*2.3. pH and Concentration Dependencies*

For detailed analysis we selected IEM-2163 and IEM-2195, as they demonstrate opposite effects (inhibition and potentiation, respectively) on the sustained currents. First, we estimated the pH-dependence of action on peak currents (Figure 3A). Figure 3B demonstrates that both compounds caused a parallel shift of activation to more acidic values without affecting maximal response. IEM-2163 at 0.5 mM shifted the pH50 value from 6.26 ± 0.02 in control to 6.17 ± 0.06. The shift caused by IEM-2195 was about equal, with the pH50 of activation being 6.17 ± 0.04 in the presence of this drug.

**Figure 3.** pH and concentration dependencies of IEM-2163 and IEM-2195 action. (**A**) Peak current inhibition was pH-dependent, the effects of both drugs disappeared under strong acidification. (**B**) IEM-2163 and IEM-2195 caused acidic shift of the ASIC3 activation curve. (**C**–**F**) Concentration dependencies of IEM-2163 (**D**,**F**) and IEM-2195 (**C**,**E**) action on peak (**C**,**D**) and sustained (**E**,**F**) currents. Fitting is shown in solid lines. Note that the concentration dependence of IEM-2163's action on sustained current was biphasic at pH 6.0 where the current inhibition was small. Low concentrations caused inhibition, but at high concentrations the effect was inverted.

We then studied the concentration dependencies of the actions of IEM-2163 and IEM-2195 on peak and sustained components of the response at pH 6.85 and 6.0 (Figure 3C–F). At pH 6.85, peak inhibition by IEM-2195 (Figure 3C)—which reflects its effect on activation—was well-fitted by the Hill equation, with optimal parameters nH = 0.82 ± 0.54, IC50 = 21 ± 15 μM. At pH 6.0, no significant effect was detected for concentrations up to 1 mM (Figure 3C). Our attempts to further increase the concentration led to poor clamp stability, resulting in highly diverging data at higher concentrations. In contrast, potentiation of the sustained current was well established at pH 6.0 (Figure 3E), where inhibition of activation was absent. The fitting resulted in EC50 = 784.2 ± 122.8 μM, nH = 1.31 ± 0.06, and maximal effect 588% ± 55% potentiation.

IEM-2163 also strongly inhibited peak current. The IC50 at pH 6.85 (Figure 3D) was 245.64 ± 16.62 μM, nH = 1.17 ± 0.08. Similar to IEM-2195, peak inhibition at pH 6.0 was not significant. Sustained currents were significantly inhibited by IEM-2163 at pH 6.85 as well (Figure 3F), IC50 = 117.3 ± 5.6 μM, nH = 1.12 ± 0.07. A peculiar concentration dependence was observed for IEM-2163's action at pH 6.0. Low concentrations caused progressive inhibition, but at around 1 mM the effect reached saturation at the level of 52% ± 16% of inhibition (*n* = 5), and at 3 mM, despite large data diversity, we saw an apparent potentiation by 84% ± 111% (*n* = 5). To ensure this was not an artifact of data variation, we performed additional experiments at 2 mM, which complied with the observed reversion of the effect resulting in 101% ± 56% (*n* = 6) potentiation.

An explanation of such concentration dependencies could be that they reflect a mixture of two distinct effects: pH-dependent inhibition of activation, which is responsible for the inhibition of peak component and window component in low concentrations; and reduction of desensitization, which determines the potentiation of the sustained current at high concentrations. Thus, analysis of concentration dependencies provided arguments in favor of the independence of drug effects on activation and desensitization.
