Figure 5.

CaM is involved in channel activation. (A) Overexpression of CaM mutants in HEK 293 cells transfected with ANO 1 channels. Wt CaM or CaM with all four Ca²⁺-binding sites disabled (CaM1234) had no significant effect on the Ca²⁺ dependent Cl⁻ current. CaM mutants with either the N-terminal (CaM12) or C-terminal (CaM34) Ca²⁺-binding sites disabled reduced the ANO 1 current by 54%. Dumbbell symbols depict the two Ca²⁺-binding lobes of the CaM molecule (green, intact; gray, disabled). ***, P < 0.001, unpaired t test. Numbers on bars indicate the numbers of experiments. Error bars indicate mean ± SEM. (B) Coprecipitation of CaM with ANO 1 and ANO 2, precipitated with an anti-GFP antiserum. YFP was fused to the ANO channels as a tag. In HEK 293 cells transfected with YFP alone, no CaM coprecipitated with YFP (control). (C) Current topology model for ANO Ca²⁺-activated Cl⁻ channels according to Yu et al. (2012). The channel protein probably has eight TMRs with cytosolic N and C termini. The structure of the pore region that includes TMRs 6 and 7 as well as a reentrant loop has not yet been definitively clarified. The N-terminal region contains a CRS motif, here referred to as regulatory CaM-binding motif (RCBM), which is encoded by exon 9 in human ANO 1 (Ferrera et al., 2009). The sequence alignment demonstrates a high degree of similarity between ANO 1 and ANO 2 in this motif. Red letters indicate residues predicted to form the hydrophobic side of the amphipathic helix. The flanking basic motifs are shown in blue. TMR1, transmembrane region 1. (D) Averaged (n = 3) and normalized CD spectra in the presence of 67 µM Ca²⁺ are shown for mixtures of CaM and the test peptides (black lines for ANO 1 and red lines for ANO 2 and ANO control [ANO ctrl]) and for their weighted averages (blue lines). Spectra for the binding partners alone (with 67 µM Ca²⁺) are shown in gray (top trace, test peptide; bottom trace, CaM). The control peptide ANO ctrl contained glutamate in positions 2, 6, 15, and 20 of the RCBM sequence. Blue asterisks denote a significant deviation of the calculated spectrum from the measured spectrum of the mixture, which is indicative of an interaction between the test peptides and CaM. (E) Control CD spectra show that neither test nor control peptides bound to Ca²⁺-insensitive CaM mutant CaM1234 in the presence of Ca²⁺.