Figure legends
Figure 1 Effect of the ENV59-GP3 peptide and KCa3.1-blocker NS6180 in DSS-induced acute colitis mice model .
Either saline (control) or ENV59-GP3 peptide (0.5mg/mg) or NS6180 blocker (2.5mg/kg) were administrated intraperitoneally every other day of DSS treatment. Weight development was monitored from day 0 to day 10. (A) daily weight development is shown for each of the treatment groups (pool of 2 independent experiments, 9-10 mice in each group). (B) The comparison of colon length. Data are expressed as means ± SEM (n = 10 per group). * p < 0.05, * * * p < 0.001, and * * * * p < 0.0001, one-way ANOVA with post hoc Tukey’s test. (C) Evaluation of inflammatory cytokines. Total distal colon tissue was isolated from mice 10 days after the induction of DSS colitis and relevant treatments and equivalent biopsy sections were taken for inflammatory mRNA analysis (RT-qPCR). All gene values were normalized to the expression of Hprt1. Data are presented as mean ± SEM (n = 10 per group).
Figure 2 Histomorphology of colon tissue and inflammation scores according to the scale described in the Materials and Methods. (A) Inflammation score for each treatment group. The histological H&E stains were scored by 2 individuals blinded to treatment and experiments. Right panel, Representative H&E staining from mouse colon. Bar = 100 μm. Differences were calculated using a one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test test. Values are presented as mean ± SD. **P  < 0.01, ***P  < 0.001.
Figure 3 A) Functional expression of KCa3.1 and Kv channels in THP-1 cells. Application of NS309 or 1 μM NS6180 occurred by switching to the appropriate tube feeding the inflow-line. From left to the right: The IV curve recorded after stabilization of the current after the initial run up (Control) and ~1 min after application of 500 nM NS309.: After NS309 wash-out and establishment of new stable baseline (NS309 wash-out) 1 μM NS6180 was applied (NS6180) which completely blocked the KCa3.1 component. Note the distinct depolarizing shift in reversal potential. : The difference curve obtained by subtracting the curves in the middle panel revealing the NS6180 sensitive component. B) Expression ofKCNN4 mRNA in THP-1 cells . Melting curve plot showing Q-PCR product specific for KCNN4 mRNA. C) Cytokine release and inhibiting effect of Env59-GP3 and of NS6180 in stimulated THP-1 cells.mRNA and protein levels for IL-6 and TNF-alpha.
Figure 4 A) Calcium influx following Ca 2+ depletion in THP-1 cells. Calcium flux to the cytosol following cell activation is reduced in the presence of Env59-GP3 or NS6180. B-C) Selective inhibition of THP-1 KCa3.1 channels by Env59-GP3 . Whole cell current recording from THP-1 cell. B shows the IV curves after current stabilization for about 15 min (a) and after perfusion with Env59-GP3 peptide at a concentration of 20µM for 5 min (b) (left figure). The IV curve after Env59-GP3 peptide wash-out (c ) (right figure) and after perfusion with 1µM NS6180 for 5 min (d). The arrows indicate -40mV, the membrane potential used for putative “pure” KCa3.1 current recording. The -40mM current recordings were used for construction of the experimental time course relation shown in C, where the letters mark the times for the trace recordings. D-E) IC50 value for ENV59-GP3 in recombinant human KCa3.1 channels stably expressed in HEK293 cells.The fraction of unblocked current at the end of compound application as a function of the test concentration. The lines are the curves resulting from fits of the data to the Hill equation described by IC50 values of 43µM for Env59-GP3 peptide.
Figure 5 A-D. ENV59-GP3 inhibition of recombinant human KCa3.1 channels stably expressed in HEK293 cells . A,B,C show the KCa3.1 currents in the absence (a,c.e) and presence (b,d,f) of compounds, with the letters also showing the times of recording at the time course in D. After establishment of the whole cell configuration KCa3.1 currents increased as expected due to equilibration with the buffered Ca2+ solution in the pipette (compare also to Fig. 4) and then ran down to a stable current (D). The reversible KCa3.1 blocking scorpion toxin peptide Charybdotoxin (10 nM) (A,D) and the KCa3.1 positive modulator NS309 (500 nM) (D) framed two additions of ENV59-GP3 (50 μM) (B,D) and the control peptide Flu ISU F#2 (50 μM) (C,D), respectively.