CaM (E/Q) can only recover SK channel activity in the presence of NS309. (A) Time course of the average SK current level at −80 mV with a patch excised from an oocyte coexpressing SK channels and CaM (E2Q). Patch was subjected to solutions containing different amount of Ca²⁺ and 20 µM WT or mutant CaM as shown by the legend. (B) Time course of the average SK current level at −80 mV with a patch from an oocyte coexpressing SK channels and CaM (E1Q). (C) A representative dose–response relationship for SK channels recovered with CaM (E2Q) and NS309 in a patch from an oocyte coexpressing SK channels and CaM (E2Q) (open circles). Initial currents were allowed to run down before the application of 20 µM CaM (E2Q) and 100 µM NS309. After recovery, the dose–response relationship was measured in the presence of 20 µM CaM (E2Q) and 100 µM NS309. Dashed line represents a fit of the data with a single Hill equation (EC₅₀ = 0.41 µM and h = 1.0). Solid line represents a fit of the data with a two-component Hill equation, I/I_max = c/(1+(EC_50a/[ Ca²⁺])^ha) + (1−c)/(1+(EC_50b/[Ca²⁺])^hb), in which c is the fraction of the first component (c = 0.41, EC_50a = 90 nM, and ha = 2.9; EC_50b = 1.14 µM and hb = 1.9). Filled circles are a representative dose–response relationship in the presence of 100 µM NS309 for CaM (WT)–recovered SK channels. Solid line is a fit with a single Hill equation (EC₅₀ = 69 nM and h = 4.2). (D) A representative experiment where NS309 was applied in the absence of CaM to a patch from an oocyte coexpressing SK channels with CaM (E/Q). (E) An example of recovery using CaM (E1Q) and NS309 without pretreatment with CaM (E1Q) alone.