Fig 2: EtOH and AA elicit TRPV6 currents and trigger Ca2+ influx in the intestinal epithelium(A) Representative whole-cell patch-clamp recording from Caco-2 cells challenged with EGTA (2 mM), EtOH (50 or 150 mM), AA (200 μM), or 50 mM EtOH and 200 μM AA (EtOH + AA) combined.(B) Mean EGTA-normalized currents evoked by EtOH, AA, or EtOH + AA in Caco-2 cells. Values are mean ± SEM (n = 3 for 50 mM EtOH, n = 5 for 150 mM EtOH and 200 μM AA, and n = 4 for EtOH + AA). **p < 0.01 and ***p < 0.005 for significant differences between indicated groups; ns, not significant.(C) Representative whole-cell patch-clamp recording from Caco-2 cells challenged with EGTA (2 mM), EtOH (50 mM), AA (200 μM), or EtOH + AA in the presence of SOR-C13 (10 μM) in the bath and recording pipette. The record is representative of at least five independent patches.(D) Caco-2 cells were transfected with non-specific RNA (NS-RNA) or Trpv6-specific shRNA (see also Figure S1). Intracellular Ca2+ ([Ca2+]i) was measured in transfected cell monolayers using Indo-1 dye and live-cell time-lapse imaging of fluorescence at wavelengths of 405 nm and 485 nm. Cells were treated with EtOH, AA, and ionomycin (Iono). Representative images are presented on the left panel (captured 20 min after AA application). Scale bar, 50 μm. Real-time changes in 405/485 nm fluorescence ratio are presented in graphs in the middle. The changes in fluorescence ratio from the basal values at 20 min after AA treatment are plotted in the bar graph on the right. Values in the bar graphs are mean ± SEM (n = 45–50 cells each from six NS-RNA monolayers and four shRNA monolayers). *p < 0.05 and ****p < 0.001 for significant differences between indicated groups; ns, not significant.(E and F) The effect of EtOH and AA (as above) on [Ca2+]i was measured in mouse enteroids (E) and colonoids (F) prepared from wild-type and Trpv6−/− mice. Images represent the heatmap images of 405/485 nm ratio measured at 20 min after AA application. Scale bars, 50 μm. Values in the bar graphs are mean ± SEM (n = 5 regions each from three different organoids per group). ****p < 0.001 for significant difference between indicated groups; ns, not significant. Real-time video recordings of Ca2+ flux in enteroids and colonoids are submitted as Videos S1 and S2.

Fig 3: TRPV6 channel plays a role in EtOH and AA-induced TJ disruption and barrier dysfunction in mouse intestinal organoids(A) GFP-Ocln/RFP-ZO1 colonoids were microinjected with 50 mM EtOH and 200 μM AA (EtOH + AA), along with AF647-dextran. Time-lapse fluorescence imaging was performed for GFP, RFP, and AF647, with differential interference contrast microscopy. Representative images captured at indicated times are presented. Scale bar, 25 μm. The graph shows the outside-to-inside (Out/In) ratio for AF647-dextran fluorescence.(B) The fluorescence images for the nucleus (Hoechst 33342 dye) and AF647-dextran were evaluated in wild-type and Trpv6−/− colonoids at 10 or 110 min after microinjection of EtOH + AA with AF647-dextran as above. Scale bar, 25 μm. The graph shows the Out/In ratio of AF647-dextran fluorescence measured at 110 min. The values are mean ± SEM (n = 6 for wild-type and n = 5 for Trpv6−/− colonoids). *p < 0.05 and **p < 0.01 for significant differences between the indicated groups; ns, not significant.(C and D) Colonoids from wild-type and Trpv6−/− mice incubated with 50 mM EtOH and 200 μM AA (EtOH + AA) in the medium. At 4 h, colonoids were fixed and co-stained for OCLN and ZO-1 (C) or E-cadherin and β-catenin (D). Scale bar, 25 μm.(E–G) GFP-Ocln/RFP-ZO1 colonoids were microinjected with EtOH + AA containing AF647-dextran, with (F) or without (E) 10 μM SOR-C13. Time-lapse imaging was performed for GFP (green), RFP (red), and AF647 (magenta) at varying times (G); phase-contrast images were captured simultaneously with fluorescence imaging. Arrows in (E) indicate some areas with reduced fluorescence compared with the 10-min image. Scale bar, 25 μm. The Out/In ratio for AF647-dextran fluorescence was calculated (G). Values are mean ± SEM (n = 28–30 regions from three different colonoids).In (A), (B), and (G), **p < 0.01 and ****p < 0.001 for significant differences between the indicated groups; ns, not significant. See also Figure S2.

Fig 4: TRPV6 channel is required for EtOH-induced neuroinflammation(A–D) Adult wild-type (WT) and Trpv6−/− (KO) mice were fed a liquid diet with EtOH (EF) or isocaloric maltodextrin (PF). Neuroinflammation was evaluated by measuring mRNA for TNFα (A), MCP1 (B), Bdnf (C), and TrkB (D) in the cerebral cortex. In all graphs, the values are mean ± SEM (n = 4 for PF and n = 6 for EF groups). *p < 0.05, **p < 0.01, and ***p < 0.005 for significant differences between the indicated groups; ns, not significant.

Fig 5: TRPV6 channel is required for EtOH-induced endotoxemia, systemic inflammation, and liver damageAdult wild-type (WT) and Trpv6−/− (KO) mice were fed a liquid diet with EtOH (EF) or isocaloric maltodextrin (PF).(A) Lipopolysaccharide (LPS) levels were measured in plasma. The values in all graphs are mean ± SEM (n = 4 for PF and 6 for EF groups); * = p < 0.05 for significant differences between the indicated groups; “ns” = not significant.(B–D) Plasma levels of TNFα (B), IL-6 (C), and MCP1 (D) were measured by ELISA. The values in all graphs are mean ± SEM (n = 4 for PF and 6 for EF groups); ** = p < 0.01, *** = p < 0.005, and **** = p < 0.001 for significant differences between the indicated groups; “ns” = not significant.(E and F) Plasma was analyzed for ALT and AST activity. The values in all graphs are mean ± SEM (n = 4 for PF and 6 for EF groups); **** = p< 0.001 for significant differences between the indicated groups; “ns” = not significant.(G) Liver histopathology was performed by H&E staining and bright-field light microscopy. Scale bar = 100 μm.(H) Steatosis was assessed by measuring liver triglyceride content. The values in all graphs are mean ± SEM (n = 4 for PF and 6 for EF groups); **** = p < 0.001 for significant differences between the indicated groups; “ns” = not significant.(I–K) Inflammatory responses in the liver were determined by measuring mRNA for IL-1β (I), TNFα (J), and CXCL1 (K). The values in all graphs are mean ± SEM (n = 4 for PF and 6 for EF groups); * = p < 0.05, ** = p < 0.01, *** = p < 0.005, and **** = p < 0.001 for significant differences between the indicated groups; “ns” = not significant.See also Figure S8.