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. 2012 May 29;31(14):3147–3156. doi: 10.1038/emboj.2012.156

Figure 3.

Figure 3

Muscarinic stimulation induces dissociation of calmodulin from the KCNQ2 channel complex in living cells. (A) Pooled FRET efficiencies. Increased FRET efficiencies were observed in the wild-type KCNQ2–YFP (Q2 (wt)–YFP) and calmodulin–CFP (CaM–CFP) pair over the control pair, KCNQ2–YFP and CFP alone. KCNQ2 (S541A)–YFP (Q2 (S541A)–YFP) and CaM–CFP pair showed equivalent FRET efficiency to the wild-type pair. KCNQ2 (S541D)–YFP (Q2 (S541D)–YFP) and CaM–CFP showed reduced FRET efficiency compared with that from the wild-type pair, but showed significant FRET efficiency over the CFP control. N indicates numbers of cells observed. ***<0.001 by nonparametric ANOVA followed by t-test. (B, C) Schematic representations of KCNQ2–YFP and calmodulin–CFP interaction in control (B) and oxo-M applied condition (C). (D) Fluorescent images from CHO hm1 cells coexpressing KCNQ2–YFP and CaM–CFP. (E) Pseudocolour images of the cells shown in (D) showing decrease in FRET efficiency after 3 μM oxo-M application. (F) Changes in FRET efficiencies of KCNQ2–YFP and CaM–CFP upon 3 μM oxo-M application. FRET efficiency of the wild-type KCNQ2–YFP+CaM–CFP (Q2 (wt), blue circles) was decreased upon stimulation with oxo-M. Pretreatment with BIS IV attenuated oxo-M responses (Q2 (wt) BIS IV, black circles). KCNQ2 (S541A)+CaM–CFP pair showed similar attenuated responses (Q2 (S541A), green circles). The black box indicates the presence of oxo-M. Error bars indicate s.e.m.