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Figure 3. Effects of CNGB3 disease-associated mutations on the absolute agonist efficacy of cGMP and the spontaneous open probability. (A, Left) Overlay of four current traces from A3+B3 channels, activated by a sub-saturating concentration of cGMP (25 μM) to produce an approximately half-maximal current amplitude. Shaded area indicates the region of interest (ROI) used for the calculation of mean current and mean isochrone variance for each concentration of cGMP. (A, Right) ROI overlays at various cGMP concentrations (μM cGMP indicated on the right). Current and variance calculations were performed as described in the Materials and Methods. The mean isochrone variance is minimal at extreme low and high open probabilities and maximizes at 50% open probability. For wild type cone CNG channels, ~50% open probability is achieved at cGMP concentrations near the K1/2. The mean isochrone variance at saturating cGMP (i.e., 1 mM) reflects the maximum open probability (PO, MAX), such that a decreased variance indicates an increased PO, MAX (provided that PO, MAX > 50%). (B) Current-variance plot for A3+B3 wild type (closed circles) and A3+B3L595F (open circles) after activation by cGMP at +80 mV. The L595F mean current and variance estimates were scaled to WT IMAX to facilitate comparison of the initial slope from zero current and the variance at saturating ligand concentrations. Continuous lines produced by fitting with a 2nd order polynomial, as described in the Materials and Methods, using the following best-fit parameters: WT (solid), i = 3.1 pA, N = 1550 channels; L595F (hashed), i = 3.2 pA, N = 1370 channels. The mean variance at saturating cGMP is reduced for L595F channels (gray arrowhead) compared to WT channels (black arrowhead), indicating an increased PO, MAX for L595F channels. (C) Summary of maximal open probabilities after activation with saturating cGMP (1 mM). Maximal open probabilities were calculated as described in the Materials and Methods. The PO, MAX was significantly increased for Y469D and L595F channels compared to wild type (p < 0.01, single-factor ANOVA, n = 4–9; Holm's t-test, *p < 0.05, **p < 0.01). (D) Summary of unitary conductance values based on best-fit polynomials described in (B). The CNGB3 disease-associated mutations did not significantly alter the unitary conductance (p = 0.28; single-factor ANOVA). (E) Relationship between the spontaneous isochrone variance (σ2SP) and estimated number of channels (N) in the membrane patch for WT (black circles), L595F (open circles) and A3 only (gray circles) containing channels. The σ2SP and N estimates were determined as described in the Materials and Methods. Data fit with linear models using the following best-fit slopes (with the units: A2/channel x10−26): A3+B3 WT = 1.53; A3+B3 L595F = 4.98; A3 WT = 0.58. The slope of the relationship between σ2SP and N (dσ2SP/dN) was elevated for L595F channels compared to WT, reflecting an increase in the spontaneous open probability (PO, SP) for L595F channels. (F) Spontaneous open probabilities were calculated from trend-line slopes (dσ2SP/dN) from E. PO, SP calculations were based on the relationship: dσ2/dN = i2Poq, as described in Materials and Methods. Error estimates of PO, SP were propagated from the standard error of slopes from (E). We observed that the spontaneous open probability is significantly lower for A3 homomeric channels (A3WT, PO, SP = 0.50 ×10−3) compared WT heteromeric channels (A3+B3 WT, PO, SP = 1.6 x10−3) (***p < 0.001, extra sum-of-squares F-test, n = 6, 7). These estimates are in reasonable agreement with previous characterization of mouse cone CNG channels (Gerstner et al., 2000). We also observed that the PO, SP is significantly increased for L595F-containing channels (A3+B3 L595F, PO, SP = 4.3 ×10−3) compared to WT heteromeric channels (**p < 0.001, extra sum-of-squares F-test, n = 7, 8), reflecting a change in the intrinsic gating properties of L595F-containing channels. 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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