Mutation of both BD-N and BD-C2 completely disrupt the binding of CaM to full-length hEAG1.
(A) Amino acid sequence of AA 147-209 of EAG1 with the two putative 1-8-14 CaM binding motifs highlighted. (B) CaM overlay blot of the 1-8-14 mutated N-terminus of rEAG1. Detection with HRP-conjugated streptavidin revealed that mutations F151S, A152S (I) completely disrupted binding of biotinylated CaM whereas V164S, L165S (II) and V178S, H179D (III) only caused a reduction in binding. (C–E) FRET measurements in cells transfected with YFP-CaM and Cerulean-labeled full-length hEAG1, mutated in BD-N (F151S, A152S) or in both BD-N and BD-C2 (F151S, A152S, F714S, F717S). Cells were treated with 1 µM ionomycin in the presence of high Ca2+. (C) Fluorescence intensity images of Cerulean-hEAG and YFP-CaM after photobleaching and the corresponding FRET efficiency images in pseudo-color (D) Cumulative FRET distribution histograms (probability density function, PDF) and (E) average FRET efficiencies (± SEM) of several cells. Scale bar: 5 µm.