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.