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Figure 1. Amino acid sequences of the S2âS3 loop of various wild-type CNG channels. Top diagram demonstrates the primary structure and membrane topology of a CNG channel. All wild-type sequences contain a glycine residue at the site equivalent to position 204 (highlighted in bold and underlined) in the Rolf channel. Residues corresponding to the equivalent Rolf residues at positions 199â207 are very highly conserved in all channels. In contrast, there is more variation in the carboxyl-terminal half of the loop sequence. Rolf, rat olfactory; Folf, fish olfactory; Bolf, bovine olfactory; Molf, mouse olfactory; Brod, bovine rod; Hrod, human rod; Bcone, bovine cone; Hcone, human cone; Ccone, chick cone; Btestis, bovine testis; and Drosant, Drosophila antenna. The original Molf clone contained a mutation at position 204 (Molf G204E), and we mutated the glutamate (E) to the wild-type glycine (G). For the Rolf channel, the glycine at 204 was replaced with either glutamate, aspartate (D), or tryptophan (W). Replacement with a lysine residue (K) gave no functional expression.
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Figure 2. Sensitivities to cGMP and cAMP of Molf G204E and Rolf G204E CNG channels were similar to those of the wild-type Molf and Rolf channels. Brod and Rolf data are from the companion article (Crary et al. 2000, in this issue). Steady state, cGMP-activated currents were measured at +100 mV. (A) cGMP doseâresponse curves for the three wild-type channels, Molf G204E, and Rolf G204E channels. SDs are indicated by error bars. Smooth curves were drawn by fitting the averaged data with the Hill equation, I/Imax = [cNMP]n/(K1/2n + [cNMP]n), where I is the cGMP- or cAMP-activated current, Imax is the cGMP-activated current obtained at saturating cGMP, K1/2 is the concentration of cGMP or cAMP giving half-maximal activation, and n is the Hill coefficient. In fitting the data, this relation was scaled to match the measured maximal I/Imax (e.g., the maximal I/Imax for the rod channel in the presence of saturating cAMP was only 0.05 [B], rather than 1, as for saturating cGMP). The Hill relation was used only for an empirical description of the data to quantify changes in the apparent affinity and maximal activation (or inhibition; see Fig. 4 and Fig. 8); it is not meant to suggest a mechanism of channel activation or inhibition by DAG. The calculated K1/2 values were 31 μM (Brod; 11 patches), 1.2 μM (Molf; 10 patches), 2.1 μM (Rolf; 18 patches), 1.9 μM (Molf G204E; 8 patches), and 1.8 μM (Rolf G204E; 6 patches). Hill fits for Brod and Rolf channels are designated by dashed lines for emphasis. (A) Doseâresponse curves for activation by cAMP; the symbols are the same as those used in A. Averaged data points are shown with the SD as error bars: Brod (8 patches), Rolf (7 patches), Molf (5 patches), Rolf G204E (1 patch), and Molf G204E (4 patches). Hill fits to channel data indicate K1/2 values for activation by cAMP of 1.83 mM for Brod and 37 μM for Rolf and are represented by dashed lines. See Table for additional information.
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Figure 3. Current families from patches at saturating cGMP showed greater inhibition of current by DAG in Molf G204E and Brod channels than in Rolf channels. Current traces were obtained from excised, inside-out patches. Leak currents in the absence of cGMP have been subtracted. Membrane potentials were held at 0 mV and jumped in increments of 50 mV from â100 to +100 mV. The families on the left were obtained at saturating cGMP (2 mM cGMP for Brod and 100 μM for Rolf and Molf G204E), and the families on the right were obtained after the addition of 1.5 μM DAG.
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Figure 4. DAG completely suppressed saturating cGMP-activated currents of Molf G204E and Rolf G204E CNG channels, but only partially inhibited the corresponding wild-type channels at saturating agonist concentrations. Brod and Rolf data from the companion article (Crary et al. 2000, in this issue) are shown for comparison. Saturating cGMP concentrations were 2 mM for Brod channels and 100 μM for all Molf and Rolf channels. Averaged data obtained at saturating cGMP were fit with the Hill equation, IN/INmax = [DAG]n/(IC50n + [DAG]n) where IN is the percent inhibition, INmax is maximal inhibition, IC50 is the concentration of DAG required to achieve half-maximal inhibition, and n is the Hill coefficient. As for Fig. 2, the relation was scaled to reflect the measured maximal IN/INmax. For the Brod, Molf G204E, and Rolf G204E channels, the maximum inhibition was 100%. For Brod (11 patches), IC50 = 0.83 μM, and n = 1.7; for Molf G204E (12 patches), IC50 = 0.99 μM, and n = 3.6; and for Rolf G204E (5 patches), IC50 = 0.73 μM and n = 2.8. For the wild-type olfactory channels, inhibition is only partial. For Rolf (13 patches), INmax = 31%, IC50 = 3.24 μM (denoting 15.5% inhibition), and n = 1.4; and for Molf (5 patches), INmax = 17%, IC50 = 3.50 μM (denoting 8.5% inhibition), and n = 0.8. Hill fits of Brod and Rolf channels are designated by dashed lines. Error bars indicate SDs.
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Figure 5. A glutamate at position 204 of the Molf or Rolf channel produced gating kinetics similar to those of the Brod channel. In the absence of DAG, the Molf channel has no detectable gating kinetics, whereas Molf G204E has obvious gating kinetics, much like the Brod channel. Currents were obtained with either 10 μM (Brod), 1 μM (Molf), or 1 μM (Molf G204E) cGMP in response to voltage jumps ranging from â100 to +100 mV in steps of 50 mV, from a holding potential of 0 mV. For clarity, only currents at â100, 0, and +100 mV are shown. At +100 mV, the observed currents had an I/Imax of 0.20, 0.15, and 0.15, respectively (from left to right).
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Figure 6. A glutamate at position 204 of the Molf or Rolf channel produced voltage-dependent gating similar to that of the Brod channel. To analyze the data from multiple patches, the doseâresponse curve from each patch was fit with the Hill relation, and each set of data was normalized to the cGMP K1/2 at +100 mV for that patch. Each different symbol represents the normalized data points from a single patch; open symbols for â100 mV and closed symbols for +100 mV. All points for each voltage were re-fit with the Hill equation, setting the normalized K1/2 for +100 mV at 1.0; a change in the normalized K1/2 at â100 mV indicates voltage-dependent gating. (A) Brod channel (six patches): at +100 mV, n = 2.0; and at â100 mV, the normalized cGMP K1/2 = 1.4 and n = 2.0. (B) Rolf channel (six patches): at +100 mV, n = 2.8; and at â100 mV, the normalized cGMP K1/2 = 1.0 and n = 2.8. (C) Rolf G204E channel (2 patches): at +100 mV, n = 1.7; and at â100 mV, the normalized cGMP K1/2 = 1.7 and n = 1.7.
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Figure 8. The Rolf G204W mutant had a slightly greater DAG sensitivity than that of the Brod channel, and its inhibition was similarly voltage-independent (compare with Fig. 4 in companion article, Crary et al. 2000, in this issue). (A) The DAG doseâresponse curve was measured in the presence of 2 mM cGMP: data were averaged from three patches and fit with the Hill equation. The maximum inhibition for the Rolf G204W mutant was 100% with an IC50 = 0.26 μM and n = 3.2. The Hill fits for the Brod and Rolf channels (from Fig. 4) are shown for comparison as dashed curves. (B) Steady state currentâvoltage relations obtained from data acquired on a single patch containing the Rolf G204W mutant. Open circles represent cGMP-activated currents at saturating concentrations in the absence of DAG. Filled circles represent currents at saturating cGMP in the presence of 0.8 μM DAG. For comparison, the triangles show the currents at lower (5 μM) cGMP concentration, without DAG.
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Figure 7. Introduction of tryptophan at position 204 produced a mutant Rolf channel with measurable gating kinetics, lower apparent agonist affinity, and decreased cAMP efficacy. (A) The current families were obtained with the designated amounts of cGMP in response to voltage jumps ranging from â100 to +100 mV in 50-mV steps, from a holding potential of 0 mV. The traces were corrected for leak by subtracting responses in the absence of cGMP. (B) The doseâresponse curves for activation by cGMP at + 100 mV and â100 mV are shown for a single patch containing the Rolf G204W mutant; the data were fit with the Hill relation, as in Fig. 2 (see legend). For this patch, at +100 mV, the cGMP K1/2 = 14 μM and n = 2.0; and at â100 mV, the cGMP K1/2 = 18 μM and n = 2.0. For averaged data from six patches, at +100 mV, the K1/2 = 14 μM and n = 1.7; and at â100 mV, the K1/2 = 19 μM and n = 1.7. The Hill fits (Fig. 2) for the cGMP doseâresponse curves of the Rolf and Brod channels (at +100 mV) are shown for comparison. (C) cAMP is only a partial agonist for Rolf G204W, unlike the Rolf channel (Hill fit from Fig. 2 is shown for comparison). A doseâresponse curve for activation by cAMP is shown for a single patch: K1/2 = 440 μM and n = 0.9. The average efficacy for two patches was 44%.
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Figure 9. All mutant olfactory channels had greater sensitivity to tetracaine than did the wild-type olfactory channels. Averaged data are reported for percent inhibition at 40 μM tetracaine for Brod (3 patches), Rolf (10 patches), Molf (5 patches), Molf G204E (4 patches), Rolf G204E (4 patches), Rolf G204D (2 patches), and Rolf G204W (4 patches). SDs are indicated by error bars. Values of percent inhibition are found in Table .
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