16A. IClCa were evoked using a pipette solution in which free Ca2+ concentration was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP, IClCa decayed to <50% of the initial current magnitude within 10 min after seal rupture. IClCa rundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. IClCa recorded after 20 min of cell dialysis with 0 ATP were more than twofold larger than those recorded with 5 mM ATP. Intracellular application of autocamtide-2-related inhibitory peptide (5 µM) or KN-93 (10 µM), two specific CaMKII inhibitors, produced a similar attenuation of TMEM16A rundown. In contrast, internal application of okadaic acid (30 nM) or cantharidin (100 nM), two nonselective PP1 and PP2A blockers, promoted the rundown of TMEM16A in cells dialyzed with 0 ATP. Mutating serine 528 of TMEM16A to an alanine led to a similar inhibition of TMEM16A rundown to that exerted by either one of the two CaMKII inhibitors tested, which was not observed for three putative CaMKII consensus sites for phosphorylation (T273, T622, and S730). Our results suggest that TMEM16A-mediated CaCCs are regulated by CaMKII and PP1/PP2A. Our data also suggest that serine 528 of TMEM16A is an important contributor to the regulation of IClCa by CaMKII.

Edited by F. Anne StephensonAccessory 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

TMEM16A (Transmembrane protein 16A or Anoctamin1) is a calcium-activated chloride channel. (CaCC),that exerts critical roles in epithelial secretion. However, its localization, function, and regulation in intestinal chloride (Cl − ) secretion remain obscure. Here, we show that TMEM16A protein abundance correlates with Cl − secretion in different regions of native intestine activated by the Ca 2+ -elevating muscarinic agonist carbachol (CCH). Basal, as well as both cAMP- and CCH-stimulated Isc, was largely reduced in Ano1 ± mouse intestine. We found CCH was not able to increase Isc in the presence of apical to serosal Cl − gradient, strongly supporting TMEM16A as primarily a luminal Cl − channel. Immunostaining demonstrated apical localization of TMEM16A where it colocalized with NHERF1 in mouse colonic tissue. Cellular depletion of NHERF1 in human colonic T84 cells caused a significant reduction of both cAMP- and CCH-stimulated Isc. Immunoprecipitation experiments revealed that NHERF1 forms a complex with TMEM16A through a PDZ-based interaction. We conclude that TMEM16A is a luminal Cl − channel in the intestine that functionally interacts with CFTR via PDZ-based interaction of NHERF1 for efficient and specific cholinergic stimulation of intestinal Cl − secretion.

There is growing interest in dietary factors, in particular micronutrients, from the perspective of disease pathogenesis and potential for treatment. Diarrheal disease in infant results in abnormally low concentrations of serum zinc. Zinc has been tested for its ability to treat and prevent diarrheal diseases in many large field trials over a period of over 4 decades and has generally been found effective. The mechanism by which zinc deficiency causes diarrhea is not known. The present study investigated the impact of zinc deficiency on the host, particularly of intestinal tight junction (TJ) integrity and proteins that are involved in intestinal absorption and secretion along with the pathogenesis of enteric infection. We have used human colonic T84 cells to study barrier function and Shigella to test susceptibility of intestinal infection due to zinc deficiency. T84 grown onto transwell inserts in zinc deficient media showed a low transepithelial electrical resistance (TEER) compare to zinc sufficient media (1014 ± 165 Ω.cm2 vs 3663 ± 293 Ω.cm2) in confluent T84 monolayers. We furthermore analysed the correlation between TEER and paracellular ionic conductance. We found that zinc deficiency altered paracellular ionic conductance. Bacterial transmigration studies showed a significant increase in apico‐basolateral transmigration of Shigella (6.58 ± 0.13 vs 7.98 ± 0.23 log10 CFU/ml) which peaked at 6hrs post infection. Transmission electron microscopy data showed widened TJ complex due to few membrane fusion proteins and distorted TJ morphology, whereas the desmosomes were still intact. Electrophysiological studies with Ussing chamber and qPCR quantification demonstrated reduced cAMP dependent electrogenic Cl− secretion (34.5 ± 1.25 μA/cm2 vs 17.75 ± 2.3 μA/cm2) along with reduced expression of CFTR in zinc deficient cells, resulting dehydrated lumen thus aiding in bacterial colonization. Our results suggest that zinc deficiency caused (1) perturbed barrier function and integrity (2) altered Cl− secretion leading to susceptibility of infection. We conclude that adequate zinc is required to maintain intestinal barrier health to avoid risk against intestinal infection.Support or Funding InformationGovt of India National Funding AgencyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.