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Figure 2. Current families for wild-type and mutant channels activated by cGMP. Current families are shown from inside-out patches excised from Xenopus laevis oocytes expressing BROD (A) and CHM15 (B) channels with mutations at position 604. Currents were elicited by 16 mM cGMP and voltage pulses from 0 mV to potentials between −80 and +80 mV in 20-mV steps. Leak currents in the absence of cyclic nucleotides were subtracted. The currents were normalized (using the +80-mV trace) to the maximum current obtained after Ni2+ potentiation (Gordon and Zagotta, 1995a) in the presence of a saturating concentration of the best agonist.
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Figure 3. Current families for wild-type and mutant channels activated by cIMP. Current families are shown from inside-out patches excised from Xenopus laevis oocytes expressing BROD (A) and CHM15 (B) channels with mutations at position 604. Currents were elicited by 16 mM cIMP and voltage pulses from 0 mV to potentials between −80 and +80 mV in 20-mV steps. Leak currents in the absence of cyclic nucleotides were subtracted. The currents were normalized (using the +80-mV trace) to the maximum current obtained after Ni2+ potentiation (Gordon and Zagotta, 1995a) in the presence of a saturating concentration of the best agonist.
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Figure 4. Current families for wild-type and mutant channels activated by cAMP. Current families are shown from inside-out patches excised from Xenopus laevis oocytes expressing BROD (A) and CHM15 (B) channels with mutations at position 604. Currents were elicited by 16 mM cAMP and voltage pulses from 0 mV to potentials between −80 and +80 mV in 20-mV steps. Leak currents in the absence of cyclic nucleotides were subtracted. The currents were normalized (using the +80-mV trace) to the maximum current obtained after Ni2+ potentiation (Gordon and Zagotta, 1995a) in the presence of a saturating concentration of the best agonist.
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Figure 5. Thermodynamic mutant cycles identify direct interactions between cyclic nucleotides and D604. (A) The effect of substituting the olfactory amino terminal region for the BROD amino terminal region was cyclic nucleotide independent. (B) The effect of mutating D604 to D604M was strongly cyclic nucleotide dependent, indicating that the amino acid at position 604 is critical for cGMP vs. cAMP discrimination by the channel.
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Figure 6. Representative single-channel traces at a saturating concentration of cGMP. Single-channel currents for all 10 constructs were recorded in the presence of 16 mM cGMP with the membrane voltage clamped at +80 mV. The upper and lower dotted lines indicated the open and closed levels, respectively, and are separated by 2.3 pA.
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Figure 8. Representative single-channel traces at a saturating concentration of cIMP. Single-channel currents for all 10 constructs were recorded in the presence of 16 mM cIMP with the membrane voltage clamped at +80 mV. The upper and lower dotted lines indicated the open and closed levels, respectively, and are separated by 2.3 pA.
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Figure 10. Representative single-channel traces at a saturating concentration of cAMP. Single-channel currents for all 10 constructs were recorded in the presence of 16 mM cAMP with the membrane voltage clamped at +80 mV. The upper and lower dotted lines indicated the open and closed levels, respectively, and are separated by 2.3 pA.
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Figure 7. Amplitude histograms for activation by a saturating concentration of cGMP. Amplitude histograms corresponding to the representative traces in Fig. 6 are shown. The histograms were fit to the sum of two Gaussians, and the peak of the closed level Gaussian was used to subtract off leak currents. The parameters for the amplitude histograms were as follows: for BROD, σclosed = 480 fA, σopen = 560 fA, Popen = 0.95; for CHM15, σclosed = 1.6 pA, σopen = 340 fA, Popen = 0.99; for BROD-D604E, σclosed = 490 fA, σopen = 430 fA, Popen = 0.71; for CHM15-D604E, σclosed = 850 fA, σopen = 240 fA, Popen = 0.97; for BROD-D604Q, σclosed = 380 fA, σopen = 890 fA, Popen = 0.06; for CHM15-D604Q, σclosed = 240 fA, σopen = 270 fA, Popen = 0.91; for BROD-D604N, σclosed = 270 fA, σopen = 560 fA, Popen = 0.07; for CHM15-D604N, σclosed = 260 fA, σopen = 290 fA, Popen = 0.92; for BROD-D604M, σclosed = 440 fA, σopen = 1.33 pA, Popen = 0.003; for CHM15-D604M, σclosed = 270 fA, σopen = 390 fA, Popen = 0.42.
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Figure 9. Amplitude histograms for activation by a saturating concentration of cIMP. Amplitude histograms corresponding to the representative traces in Fig. 8 are shown. The histograms were fit to the sum of two Gaussians, and the peak of the closed level Gaussian was used to subtract off leak currents. The parameters for the amplitude histograms were as follows: for BROD, σclosed = 240 fA, σopen = 360 fA, Popen = 0.74; for CHM15, σclosed = 530 fA, σopen = 270 fA, Popen = 0.96; for BROD-D604E, σclosed = 300 fA, σopen = 360 fA, Popen = 0.62; for CHM15-D604E, σclosed = 930 fA, σopen = 250 fA, Popen = 0.96; for BROD-D604Q, σclosed = 250 fA, σopen = 610 fA, Popen = 0.011; for CHM15-D604Q, σclosed = 320 fA, σopen = 360 fA, Popen = 0.81; for BROD-D604N, σclosed = 260 fA, σopen = 650 fA, Popen = 0.044; for CHM15-D604N, σclosed = 290 fA, σopen = 320 fA, Popen = 0.80; for BROD-D604M, σclosed = 320 fA, σopen = 610 fA, Popen = 0.009; for CHM15-D604M, σclosed = 210 fA, σopen = 340 fA, Popen = 0.59.
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Figure 11. Amplitude histograms for activation by a saturating concentration of cAMP. Amplitude histograms corresponding to the representative traces in Fig. 10 are shown. The histograms were fit to the sum of two Gaussians, and the peak of the closed level Gaussian was used to subtract off leak currents. The parameters for the amplitude histograms were as follows: for BROD, σclosed = 290 fA, σopen = 780 fA, Popen = 0.004; for CHM15, σclosed = 330 fA, σopen = 410 fA, Popen = 0.31; for BROD-D604E, σclosed = 230 fA, σopen = 630 fA, Popen = 0.05; for CHM15-D604E, σclosed = 300 fA, σopen = 390 fA, Popen = 0.61; for BROD-D604Q, σclosed = 360 fA, σopen = 580 fA, Popen = 0.009; for CHM15-D604Q, σclosed = 270 fA, σopen = 340 fA, Popen = 0.62; for BROD-D604N, σclosed = 250 fA, σopen = 660 fA, Popen = 0.015; for CHM15-D604N, σclosed = 300 fA, σopen = 390 fA, Popen = 0.71; for BROD-D604M, σclosed = 470 fA, σopen = 770 fA, Popen = 0.082; for CHM15-D604M, σclosed = 330 fA, σopen = 370 fA, Popen = 0.95.
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Figure 12. Box plot summaries for k01 and k10. Box plots of the k01 (A) and k10 (B) rate constants for the C0 ↔ O1 ↔ C2 scheme are shown for all 10 constructs for cGMP, cIMP, and cAMP. Values for the rate constants were determined by HMM analysis. The horizontal line within each box indicates the median of the data; boxes show the 25th and 75th percentiles of the data; whiskers show the 5th and 95th percentiles. Extreme data points are also indicated.
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Figure 13. Box plot summaries for k12 and k21. Box plots of the k12 (A) and k21 (B) rate constants for the C0 ↔ O1 ↔ C2 scheme are shown for all 10 constructs for cGMP, cIMP, and cAMP. Values for the rate constants were determined by HMM analysis.
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Figure 14. Box plot comparison between the values for k01/k10 and for L calculated from macroscopic experiments using Ni2+ potentiation. Box plots comparing the values for ΔG0 for the allosteric transition from singles and macroscopic current experiments are shown for the BROD (A) and CHM15 (B) channels.
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Figure 15. Free energy profile for the allosteric transition. The free energy profiles for the allosteric transition (C0 ↔ O1) are illustrated based on the median values of the rate constants determined from HMM analysis of the single-channel currents. The rate constants were converted to energies using simple transition-state theory, with the highest energy intermediate postulated to break down to product at the vibrational frequency of a covalent bond. (A) Free energy profiles for cGMP, cIMP, and cAMP on BROD channels. (B) Free energy profiles for cAMP with and without Ni2+. (C) The free energy profiles of the allosteric transition for BROD and BROD-D604M channels activated by a saturating concentration of cGMP. (D) The free energy profiles of the allosteric transition for BROD and CHM15 channels activated by a saturating concentration of cIMP.
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Figure 16. Fraction of energetic effect occurring after the allosteric transition. The fraction of the energetic effect occurring after the allosteric transition was calculated as ΔΔG‡/ΔΔG0 = |(ΔG‡1 − ΔG‡2)/(ΔG01 − ΔG02)|, where ΔG‡ is the activation energy for the closing transition, ΔG0 is the standard free energy for the allosteric transition, and the subscript indicates the different conditions of allosteric modulation. ΔGs were the median values determined from an HMM analysis of the single-channel currents. The ΔΔG‡/ΔΔG0 for the interactions with different cyclic nucleotides was the average of the values for BROD channels between cGMP and cIMP, cGMP and cAMP, and cIMP and cAMP. The ΔΔG‡/ΔΔG0 for the interactions with D604 was the average of the values between BROD channels and D604M channels and between CHM15 channels and CHM15-D604M channels for all three cyclic nucleotides. The ΔΔG‡/ΔΔG0 for the interactions with Ni2+ was the average of the values between BROD channels with and without Ni2+ for cIMP and cAMP (Sunderman and Zagotta, 1999). The ΔΔG‡/ ΔΔG0 for the interactions with the amino terminal region was the average of the values between BROD channels and CHM15 channels for all three cyclic nucleotides.
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