*2.2. Estimation of Drug Activities*

For the sixteen compounds presented in Figure 1 we estimated the effects on peak and sustained currents evoked by acidification from pH 7.4 to 6.85, which caused 10% ± 7% (*n* = 11) of maximal peak response, and to pH 6.0, which caused 74% ± 16% (*n* = 11) maximal peak response. The compounds were applied simultaneously with acidification at a concentration of 0.5 mM. These applications were repeated 3–7 times to reach the effect's equilibrium point and then 3–10 washout acidifications were done until complete recovery was achieved.

**Figure 1.** Chemical structure of the tested compounds. The first row represents common hydrophobic moieties (Ph-Ch, Ad, and dimet-Ad) of IEM compounds and memantine, with their terminal radicals (R) shown directly below.

The results are presented in Figure 2. At pH 6.85 (Figure 2A, with sample traces shown in Figure 2B,C) the peak component of the response was strongly inhibited by a number of compounds, the most potent being IEM-2195 at 85% ± 7% (*n* = 6) inhibition, and only IEM-2117 slightly potentiated the peak response by 42% ± 21% (*n* = 5). On the other hand, sustained current was typically potentiated, with the strongest effect by IEM-2117 at 382% ± 84% (*n* = 5). IEM-2163 and IEM-2151 were the only compounds that reduced the sustained current by 42% ± 21% (*n* = 9) and 29% ± 4% (*n* = 5), respectively. At pH 6.0 (Figure 2D, with sample traces shown in Figure 2E,F) the drugs' effect on peak current disappeared, while for sustained current the overall picture stayed the same and the effects even somewhat increased in magnitude, with maximal potentiation by IEM-2117 reaching 498% ± 196% (*n* = 5) and inhibition by IEM-2163 at 54% ± 23% (*n* = 8). We can conclude from the data that (1) structural determinants of the effects on peak and sustained components of the response did not coincide and (2) only the effect on peak component demonstrated pronounced pH-dependence.

**Figure 2.** Estimation of the drug activities. Number of \* denotes statistical significance at \* *p* < 0.05, \*\* *p* < 0.01, or \*\*\* *p* < 0.001, respectively, *n* ≥ 5. (**A**) Effects of 500 μM of the compounds studied on the ASIC3 activated by pH drop from 7.4 to 6.85. Compounds were applied simultaneously with activation. (**B**,**C**) Representative examples of ASIC3 responses in control and in the presence of IEM-2044 (**B**) and IEM-2174 (**C**) when activated by pH drop from 7.4 to 6.85. (**D**) effects of 500 μM of the compounds studied on the ASIC3 activated by pH drop from 7.4 to 6.0. Compounds were applied simultaneously with activation. (**E**,**F**) Representative examples of ASIC3 responses in control and in the presence of IEM-2044 (**E**) and IEM-2174 (**F**) when activated by pH drop from 7.4 to 6.0.

Typically, potentiation of the sustained current was accompanied by deceleration of the response decay, reflecting an effect on desensitization. In control experiments the decay time constant was 462 ± 143 ms (*n* = 8). The most drastic increase was seen for IEM-2195, which changed the decay time constant of the response to 4891 ± 1996 ms (*n* = 6). Inhibition of sustained current by IEM-2163 or IEM-2151 did not elicit significant changes of the response kinetics.
