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J Biol Chem
2019 Nov 22;29447:17978-17987. doi: 10.1074/jbc.RA119.010246.
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N6-modified cAMP derivatives that activate protein kinase A also act as full agonists of murine HCN2 channels.
Leypold T
,
Bonus M
,
Spiegelhalter F
,
Schwede F
,
Schwabe T
,
Gohlke H
,
Kusch J
.
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cAMP acts as a second messenger in many cellular processes. Three protein types mainly mediate cAMP-induced effects: PKA, exchange protein directly activated by cAMP (Epac), and cyclic nucleotide-modulated channels (cyclic nucleotide-gated or hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels). Discrimination among these cAMP signaling pathways requires specific targeting of only one protein. Previously, cAMP modifications at position N6 of the adenine ring (PKA) and position 2'-OH of the ribose (Epac) have been used to produce target-selective compounds. However, cyclic nucleotide-modulated ion channels were usually outside of the scope of these previous studies. These channels are widely distributed, so possible channel cross-activation by PKA- or Epac-selective agonists warrants serious consideration. Here we demonstrate the agonistic effects of three PKA-selective cAMP derivatives, N6-phenyladenosine-3',5'-cyclic monophosphate (N6-Phe-cAMP), N6-benzyladenosine-3',5'-cyclic monophosphate (N6-Bn-cAMP), and N6-benzoyl-adenosine-3',5'-cyclic monophosphate (N6-Bnz-cAMP), on murine HCN2 pacemaker channels. Electrophysiological characterization in Xenopus oocytes revealed that these derivatives differ in apparent affinities depending on the modification type but that their efficacy and effects on HCN2 activation kinetics are similar to those of cAMP. Docking experiments suggested a pivotal role of Arg-635 at the entrance of the binding pocket in HCN2, either causing stabilizing cation-π interactions with the aromatic ring in N6-Phe-cAMP or N6-Bn-cAMP or a steric clash with the aromatic ring in N6-Bnz-cAMP. A reduced apparent affinity of N6-Phe-cAMP toward the variants R635A and R635E strengthened that notion. We conclude that some PKA activators also effectively activate HCN2 channels. Hence, when studying PKA-mediated cAMP signaling with cAMP derivatives in a native environment, activation of HCN channels should be considered.
Figure 1. cAMP effects on steady-state and non-steady state parameters of mHCN2 channel activation. A) Protocol and representative current traces to study concentration-dependent gating at a fixed command voltage. Tail currents were obtained from a -100 mV pulse following an activating -130 mV pulse. B) Protocol and representative current traces (exemplary for a saturating concentration of 10 µM cAMP) to study voltage-dependent gating at a fixed agonist concentration. A voltage family from -70 mV to -150 mV was applied with 10 mV increments. Tail currents were obtained from a -100 mV pulse following the variable test pulse. C) Concentration-response relationship for cAMP. Mean values for relative current
amplitudes (I/Imax) were obtained from 6 to 13 recordings and plotted against the cAMP concentration. The Hill equation (equation 1) was approximated to the data, yielding the concentration of half-maximum activation, EC50 (21.3±3.3 nM), and the Hill coefficient H (1.1±0.2), respectively. D) Steady-state activation relationship at zero and saturating [cAMP]. Mean values for relative current amplitudes (I/Imax) for zero cAMP (n=14) and for saturating cAMP of 10 µM (n=13) were plotted against the command voltage. The Boltzmann equation
was approximated to the data, yielding V1/2= -117.8±1.5 mV and a slope of zδ=4.2±0.3 for zero and V1/2= -97.9±1.9 mV and a slope of zδ=4.2±0.3 for 10 µM cAMP. E) Activation kinetics at different cAMP concentrations. Activation time constants were obtained from approximating a monoexponential function (equation 3) to the current time courses, yielding τa,conc. Mean values were obtained from 3 to 7 recordings. The protocol used is shown in A). F) Activation kinetics at different command voltages at zero and saturating [cAMP]. Activation time constants were obtained from approximating a monoexponential function (equation 3) to the current time courses, yielding τa,voltage. Mean values were obtained from 14 recordings for zero and 13 recordings for 10 µM cAMP. The protocol used is shown in B).
Figure 2. Structure and apparent affinities of the three tested derivatives.
A) Molecular formulas for three N6 -modified derivatives known to activate PKA. B) Representative current responses before and after application of the respective ligand. Black traces represent recordings in the absence of the ligand, colored traces represent recordings during application of a saturating concentration of the respective ligand. Each ligand turned out to be an activator of heterologously expressed HCN2 channels. C) Concentration-response relationships for the three derivatives (colored symbols and fits) in comparison to mHCN2 wildtype data (black symbols and fits). The Hill equation (equation 1) was approximated to the data to obtain the half-maximum concentration, EC50, and the Hill coefficient, H. Error bars indicate S.E.M. D) Box plot of EC50 values obtained from C. Filled circles indicate individual recordings. Error bars indicate S.D. E) Box plot of Hill coefficients obtained from C. Filled circles indicate individual recordings. Error bars indicate S.D.
Figure 3. Efficacy of the three tested derivatives.
A) Steady-state activation relationships at zero and saturating agonist concentrations. The graphs show the relationships for the tested derivatives at saturating concentrations and in the absence of any ligand. Except for N6 -Bnz-cAMP, for which 100 µM was used as saturating concentration, the concentration was 10 µM for saturation. The relative currents (I/Imax) are mean values obtained from 3 to 6 recordings. The Boltzmann equation was approximated to the relative current values to yield V1/2. The results of the fits were the following: -97.5±2.0
mV for N6 -Phe-cAMP, -104.9±2.7 mV for N6 -Bn-cAMP, and -98.3±1.4 mV for N6
-Bnz cAMP. The slopes were 3.4±0.2, 4.1±0.2, and 4.4±0.3 respectively. B, C) Comparison of the efficiency of the three derivatives in comparison to cAMP in two measures. B) Box plots of relative maximum current amplitude Imax / Imax, cAMP. Filled circles indicate individual recordings. Error bars indicate S.D. C) Box plots of maximum agonist-induced shift of halfmaximum voltage ∆V1/2. Filled circles indicate individual recordings. Error bars indicate S.D.
Figure 4. Activation kinetics for the tested cAMP-derivatives.
A) Activation kinetics in dependence on agonist concentration at a given command voltage of -130 mV. τa,conc values, obtained from approximating a monoexponential equation (Equation 3) to the current time courses, are plotted against the cAMP concentration. Mean values were obtained from 3 to 9 recordings. Black symbols in each plot illustrate the case for cAMP. The protocol used is shown in 1A). B) Activation kinetics in dependence on command voltage at a saturating agonist concentration. τa,voltage values were plotted against the command voltage. Open symbols represent recordings in the absence of ligands, filled symbols in the presence of saturating agonist concentrations. Mean values were obtained from 3 to 19 recordings. The protocol used is shown in 1B).
Figure 5. Predicted binding modes of the tested cAMP-derivatives.
Predicted binding modes of A) cAMP (grey); crystallographic pose (PDB ID: 1Q5O (25) shown in gold, B) N6 -Phe-cAMP (blue), C) N6 -Bn-cAMP (orange), and D) N6
-Bnz-cAMP (magenta). In panel A)-C), hydrogen bonds to and from protein side chains are depicted as dashed yellow lines, hydrogen bonds to and from the protein main chain as dashed green lines, and cation-π interactions as dashed blue lines. E) Predicted binding mode of the cAMP fragment in N6 -Bn-cAMP extended with a benzoyl group as found in N6 -Bnz-cAMP. The steric clash with R635 resulting from this extension is highlighted (red box), explaining the inverted binding mode of N6 -Bnz-cAMP shown in panel D).
Figure 6. Response to N6
-Phe-cAMP after charge neutralization or reversal at position R635 in the CNBD.
A) Representative current traces for R635A and R635E at zero and saturating cAMP. B) Concentration-activation relationships for mHCN2, R635A, and R635E. The Hill equation (equation 1) was approximated to the relative currents to obtain EC50 and the Hill coefficient. C) Box plot of EC50 values. Filled circles indicate individual recordings. Error bars indicate S.D. EC50 values obtained with N6
-Phe-cAMP for R635A and R635E, respectively, are not different from the EC50 value obtained for cAMP and mHCN2 (Student t-test).
Figure 7. Comparison of relative apparent affinity for PKA and HCN2.
A) Apparent affinities for cAMP derivatives were related to apparent affinities for unmodified cAMP. Amongst the tested derivatives the best discrimination can be realized with N6 -BnzcAMP. Data for PKA were obtained from: (29,34-36). Filled symbols represent PKAI, empty symbols PKAII. B) Sequence alignment comparing the distal parts of the C-helix sequences of mouse and human HCN isoforms with those from A and B sites in regulatory subunits of human PKAI and II. Alignments and structure predictions (α-helix C marked in green) are shown as proposed by Berman and co-workers (2005) (39), who used a structure and
transformation method (SAT). Prediction of the HCN C-helix follows a suggestion of Lee and MacKinnon (2017) (40). The asterisks mark R635 mutated herein. Positively charged residues are shown in red, negatively charged in blue.
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