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The FRET donor was monomeric cerulean, a brighter version of CFP (Shaner et al, 2005), tagged calmodulin (CaM�CCFP). The FRET acceptor was monomeric citrine, a brighter version of YFP (Shaner et al, 2005), fused to KCNQ2 (KCNQ2�CYFP). Fluorescent signals in the vicinity of the plasma membrane were detected by total internal reflection fluorescence (TIRF) microscopy (Steyer and Almers, 2001). These measurements revealed a higher FRET efficiency from the wild-type KCNQ2 (wt)�CYFP and CaM�CCFP pair over the control pair of KCNQ2 (wt)�CYFP and CFP alone (Figure 3A and B; n=96 cells, n=92 cells, respectively). KCNQ2 (S541A) and CaM�CCFP pair showed equivalent FRET signals to that of the wild-type pair (Figure 3A, middle; 104��8.2% of wild-type, n=101). Importantly, a lower FRET efficiency was measured with the KCNQ2 (S541D)�CYFP http://www.selleck.cn/products/sunitinib.html and CaM�CCFP pair compared with wild-type KCNQ2�CYFP (Figure 3A, right; 68.2��9.8% http://www.selleckchem.com/products/q-vd-oph.html of wild-type, n=123, P http://www.selleckchem.com/products/bay-61-3606.html Since our coimmunoprecipitation experiments indicated that less KCNQ2 subunit bound CaM after oxo-M treatment (Figure 2C), we reasoned that the oxo-M responsive decrease in FRET signal was due to dissociation of CaM rather than change in dipole orientation. We next examined PKC involvement in calmodulin dissociation by suppressing PKC phosphorylation either by treating cells with BIS IV or by using KCNQ2 (S541A). Oxo-M induced FRET responses were suppressed to a similar degree in both KCNQ2 (S541A)�CYFP+CaM�CCFP (Figure 3F, green circles; n=78, P