Fig. 7. CLIC1 point mutation impairs metformin effects.
A) Whole-cell current time-course of CHO cells transfected with CLIC1-wt, stimulated every 5 seconds with 800 ms, 100 mV test potential from −40 mV holding voltage. Metformin (10mM) inhibits membrane current, which was not further decreased by IAA94 (100μM). B) Similar experiments as in A, using CHO cells transfected with CLIC1 R29A show that metformin (10mM) achieves only a partial block of IAA94 (100μM)-sensitive current. C) Representative dose-response curves of IAA94-dependent current inhibition demonstrate that both CLIC1 constructs transfected in CHO cells have a similar EC50: 27.3 μM for CLIC1-wt (squares, n=4, data expressed as mean±s.e.m.), and 29.2 μM for CLIC1-R29A (circles, n=4). Data are reported as inhibition of IAA94-sensitive current, over maximal inhibition. D) Box-plot quantifying metformin-insensitive portion of IAA94-sensitive current. Ratio between metformin (10mM) and IAA94 (100μM) is reported for native CHO cells (n=4, mean 0.83±0.06), cells transfected (Tx) with CLIC1-wt (n=8, mean 0.83±0.03), CLIC1-R29A (n=9, mean 0.54±0.06) and CLIC1 K37A (n=5, mean 0.87±0.028). Only the trials with CLIC1-R29A produced a value significantly different from the others (**p<0.01 vs. native CHO; ***p<0.001 vs. CLIC1 wt and K37A, one-way ANOVA, followed by Tukey test). E-F) Currents from transfected CLIC1 constructs are functionally identified using anti-FLAG antibody acting as current blocker (see text). Both CLIC1 wt- (E) and R29A-transfected cells (F) show partial inhibition of membrane current by anti-FLAG antibody while the remaining current, representing endogenous CLIC1, was blocked by both metformin (10mM) and IAA94 (100μM). G-H) Metformin induces a total membrane current knockdown in CLIC1 wt-transfected cells, without a further decrease caused by anti-FLAG antibody or IAA94 (G). In R29A-expressing cells, metformin only partially blocks the current, which is fully inhibited by anti-FLAG antibody or IAA94 (H). I) Summary box-plots demonstrating that in CLIC1 R29A-transfected cells a consistent part of the current is insensitive to metformin (Met). Mean Imet/IIAA94 ratios are 0.84±0.02 (n=3), 0.76±0.08 (n=5), 0.52±0.07 (n=5) for native, transfected wt, and R29A mutant CLIC1, respectively. The current ratio in R29A transfected cells is statistically different from both native and CLIC1 wt-transfected cells. Further perfusion with anti-FLAG antibody equalizes IAA94-sensitive current inhibition showing a mean Ianti-FLAG/IIAA94 ratio of 0.96±0.03 (n=3), 0.9±0.08 (n=5), 0.87±0.05 (n=5) for native, wt and R29A CLIC1, respectively. In R29A-transfected cells metformin and anti-FLAG current inhibitions are statistically different. **p<0.01 and ***p<0.001 (one way ANOVA followed by Tukey test).