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. 2012 Aug 30;287(43):36312–36321. doi: 10.1074/jbc.M112.392811

FIGURE 5.

FIGURE 5.

FRET between wild type CaBP4 and CaBP4 mutants and C-terminal variants of Cav1.4 channels. A, schematic of the constructs used for FRET experiments. Top, CFP-tagged wild type CaBP4 and CaBP4 mutants. Boxes represent EF-hands 1–4. Gray boxes, functional EF-hands 1, 3, and 4; black box, nonfunctional EF-hand 2. NT, N terminus of CaBP4; CT, C terminus of CaBP4. Middle, C-terminal variants of Cav1.4 tagged by YFP or double-labeled by YFP and CFP. Boxes represent EF-hand, IQ motif, and ICDI domain; 5A represents the mutated IQ motif. 1.4DL, C terminus of Cav1.4 double-labeled by YFP and CFP. Bottom, N terminus of Cav1.4 (1.4 NT). B, interaction of CFP-CaBP4 with YFP-tagged fragments of the C terminus or with the N terminus of Cav1.4 as indicated. C, interaction of the ICDI domain with the C terminus of Cav1.4 lacking the ICDI domain (ΔICDI) in the absence and the presence of CaBP4 variants (group of four bars from the left). Intramolecular FRET for the C terminus of Cav1.4 double-labeled by YFP and CFP (1.4 DL) in the absence and presence of CaBP4 (group of 4 bars from the right). D, Western blot demonstrating the expression of mutant and wild type CaBP4 coupled to CFP. control: nontransfected HEK cells. E, interaction of CFP-tagged wild type and mutant variants of CaBP4 with the C terminus of Cav1.4. FR: FRET ratio; the number of cells is given in parentheses. Error bars indicate S.E. *, p ≤ 0.05, ***, p ≤ 0.001. Statistical significance is given in comparison with 1.4/5A and 1.4 NT (B), -WT (C), and NT (D).