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Figure 1. Current measurement and concentration-activation relationships in olfactory CNG channels. (a) Representative current recording from CNGA2 channels activated by saturating cGMP (1G, 100 µM) according to the indicated voltage protocol. The amplitude of the late current at +10 mV was evaluated for the concentration-activation relationships (black arrow). (b) Concentration-activation relationship for CNGA2 channels. The continuous curve was obtained by fitting equation (1) yielding EC50 = 1.47 µM and H = 2.52. (c) Concentration-activation relationship for heterotetrameric CNGA2:CNGA4:CNGB1b channels. EC50 = 1.24 µM and H = 2.09.
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Figure 2. Box plot of the effect of cNMP derivatives on the EC50 value for CNG channels. Shown are mean as a horizontal line within each box, the boxes as 25th and 75th percentiles and the whiskers as 10th and 90th percentiles of the data. The numeric mean values are indicated above each box. The asterisks indicate EC50 values that were significantly smaller than the respective natural cyclic nucleotide 1G/A (p < 0.01). (a) CNGA2 channels with cGMP derivatives. (b) CNGA2 channels with cAMP derivatives. (c) CNGA2:CNGA4:CNGB1b channels with cGMP derivatives. (d) CNGA2:CNGA4:CNGB1b channels with cAMP derivatives. Independent of the channel type and the cNMP, the most hydrophobic residues produce the highest apparent affinity whereas the hydrophilic residues, though similar by size, leave the apparent affinity approximately unaffected.
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Figure 3. Comparison of the effects of N-acetylated with non-acetylated thioalkylamine residues in CNGA2 channels. Box plot of the EC50 values. Generally, the longer residues cause a lower EC50 value (c.f. Supplementary Table 1a,b). The EC50 values of all cGMP and cAMP derivatives were significantly smaller (asterisks) than those of the natural cyclic nucleotides 1G and 1A, respectively (p < 0.01). The “#” indicates that compound 2A is a partial agonist.
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Figure 4. The hydrophobic residue in 8-position slows down the unbinding of 8-(Ac)AHT-cGMP (5G) compared to cGMP (1G). The channels were activated and deactivated by concentration jumps evoked by a piezo actuator switching between a control solution and a test solution containing a defined ligand concentration. (a) Superimposition of a current time course at 10 µM of 5G and 1G in CNGA2 channels. (b) Activation time constant τa as function of the 5G and 1G concentration in CNGA2 channels. (c) Deactivation time constant τd as function of the 5G and 1G concentration in CNGA2 channels. (d,e,f) Analog to a, b, c for CNGA2:CNGA4:CNGB1b channels. Error bars indicate SEM.
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Figure 5. Effects of cAMP derivatives on HCN2 channels. (a) Current traces in the absence and presence of saturating cAMP (20 µM) evoked by the indicated pulse scheme. The increase of the instantaneous current at 130 mV by cAMP (arrows) was evaluated. (b) Concentration-activation relationship for the cAMP-induced current increase of an individual experiment. The continuous curve was obtained by fitting the Hill equation (equation (1)) yielding EC50 = 25.42 nM and H = 1.28. (c) Box plot of the EC50 values for the indicated cAMP derivatives. Shown are mean as a horizontal line within each box, the boxes as 25th and 75th percentiles and the whiskers as 10th and 90th percentiles of the data. The numeric mean values are indicated above each box. The EC50 values were not significantly different from 1A apart from 3A and 8A which were significantly larger (asterisks; p < 0.01).
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Figure 6. Calculated relative binding free energies (ΔΔGbind) and conformational heterogeneity of cAMP derivatives in CNGA2 channels. (a) ΔΔGbind with respect to 8-AET-cAMP (2A) for cAMP derivatives of the congeneric series carrying C2-, C6-, and C10-substituents (3A–7A) for internal dielectric constants of ε = 1.0 (green circles) and ε = 4.0 (black, filled circles). Linear regression lines are drawn as solid lines in the respective colors, and the standard error of the estimate is indicated by the surrounding dashed lines. (b) Binding pose of 8-AHT-cAMP (4A; grey sticks, center) in a single CNBD. Residues within 5 Å of the ligand are depicted explicitly. Residues forming hydrogen bonds and/or electrostatic interactions to the ligand are depicted as sticks, other residues are depicted as lines. Lysines K486, K533 and K582 are colored in blue. A representative position in which the positively charged nitrogen of a C2 ligand would be is depicted as blue sphere; the spatial proximity to the surrounding lysines indicates a potential electrostatic repulsion in C2 derivatives that opposes binding. (c) Visualization of the calculated ADPs (ellipsoid scaling factor: 0.5) for the six cAMP derivatives 2A–7A. (d) Conformational heterogeneity of the tail in the respective cAMP derivatives in the complex with a CNGA2 channel. Bar heights represent the quadratic means of the pairwise root-mean-square deviation (2D-RMSD) of the atomic coordinates of the ligand tail region after root-mean-square fitting of the core/head region.
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Figure 7. Decomposition of the binding free energy of cAMP derivatives. Binding free energies were decomposed into their respective energy (a,c,e; electrostatic (elec), van der Waals (vdw), polar solvation (pol), and nonpolar solvation (nonpol)) and configurational entropy (b,d,f; translational (trans), rotational (rot), and vibrational (vib)) terms for ε = 1. (a,b) Difference between 8-AET-cAMP (2A) and 8-AHT-cAMP (C6; 4A) or 8-ADT-cAMP (C10; 6A). (c,d) Difference between 8-(Ac)AET-cAMP (3A) and 8-(Ac)AHT-cAMP (C6; 5A) or 8-(Ac)ADT-cAMP (C10; 7A). (e,f) Difference between 8-AET-cAMP (2A) and 8-(Ac)AET-cAMP (C2; 3 A), 8-AHT-cAMP (4A) and 8-(Ac)AHT-cAMP (C6; 5A), and 8-ADT-cAMP (6A) and 8-(Ac)ADT-cAMP (C10; 7A).
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S1. Ligand parameterization scheme. Groups for which inter-molecular charge constraints were applied are indicated by rounded boxes and a connecting line.
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S2. Energy profile of the rotation about the na-cc-ss-c3 torsion in 2-(methylthio)-1H-imidazole. The new parameters (black) improve the agreement with the QM-derived energies (green) by, on average, 0.80 kcal mol-1 with respect to the parameters derived from the parmchk2 tool (grey).
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S3. Root-mean-square deviation (RMSD) of the atomic coordinates of the (a) head region, (b) tail region, and (c) complete ligand of the investigated cAMP derivatives after root-mean-square fitting of the protein backbone. RMSD values were calculated for all four ligands separately with respect to the starting structure and were averaged over the whole MD trajectory. Error bars display sample standard deviations.
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S4.Conformational heterogeneity of the ligand in complexes of rCNGA2 and cAMP-derivatives. Pairwise root-mean-square deviations (2D-RMSD) of the atomic coordinates of the tail region of the investigated cAMP- derivatives after root-mean-square fitting of the core/head region are color-coded from blue (RMSD=0 A) over white (RMSD= 5A) to red (RMSD= 10A). (a) 8-AET-cAMP (2A), (b) 8-(Ac)AET-cAMP (3A), (c) 8-(Ac)AHT-cAMP (5A), (e) 8-ADT-cAMP (6A), (f) 8-(Ac)ADT-cAMP (7A). Each of the four subpanels represents one binding site (BS01-BS04).
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S5. Sequence alignment of human, mouse, and rat HCN1/2 and CNGA1/1. Top residue numbers correspond to HCN1/2, bottom residue numbers to rCNGA2. Secondary structure elements are indicated as rods (helices) and orange arrows (sheets). Residues within 5A of cNMPs are highlighted with green triangles. Identical regions are highlighted with white letters on black background, similar regions are highlighted with a surrounding box. Residues highlighted in blue have been described in other studies as important for cNMP binding in HCN2 channels.
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