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Figure 4. N-terminal region of CNGA3 can confer a PIPn-dependent change in apparent cGMP affinity after C-terminal truncation. (A) Subunit diagram and sequence alignment of the N-terminal region of human CNGA3 and rat CNGA2 subunits. The residues highlighted in yellow are conserved arginines in this region of the channel. P99 and A105 of CNGA3, and the corresponding L97 and P103 of CNGA2, are in red. Bold nucleotides indicate residues in human A3 that were mutated in our study as well as related residues in the paralogous rat A2. (B) Representative cGMP concentration–response plots for activation of four different homomeric CNGA3 channels before and after 1 µM PIP3: C-terminal deleted (A3-613X), combined N and C deletions (A3ΔN (51–107), 613X), N-terminal charge neutralizations (R72A, R75A, R81A, R82A, R86A, R101A, R103A) combined with C-terminal truncation (A3-N7R-A, 613X), or P99L, A105P (PA-LP) mutations. Currents were measured at +80 mV and were normalized to the maximum current elicited by a saturating concentration of cGMP. Continuous curves represent fits of the cGMP concentration–response relationship with the Hill equation. Hill parameters of the representative patches were as follows. For A3-613X, K1/2 = 14.7 µM, h = 1.55 before PIP3; K1/2 = 50.9 µM, h = 1.4 after PIP3. For A3 PA-LP, K1/2 = 5.7 µM, h = 1.7 before PIP3; K1/2 = 11.4 µM, h = 1.7 after PIP3. (C) Summary of PIP3-dependent changes in K1/2 cGMP for CNGA3 channels having the specific deletions and/or mutations shown; *, P < 0.05 compared with wild-type A3. (D) N-terminal domain of CNGA3 was expressed as a GST fusion protein and tested for PIP3 binding in vitro by using PIP3-agarose beads. A3N, N-terminal region (amino acids 1–164) of human CNGA3; A3N7R-A, seven arginines were neutralized to alanines within the N-terminal region; A3NΔ51-107, N-terminal region with amino acids 51–107 deleted. Molecular weight markers (in kilodaltons) are shown on the left. Data are representative of four independent experiments. (E) Deletions or charge neutralizations within the N-terminal region of CNGA3, A3 ΔN(2–156) or A3 N7R-A attenuated but did not eliminate the PIP3-induced increase in relative cAMP efficacy (Imax cAMP/cGMP), whereas P99L, L105P (PA-LA) mutations in the N-terminal region eliminated this increase; *, P < 0.05 compared with wild-type A3. The broken horizontal lines refer to a ratio of one, equivalent to no change in the parameter after PIPn application. 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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