tannins and digallic-acid

Cholera pathogenesis occurs due to synergistic pro-secretory effects of several toxins, such as cholera toxin (CTX) and Accessory cholera enterotoxin (Ace) secreted by Vibrio cholerae strains. Ace activates chloride channels stimulating chloride/bicarbonate transport that augments fluid secretion resulting in diarrhea. These channels have been targeted for drug development. However, lesser attention has been paid to the interaction of chloride channel modulators with bacterial toxins. Here we report the modulation of the structure/function of recombinant Ace by small molecule calcium-activated chloride channel (CaCC) inhibitors, namely CaCCinh-A01, digallic acid (DGA) and tannic acid. Biophysical studies indicate that the unfolding (induced by urea) free energy increases upon binding CaCCinh-A01 and DGA, compared to native Ace, whereas binding of tannic acid destabilizes the protein. Far-UV CD experiments revealed that the α-helical content of Ace-CaCCinh-A01 and Ace-DGA complexes increased relative to Ace. In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure. The modulation of Ace structure induced by CaCC inhibitors was also analyzed using docking and molecular dynamics (MD) simulation. Functional studies, performed using mouse ileal loops and Ussing chamber experiments, corroborate biophysical data, all pointing to the fact that tannic acid destabilizes Ace, inhibiting its function, whereas DGA stabilizes the toxin with enhanced fluid accumulation in mouse ileal loop. The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders.

Topics: Animals; Chloride Channels; Cholera; Cholera Toxin; Circular Dichroism; Depsides; Diarrhea; Gallic Acid; Male; Mice; Mice, Inbred C57BL; Molecular Docking Simulation; Recombinant Proteins; Spectrometry, Fluorescence; Tannins; Thiophenes; Vibrio cholerae

Migration of dendritic cells (DCs), antigen presenting cells that link innate and adaptive immunity, is critical for initiation of immune responses. DC migration is controlled by the activity of different ion channels, which mediate Ca(2+) flux or set the membrane potential. Moreover, cell migration requires local volume changes at the leading and rear end of travelling cells, which might be mediated by the fluxes of osmotically active solutes, including Cl(-). The present study explored the functional expression, regulation and role of Cl(-) channels in mouse bone marrow-derived DCs.. In whole-cell patch clamp experiments we detected outwardly rectifying Cl(-) currents which were activated by elevation of cytosolic Ca(2+), triggered either by ionomycin in the presence of extracellular Ca(2+) or mobilization of Ca(2+) by IP(3) Most importantly, Ca(2+)-activated Cl(-) channels (CaCCs) were activated by CCL21 (75 ng/ml), an agonist of the chemokine receptor CCR7. The currents showed sensitivity to Cl(-) channel blockers such as tannic acid (10 µM), digallic acid (100 µM) and more specific CaCC blockers niflumic acid (300 µM) and AO1 (20 µM). According to RT-PCR and Western blot data, Anoctamin 6 (ANO6) is expressed in DCs. Knock-down of ANO6 with siRNA led to inhibition of CaCC currents in DCs. Moreover, chemokine-induced migration of both immature and LPS-matured DCs was reduced upon ANO6 knock-down.. Our data identify ANO6 as a Ca(2+)-activated Cl(-) channel in mouse DCs, show its activation upon chemokine receptor ligation and establish an important role of ANO6 in chemokine-induced DC migration.

Topics: Animals; Anoctamins; Calcium; Chloride Channels; Dendritic Cells; Depsides; Gallic Acid; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Niflumic Acid; Phospholipid Transfer Proteins; RNA, Small Interfering; Structure-Activity Relationship; Tannins