*2.4. Dependence of Action on the Application Protocols*

Next, we compared drug effects in different application protocols. In addition to the protocol of simultaneous application (see above) we applied the drugs continuously or during 30 s immediately before activation by pH drop. The results are shown in Figure 4. The peak response evoked by pH 6.0 was not strongly affected, regardless of the application protocol for both compounds. More interestingly, peak response evoked by pH 6.85 was inhibited only if the drug was present during acidification and not only before it (Figure 4A,E). This finding can be explained by two different mechanisms: (1) the compounds interact only with the open channels and/or (2) the kinetics of their action is very fast. The effects on the sustained currents also depended on the application protocol. Application of IEM-2163 before activation by pH 6.0 resulted in 143% ± 41% (*n* = 5) potentiation, while under other conditions the drug caused inhibition (Figure 4B). For IEM-2195, in all protocols potentiation at pH 6.0 was higher than at pH 6.85 (two-way ANOVA for "protocol" and "pH" as factors, F(1,36) = 25.838, *p* < 0.001). This complex behavior is readily explained by the existence of two separate effects: pH-dependent inhibition of activation, which was also seen as peak inhibition; and pH-independent reduction of desensitization, with the total effect on the sustained currents being a sum of them.

As we diminished inhibition by the use of pH 6.0 for activation or by drug application only at neutral pH before activation, the anti-desensitizing effect on the sustained current increased (for IEM-2195) or became apparent (in the case of IEM-2163).
