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Figure 2. Macular degeneration-associated Y469D and L595F mutations in CNGB3 each increase the sensitivity of heteromeric A3+B3 channels to physiological ligands. (A) Representative dose-response curves for activation of A3+B3 wild type (WT; filled circles), A3+B3Y469D (Y469D; open circles), A3+B3G558C (G558C; open triangles), and A3+B3L595F (L595F; open squares) channels by cGMP. Currents were normalized to the maximum cGMP current (IMAX = 1.0). Continuous curves represent fits of the dose-response relationship to the Hill equation as indicated in the Materials and Methods. Best-fit parameters are as follows: WT, K1/2 = 15 μM, nH = 2.4; Y469D, K1/2 = 10 μM, nH = 1.4; G558C, K1/2 = 13 μM, nH = 2.1; L595F, K1/2 = 5.7 μM, nH = 2.1. Shaded area represents approximate physiological cGMP concentration in photoreceptors in the dark (Pugh and Lamb, 1993). (B) Summary of apparent cGMP affinity (K1/2) for channels formed by CNGA3 with wild-type CNGB3 or CNGB3 having indicated disease-associated mutations. Data are based on best-fit Hill curves and expressed as mean K1/2 (± S.E.M.). The K1/2 cGMP was significantly reduced for the Y469D and L595F groups compared to wild type (p < 0.001, Kruskal-Wallis, n = 12–22; ***p < 0.001, Mann-Whitney U-test). (C) Summary of mean Hill coefficients (nH) for channels formed by CNGA3 with wild-type CNGB3 or CNGB3 having disease-associated mutations. The nH was significantly reduced for the Y469D, G558C, and L595F groups compared to wild type (p < 0.001, single-factor ANOVA, n = 5–9; ***p < 0.001, **p < 0.01 Holm's t-test). (D) Representative current traces of heteromeric human A3+B3 channels after activation by a sub-saturating concentration of cGMP (5 μM). Currents were scaled to the maximum current (IMAX) in a saturating concentration of cGMP (1 mM) as illustrated by the WT current traces. Relative sub-saturating current amplitudes (I5μM/IMAX) are as follows: WT = 0.05; Y469D = 0.25; G558C = 0.13; L595F = 0.43. (E) Representative current traces after activation by a saturating concentration of cGMP (1 mM, black line) or a saturating concentration of cAMP (10 mM, gray line). (F) Summary of relative agonist efficacy (IcAMP/IcGMP) for channels containing wild-type CNGB3 or disease-associated mutations, expressed as mean (± S.E.M.). The efficacy of cAMP was significantly enhanced for the Y469D and L595F channels compared to wild type (p < 0.001, Kruskal-Wallis, n = 5–12; ***p < 0.001, Holm's t-test). Image published in: Meighan PC et al. (2015) Copyright © 2015 Meighan, Peng and Varnum. 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 license Larger Image Printer Friendly View

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