Figure 1.
Interaction of CaM with KCNQ subunits required distinct calcium conditions.
A, Distinct calcium requirement for CaM binding to the KCNQ2, KCNQ3 and KCNQ2/KCNQ3 channels. Representative immunoblots (top) of immunoprecipitation experiments and summary histogram (bottom) are shown. B, Time course of CaM dissociation from the KCNQ2 subunit upon exposure to 500 µM Ca2+. C, Molecular structure from the PDB file (4GOW) depicting the interaction between calcium-bound CaM(E88) and KCNQ4(R538). D, Sequence alignment of Helix B, the distal CaM binding domain, of KCNQ1-5 subunits. Arrow shows the cysteine residue on KCNQ2, rKCNQ2(C527), that corresponds to the conserved arginine residue in other KCNQ subtypes, such as KCNQ4(R538); r – rat, h – human. E, KCNQ2(C527R) mutation regained CaM binding in the presence of Ca2+. * < 0.05, ** < 0.01 by Mann-Whitney test. Error bars show S.E.
Figure 2.
HoloCaM was retained in the KCNQ2 channel complex via AKAP150.
A, Calcium (100 µM) dependent binding of CaM to AKAP79. B, In vitro binding of KCNQ2 and CaM with or without AKAP150. KCNQ2-FLAG was immunopurified by anti-FLAG conjugated resin. CaM was co-purified in calcium (+) condition only in the presence of AKAP150. C, Top – the summary of quantification of relative KCNQ2-bound CaM from five independent experiments shown in B. Bottom – the summary of quantification of relative KCNQ2-bound AKAP150 from five independent experiments shown in B. Bars are labeled corresponding to the lane numbers on the immunoblot. *<0.05 non-parametric ANOVA followed by Dunn’s multiple comparisons test, ** < 0.01 by Mann-Whitney test. Error bars show S.E.
Figure 3.
Ionomycin treatment and PIP2 depletion.
TIRF analysis indicating membrane localization of the CFP-PH probe. Top panels show epifluorescent cell images (epi) indicating total fluorescence, and TIRF images (TIRF) showing plasma membrane localization. The lower panel shows pooled data from TIRF analyses. 10 µM ionomycin induced translocation of CFP-PH, which indicates depletion of PIP2. In contrast, 1 µM or 3µM ionomycin did not alter plasma membrane localization of CFP-PH. TIRF signal is normalized to that at t = 0. Black box indicates the presence of ionomycin. Error bars show S.E.
Figure 4.
Ionomycin suppressed KCNQ currents via elevation of intracellular calcium.
A, A representative KCNQ2 current response to 1 µM ionomycin with 1 mM EGTA in the pipette solution. The lower panel summarizes ionomycin-induced suppression of the KCNQ2 current, which was dependent on EGTA concentration in the pipette solution. Gray box indicates the presence of 1 µM ionomycin. 1 mM EGTA was used for the rest of the experiments. B, Ionomycin-induced KCNQ2 current suppression was prevented by co-expression of a dominant negative CaM, CaM(4DA). Control ionomycin response of KCNQ2 channel from panel A is also shown. C, Ionomycin induced augmented suppression of the KCNQ2(R353G) current. KCNQ2 response from panel A is also shown. D, Ionomycin response of the heteromeric KCNQ2/KCNQ3 channels was similar to that of the homomeric KCNQ2 channels. E, Ionomycin response of the KCNQ2(C527) channel. F, Summary of ionomycin responses. Relative KCNQ current at t = 1 min is shown. Black bars indicate responses significantly different (p < 0.01, nonparametric ANOVA followed by Dunnett’s multiple comparisons test) from the wild-type KCNQ2 response. Error bars show S.E.
Figure 5.
Ionomycin-induced suppression of KCNQ2 current was accompanied by a lower PIP2 affinity.
A, Representative current traces showing a voltage dependent KCNQ2 current decay due to the activation of Ci-VSP. A brief voltage step to –60 mV was applied to calculate the linear leak. B, Scaled KCNQ2 current traces at +10 mV showing an identical Ci-VSP-mediated current decay without ionomycin at 2-min interval. Gray and shaded red areas show S.E. C, Scaled KCNQ2 current traces showing the facilitation of KCNQ2 current decay 1 min after the application of 1 µM ionomycin. Control traces were obtained 1 min before ionomycin application. D, Ci-VSP-mediated current decay of the KCNQ2(C527R) current. Scaled current traces indicate the facilitation of current decay by 1 µM ionomycin at +10 mV. E, Summary of the Ci-VSP-induced current decay at control (t = –1 min) and t = 1 min for indicated conditions. KCNQ2(wt) and KCNQ2(C527R) showed an equivalent Ci-VSP-mediated current decay both with and without 1µM ionomycin. **<0.01 by paired t-test. Error bars show S.E.