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Figure 3. Change in relative cAMP efficacy after PIP3 is dependent on the C-terminal region of CNGA3. (A) Schematic diagram showing sites of truncations and charge neutralizations within the human CNGA3 C-terminal region, and a sequence alignment of this region with orthologous CNGA3 proteins (canine, mouse, bovine, and gallus). Seven conserved lysines and arginines are highlighted in yellow. Bold nucleotides indicate residues in human A3 that were mutated in our study as well as related residues in A3 orthologues. The numbers above the sequence alignment indicate sites for introduced channels. Green highlighting indicates the distal region of the Cα helix. The asterisk indicates D609, as described in Fig. 2. (B) Representative current traces (left), elicited by 1 mM cGMP (green) or 10 mM cAMP (red) before and after 1 µM PIP3, for CNGA3 675X and 635X channels. The right panel summarizes the effects of CNGA3 C-terminal deletions on PIP3-induced increases in cAMP efficacy; *, P < 0.05 compared with wild-type channels, n = 4–9. The arrowhead indicates Imax elicited by cAMP in the presence of PIP3. (C) Representative current traces (left), elicited by 1 mM cGMP (green) or 10 mM cAMP (red) before and after 1 µM PIP3, for A3 C7K/R-A (K624A, K629A, R643A, R646A, K657A, K659A, R661A) and RR-QQ (R643Q, R646Q) channels. The right panel summarizes effects of mutations within the A3 C-terminal region on PIP3-induced increases in cAMP efficacy; *, P < 0.05 compared with wild-type channels, n = 4–6. The broken horizontal lines refer to a ratio of one, equivalent to no change in the parameter after PIPn application. (D) In vitro pull-down assay using PIP3-bound or control agarose beads combined with GST fusion proteins representing the C-terminal region of CNGA3. Input proteins (bottom) and bound proteins (top) were identified by immunoblotting with anti-GST antibodies. A3C, C-terminal region of human CNGA3 from amino acids 612–694; A3C7K/R-A, C-terminal region with seven lysines and arginines neutralized to alanines, as highlighted in A; A3C-635X, C-terminal region of CNGA3 truncated at amino acid 635. Molecular weight markers (in kilodaltons) are shown on the left. Data are representative of four independent experiments. Error bars indicate mean ± SEM. Image published in: Dai G et al. (2013) © 2013 Dai et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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