Fig. 4.

Ca²⁺-CaM and PIP₂ are required for I_KS channel function. (A, Upper) Representative WT I_KS currents recorded from transfected CHO cells with pipette solution containing 3 µM purified recombinant WT CaM with either 5 µM free Ca²⁺ or 5 mM BAPTA. From a holding potential of −90 mV, cells were stepped for 3 s from −60 to +60 mV in 10-mV increments and repolarized for 1.5 s to −60 mV. (A, Lower) Shown are current density–voltage relations and normalized conductance–voltage relations. Activation curves were fitted by a single Boltzmann distribution. (B, Left) Normalized conductance–voltage relations of WT I_KS currents expressed in CHO cells and recorded in the presence of different concentration of internal free Ca²⁺ (0, 30, 100, 300, and 5,000 nM) in the pipette solution titrated with BAPTA. (B, Right) ΔV₅₀ values were normalized and plotted as a function of the log of free Ca²⁺ concentrations (ΔV₅₀ = 1; corresponds to the left shift obtained for free Ca²⁺ = 5,000 nM vs. 0 nM free Ca²⁺). The apparent EC₅₀ determined from a Hill fit was 96 nM, with a hill slope of 1.88. (n = 5). (C) I_KS channels (Kv7.1 and KCNE1) and Dr-VSP were expressed in CHO cells, and currents were recorded with pipette solution containing one of the following conditions: 5 mM BAPTA alone, 5 mM EGTA alone, or 5 mM EGTA and 3 µM purified recombinant WT CaM and then, in the presence of 5 µM free Ca²⁺ (titrated with EGTA), no CaM or 3 µM WT CaM, CaM₁₂, or CaM₃₄. The triple-pulse protocol is shown in Top. (Middle) Representative current traces of WT I_KS before and after activation of Dr-VSP under conditions of pipette solution containing 5 µM free Ca²⁺ without and with 3 µM WT CaM. After pipette content dialysis and rupture of the patch (∼2 min), membrane potential was first stepped for 2 s from −90 to +10 mV to open the I_KS channel (black traces). Then, PIP₂ depletion was induced by activating Dr-VSP using a 200-ms step depolarization from −90 to +90 mV 100 times. To measure the consequences of PIP₂ depletion induced by Dr-VSP activation on I_KS current amplitude, a 2-s step depolarization from −90 to +10 mV was reapplied (red traces). (Bottom) For the various experimental conditions, I_KS current amplitudes before and after Dr-VSP activation were determined and expressed as percentages of current inhibition (n = 7–33). Asterisks indicated significance level by one-way ANOVA and Bonferroni's posttest. **P < 0.01; ***P < 0.001. ns, not significant.