Cyclic Nucleotide Mapping of Hyperpolarization-Activated Cyclic

Mar 7, 2014 - Cardiovascular Research Institute, University of California San Francisco, 555 Mission Bay Boulevard South, Rm 482, San Francisco,...
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Cyclic Nucleotide Mapping of Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels Stefan Möller,† Andrea Alfieri,‡ Daniela Bertinetti,† Marco Aquila,‡ Frank Schwede,§ Marco Lolicato,∥ Holger Rehmann,⊥ Anna Moroni,‡ and Friedrich W. Herberg†,* †

Department of Biochemistry, University of Kassel, Heinrich-Plett-Straße 40, 34132 Kassel, Germany Department of Biosciences, University of Milan, Via Celoria 26, 20133 Milano, Italy § Biolog Life Science Institute, Flughafendamm 9a, 28199 Bremen, Germany ∥ Cardiovascular Research Institute, University of California San Francisco, 555 Mission Bay Boulevard South, Rm 482, San Francisco, CA 94158, United States ⊥ Molecular Cancer Research, Centre of Biomedical Genetics and Cancer Genomics Centre, University Medical Center Utrecht, Universiteitsweg 100, 3584CG Utrecht, The Netherlands ‡

S Supporting Information *

ABSTRACT: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a central role in the regulation of cardiac and neuronal firing rate, and these channels can be dually activated by membrane hyperpolarization and by binding of cyclic nucleotides. cAMP has been shown to directly bind HCN channels and modulate their activity. Despite this, while there are selective inhibitors that block the activation potential of the HCN channels, regulation by cAMP analogs has not been well investigated. A comprehensive screen of 47 cyclic nucleotides with modifications in the nucleobase, ribose moiety, and cyclic phosphate was tested on the three isoforms HCN1, HCN2, and HCN4. 7-CH-cAMP was identified to be a high affinity binder for HCN channels and crosschecked for its ability to act on other cAMP receptor proteins. While 7-CH-cAMP is a general activator for cAMP- and cGMPdependent protein kinases as well as for the guanine nucleotide exchange factors Epac1 and Epac2, it displays the highest affinity to HCN channels. The molecular basis of the high affinity was investigated by determining the crystal structure of 7-CH-cAMP in complex with the cyclic nucleotide binding domain of HCN4. Electrophysiological studies demonstrate a strong activation potential of 7-CH-cAMP for the HCN4 channel in vivo. So, this makes 7-CH-cAMP a promising activator of the HCN channels in vitro whose functionality can be translated in living cells.

The topology shows following framework: An intracellular Nterminus is followed by six α-helical transmembrane domains (S1−S6), whereas a loop between S5 and S6 builds the ion selective pore. The S4 segment, which has the characteristic positively charged amino acids repeat acts as a voltage sensor. The intracellular C-terminal part contains a cyclic nucleotide binding domain (CNBD), which is linked to S6 by the so-called C-linker. We, as well as other groups, have determined the crystal structures of the intracellular C-terminal parts of the three isoforms HCN1, HCN2, and HCN4 both in the presence and absence of cAMP18−21 and also the HCN2 isoform in the presence of cGMP.22 The first two α-helices of the C-linker form an antiparallel helix-turn-helix motif, which interacts with the next two helices of the neighboring subunit, and are located on the top of the CNBD near the S6 transmembrane domain. The structure of the CNBD is very similar to other known eukaryotic CNBDs like in cAMP-dependent protein kinase (PKA), cGMP-

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of the six-transmembrane domain voltage gated K-channels and were discovered nearly 16 years ago.1 In mammals there are four different isoforms (HCN1−4), and these are differentially expressed in the heart and brain tissues.1−6 HCN2 is the main isoform in brain, whereas HCN4 is mainly found in the sinoatrial node of the heart.7−9 HCN channels play an important role in the regulation of neuronal and cardiac firing rate and function as “pacemaker” channels for the If and Iq/Ih currents in both heart and brain.10,11 These channels are dually activated by membrane hyperpolarization and binding of cyclic nucleotides (predominantly cAMP and cGMP).1,3,12 Currents generated by HCN channels have characteristic profiles where membrane hyperpolarization is followed by sigmoidal activation kinetics.13 HCN1 activation shows the fasted kinetics; this is followed by HCN2, HCN3, and then by HCN4.4,14−16 Furthermore, HCN1 channels are activated at more positive potentials compared to HCN2 and HCN4.17 HCN channels form either homotetrameric (identical subunits) or heterotetrameric (different subunits) complexes. © 2014 American Chemical Society

Received: December 9, 2013 Accepted: February 25, 2014 Published: March 7, 2014 1128

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Figure 1. Chemical structures of all 47 cyclic nucleotide analogs (group I−VIII). Analogs are numbered according to Table 1. Replacement of atoms is marked by a big white arrow, substitutions by a black arrow.

we reveal a HCN specific binder and explain its remarkable high affinity based on a newly determined crystal structure.

dependent protein kinase (PKG), and the guanine nucleotide exchange factor (Epac)23,24 consisting of three α-helices and eight β-strands. A phosphate binding cassette (PBC) lies between β-strand 6 and 7 and contains an additional α-helix called P-helix. Within the binding pocket cAMP is bound in the anti conformation whereas cGMP binds in syn.22 This syn conformation enables the formation of a hydrogen bond between a threonine and the nitrogen of the amino group at the purine ring. Although this interaction is specific for cGMP-regulated proteins,25,26 HCN channels are nevertheless cAMP specific due to three amino acids (arginine, isoleucine, and lysine) in the Chelix, which selectively stabilize the cAMP-bound relative to the cGMP-bound form.27 The three isoforms show a high similarity and differ only in some parts of the C-linker and within the β4-β5 loop of the CNBD.18 Until now, different studies were undertaken to discover isoform selective HCN channel blockers or agonists, which influence the activation potential of these channels.28−30 However, the activation by cyclic nucleotides and especially by analogs has not been thoroughly investigated. To date, very few studies address the influence of different modifications in cAMP or cGMP for the binding to HCN channels.31−33 Therefore, we initiated a comprehensive cyclic nucleotide screen, analyzing the binding of 47 cyclic nucleotide analogs to the C-terminal part comprising the C-linker and the CNBD (henceforth called CB) of the three isoforms HCN1, HCN2, and HCN4. We have systematically analyzed the effects of modifications in the nucleobase, the ribose moiety and the cyclic phosphate, respectively, in order to reveal new protein-nucleotide interaction sites. Some analogs were also tested against wellknown cyclic nucleotide binding proteins such as PKA, PKG, and Epac to determine the specificity of binding. Within this study,



RESULTS AND DISCUSSION Since the cytosolic fragment of the HCN channels tends to oligomerize, a N-terminal fused MBP-tag was used to avoid oligomerization. Therefore, all HCNCB were purified as monomers, not containing cAMP as shown in Lolicato et al. 2011.18 Using a fluorescence polarization (FP) assay, direct binding of fluorescent labeled cAMP and cGMP was determined first and subsequently the binding affinities of the analogs were analyzed in a solution competition assay.34 HCN1CB, HCN2CB, and HCN4CB Show High Affinity Binding to 8-Fluo-cAMP and 8-Fluo-cGMP. cAMP and cGMP with a fluorescein group at the 8-position (Figure 1) showed high affinity binding to HCN1CB, HCN2CB, and HCN4CB with KD values of 114 nM, 211 nM, and 189 nM for 8-Fluo-cAMP, respectively, and 169 nM, 459 nM, and 387 nM for 8-Fluo-cGMP (Table 1, Figure 2). All three isoforms show a higher affinity for the cAMP analog compared to the cGMP analog, with the lowest KD values for the HCN1 channel. For the FP-assays, used to determine binding affinities, both the concentrations of protein and 8-Fluo-cAMP were fixed while the competitive cyclic nucleotide was varied over a broad range. EC50 values about 3 μM for cAMP and about 11 μM for cGMP were determined in agreement with published data17,27,35 (Table 1, Figure 2). These data reflect the preference of HCN channels for cAMP.27,36 Other Naturally Occurring Cyclic Nucleotides Poorly Interact with HCN. cIMP shows a clearly reduced affinity (about 50 μM) compared to cAMP and cGMP and is generally not considered as a physiological agonist of HCN-channels. cCMP, cUMP, and cTMP contain a pyrimidine base instead of a 1129

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Table 1. List of all Measured KD and EC50 Values of the Three Isoforms and Comparison of the Data Relative to cAMPa EC50 ± SEM [μM] structure I

II

III

IV

V VI

VII

VIII

relative affinity compared to cAMP

no.

analog

MBP-HCN1

n

MBP-HCN2

n

MBP-HCN4

n

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

cAMP 1-NO-cAMP 2-DMA-cAMP 2-AHA-cAMP 3′-NH-cAMP 5′-NH-cAMP 6-MB-cAMP 6-AH-cAMP 7-CH-cAMP 8-Br-cAMP 8-N-cAMP 8-N-7-CH-cAMP 8-Cl-7-CH-cAMP 8-CPT-cAMP 8-AHA-cAMP 8-AHT-cAMP 8-[Fluo]-cAMPb 2′-O-Me-cAMP 8-pCPT-2′-O-MecAMP Sp-cAMPS Sp-8-Br-cAMPS Sp-8-Br-7-CH-cAMPS Sp-7-Br-7-CH-cAMPS Rp-cAMPS Rp-8-Br-7-CH-cAMPS Rp-7-CH-cAMPS Rp-7-CH-7-Br-cAMPS cPuMP 6-DMA-cPuMP 2-NH2-cPuMP cIMP cGMP 1-Me-cGMP 2-DM-cGMP N2-3-etheno-cGMP 7-CH-cGMP 8-Br-cGMP 8-pCPT-cGMP 8-AHT-cGMP 8-[Fluo]-cGMPb 2′-O-Me-cGMP Sp-cGMPS Sp-8-Br-cGMPS Rp-cGMPS cCMP cUMP cTMP

2.4 ± 0.2 x x 36 ± 5 x x 48 ± 2 9.6 ± 0.7 0.024 ± 0.006 0.116 ± 0.031 8.4 ± 0.9 17 ± 3 0.045 ± 0.008 x 1.6 ± 0.1 0.587 ± 0.041 0.114 ± 0.005 n.b. x

3 x x 4 x x 4 4 4 3 3 3 4 x 4 3 3 x x

2.0 ± 0.1 57 ± 5 17 ± 2 21 ± 2 n.b 378 ± 83 34 ± 5 7.1 ± 0.09 0.031 ± 0.004 0.102 ± 0.017 6.5 ± 0.8 4.2 ± 0.08 0.037 ± 0.0003 3.5 ± 0.1 3.2 ± 0.2 0.858 ± 0.152 0.211 ± 0.011 n.b. n.b.

8 6 3 2 x 3 9 2 6 10 2 2 2 3 2 2 7 x x

3.7 ± 0.6 122 ± 30 17 ± 4 41 ± 3 n.b 7063 ± 6133 27 ± 4 4.0 ± 0.2 0.034 ± 0.002 0.310 ± 0.064 9.1 ± 0.2 10.6 ± 0.9 0.072 ± 0.011 3.1 ± 0.6 4.3 ± 0.5 0.948 ± 0.242 0.189 ± 0.034 n.b. n.b.

13 6 3 2 x 3 9 2 6 11 2 2 2 3 2 2 9 x x

32 ± 5 1.5 ± 0.3 0.052 ± 0.013 1.3 ± 0.04 70 ± 10 0.938 ± 0.104 1.2 ± 0.01 4.8 ± 0.2 12 ± 0.9 9.7 ± 0.8 x 48 ± 2 6.8 ± 0.6 x x x 0.453 ± 0.013 0.388 ± 0.021 x 2.9 ± 0.4 0.169 ± 0.012 x 24 ± 3 2.0 ± 0.4 49 ± 5 57 ± 6 67 ± 13 x

4 4 4 3 3 2 3 3 3 3 x 4 3 x x x 3 3 x 3 3 x 4 3 3 4 3 x

32 ± 5 0.782 ± 0.042 0.053 ± 0.007 0.453 ± 0.004 274 ± 82 0.677 ± 0.040 1.1 ± 0.1 5.5 ± 0.5 5.6 ± 0.5 7.2 ± 0.4 4.9 ± 0.5 55 ± 4 14 ± 1 23 ± 2 66 ± 14 104 ± 26 0.409 ± 0.029 3.0 ± 0.6 4.1 ± 0.5 5.8 ± 1.1 0.459 ± 0.025 246 ± 69 32 ± 5 2.6 ± 0.08 283 ± 124 53 ± 9 53 ± 10 n.b

9 2 2 2 8 2 2 2 6 6 6 9 11 6 3 3 5 5 7 2 7 6 7 2 7 8 7 x

99 ± 31 1.8 ± 0.2 0.120 ± 0.010 1.5 ± 0.06 227 ± 60 1.9 ± 0.3 2.7 ± 0.2 6.8 ± 0.3 9.7 ± 0.9 3.7 ± 0.6 8.7 ± 1.3 46 ± 3 16 ± 1 17 ± 3 52 ± 5 173 ± 93 0.486 ± 0.065 3.5 ± 0.6 3.6 ± 0.5 9.2 ± 2.3 0.387 ± 0.034 574 ± 245 42 ± 4 2.2 ± 0.1 229 ± 108 191 ± 18 199 ± 26 n.b

9 2 2 2 6 2 2 2 6 6 7 9 10 6 2 3 5 6 7 2 9 6 7 2 4 9 10 x

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

MBP-HCN1 1.0

14.78

19.79 3.93 0.0097 0.0476 3.47 6.96 0.0183 0.665 0.242 0.0468

13.13 0.600 0.0214 0.531 28.89 0.386 0.476 1.99 5.05 3.99 19.67 2.79

0.186 0.160 1.21 0.069 10.02 0.838 20.03 23.27 27.69

MBP-HCN2

MBP-HCN4

1.0 29.11 8.41 10.65

1.0 33.32 4.77 11.34

192.51 17.29 3.60 0.0157 0.0522 3.31 2.15 0.0186 1.78 1.640 0.437 0.1074

1935.07 7.48 1.10 0.0093 0.0850 2.50 2.90 0.0198 0.86 1.189 0.260 0.0519

16.06 0.398 0.0271 0.231 139.94 0.345 0.546 2.78 2.87 3.69 2.49 28.12 7.19 11.88 33.74 52.93 0.209 1.504 2.073 2.93 0.234 125.47 16.44 1.303 144.01 27.06 26.95

27.02 0.500 0.0328 0.398 62.14 0.524 0.725 1.86 2.65 1.01 2.37 12.56 4.26 4.64 14.19 47.45 0.133 0.967 0.987 2.52 0.106 157.37 11.47 0.609 62.74 52.44 54.44

a 47 cyclic nucleotide analogs were grouped (I−VIII) accordingly to their chemical structure. bKD values determined by direct measurements. n.b., no binding detectable. x, not determined.

Cyclic Phosphate Contributes to High Affinity Binding. In Sp- and Rp-analogs (Figure 1), the axial and equatorial oxygen, respectively, were changed to sulfur. This modification reduced the affinity by several orders of magnitude. Both, Sp-cAMPS and Sp-cGMPS bind in the micromolar range (24−100 μM; Table 1). Rp-analogs show further reduction in affinity in comparison to the Sp-analogs to certain isoforms of HCN. While Rp-cAMPS and Rp-cGMPS bind to HCN2 and HCN4 in the high

purine base (Figure 1). The affinities for cCMP and cUMP lie in the range 50−200 μM, as published previously.37 Interestingly, cCMP and cUMP have a 4-fold-reduced affinity for HCN4 compared to HCN2, suggesting some selectivity between the main HCN isoforms (Figure 2). No binding could be detected for cTMP, which differs from cUMP only by an additional methyl-group. 1130

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Figure 2. Radarplot of binding data. EC50 values of HCN1 (red), HCN2 (green), and HCN4 (blue) are plotted in a logarithm scale. The values are sorted for HCN2 from low to high affinities ranging from nanomolar to micromolar. Modifications at the 7- and 8-position result in a shift to the low nanomolar range.

micromolar range (approx. 250 μM), both bind to HCN1 with affinities (approx. 60 μM) comparable to the Sp-analogs (Figure 2). Based on recent HCN crystal structures, the cyclic phosphate is involved in several interactions with the CNBD. The equatorial oxygen forms a salt bridge with Arg538/Arg618/Arg669 (HCN1/2/4) and a hydrogen bond with a backbone amide of Cys531/Cys611/Cys662. Mutations of this arginine result in a drastic reduction in binding affinity.17 The axial oxygen is only engaged in a hydrogen bond with Thr539/Thr619/Thr670 (Figure 3). In agreement with this binding mode, a sulfur in the equatorial position has a much stronger affinity reducing effect than a sulfur in the axial position. Interaction with the 2′-Hydroxyl Group of the Ribose Is Necessary for HCN Binding. The 2′-hydroxyl group is engaged in an extensive hydrogen bonding network, which include Gly528/Gly608/Gly659 and the highly conserved Glu529/Glu609/Glu660 (Figure 3) and mutations of the glutamate reduce affinity.27 Under experimental conditions used here, the methyl group in 2′-O-Me-cAMP weakens binding to all HCN isoforms (Table 1, Figure 2). The 2′-O-Me-group would prevent the formation of the hydrogen bonding network and may in addition incompatible with binding due to its steric demands. Similarly, 8-pCPT-2′-O-Me-cAMP is unable to bind HCN channels (Table 1, Figure 2). 8-pCPT-2′-O-Me-cAMP is widely used in biological research as a selective Epac activator,38,39 which does not act on PKA. However, the effect

of 8-pCPT-2′-O-Me-cAMP on HCN channels is unknown. Our results show that 8-pCPT-2′-O-Me-cAMP mediated effects on HCN channels can be excluded and support superior specificity of this analog to Epac. Modifications at the 7- and 8-Position Increase the Affinity for HCN. Several substitutions at the 8-position such as in 8-Br-cAMP or 8-AHT-cAMP increase the affinity of cAMP and cGMP (Table 1, Figure 2). This is in line with the high affinity of 8-Fluo-cAMP and 8-Fluo-cGMP described above. Similar effects are observed for PKA, PKG, and Epac, suggesting that this increase in affinity is a common property of CNBDs. Next to modifications at the 8-position the replacement of the heterocyclic nitrogen at position 7 to a CH-moiety drastically increases affinity to the low nanomolar range (Table 1, Figure 4a). In fact, 7-CH-cAMP displays the highest affinity of all analogs tested, with a 100-fold increased affinity compared to cAMP. The 7-CH- or the 8-Br-modification even compensate the affinity reducing effect of modifications in the cyclic phosphate for all three HCN isoforms. For example, Sp-8-Br7-CH-cAMPS binds HCN2 with an EC50 of 53 nM (600-fold increase compared to Sp-cAMPS) whereas the affinity of Sp-8Br-cAMPS was reduced from 32 μm to 782 nM (Figure 4b). To determine the binding and kinetics of 7-CH-cAMP we performed studies based on surface plasmon resonance (SPR). Experiments were performed with two set-ups. First, HCNproteins were competed off with 7-CH-cAMP from chips with immobilized cAMP analogs. Second, HCN-proteins were 1131

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Figure 3. Protein−ligand interactions in HCN channels. (a) Overlay of the CNBDs of mHCN1 (red, Swiss-Prot: O88704), hHCN2 (green, Swiss-Prot: Q9UL51) and hHCN4 (blue, Swiss-Prot: Q9Y3Q4) with cAMP. Important amino acids (bold and underlined), which interact with cAMP in all isoforms are presented in black lines, isoform-specific in colored lines. (b) CNBD of hHCN2 with cAMP (orange) in anti- and cGMP (green) in syn conformation. Protein−ligand interactions with cGMP are shown on the right.

covalently immobilized on chips and binding of 7-CH-cAMP was detected directly. In agreement with the data obtained by FP, affinities of about 20 nM were measured for 7-CH-cAMP in the competition experiments (for comparison FP data: see Table 1). Compared to cAMP, kon is slightly and koff is strongly reduced (Supporting Information (SI) Figure 1). Overall the change of koff is dominating and causes the increase in affinity. To further investigate the interaction of 7-CH-cAMP with HCN channels in detail, we only choose HCN4 as main isoform due to its highly interesting potential in treatment of heart failure. Particular Binding Mode Causes the Higher Affinity for 7-CH-cAMP. The thermodynamic properties of the 7-CHcAMP interaction were further investigated by Isothermal Titrations Calorimetry (Figure 4c, SI Figure 2). For the interaction of 7-CH-cAMP with HCN4 a KD of 30 nM was found again confirming the data obtained by FP and SPR. 7-CHcAMP shows a reduced binding enthalpy compared to cAMP (Figure 4c). While binding of cAMP is disfavored by a negative

binding entropy, a positive binding entropy is observed for 7CH-cAMP. The gain in affinity of 7-CH-cAMP is thus attributed to the favorable entropic contribution, at the same time overcompensating the loss in binding enthalpy. Structure of HCN4 in Complex with 7-CH-cAMP. To elucidate the structural determinants underlying the high affinity of 7-CH-cAMP for HCN proteins, the structure of HCN4 (aa 521−723) in complex with 7-CH-cAMP was solved at 2.5 Å resolution (SI Table 1). The structure displays a remarkable overlap with previously published structures of HCN4 in complex with cAMP,18,21,35 confirming the inherent flexibility of two distinct parts of the protein: the C-linker and the β4-β5 loop (Figure 5a). 7-CH-cAMP binds as cAMP in the anti conformation, and its binding pose is indistinguishable from that of cAMP (Figure 5b). The interactions between the protein and the phosphoribose moiety are highly conserved compared to the previous structures (SI Table 2). Even though the heterocyclic nitrogen at position 7 of cAMP has a weak potential to form 1132

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Figure 4. Effects of the modification at position 7 of the nucleobase. (a) Determination of the affinity by a competition fluorescence polarization assay. 7CH-cAMP (open circles) increases the affinity for all three isoforms to nanomolar EC50 values compared to cAMP (filled circles). (b) The combination of a modification at the 7- and 8- position shifts the affinity of Sp-analogs (low binders) to nanomolar affinity. (c) 7-CH-cAMP shows a reduced binding enthalpy compared to cAMP. The gain in affinity is due to the entropically favored binding mode, which overcompensates the loss in enthalpy.

Figure 5. Structure of HCN4 CNBD:7-CH-cAMP complex. (a) Superposition of HCN4 CNBD:7-CH-cAMP (orange), HCN4 CNBD:cAMP (PDB code 3OTF, black;21 PDB code 3U11_B, cyan18) and HCN1 CNBD:cAMP (PDB code 3U0Z, red18) performed on the cofactor atoms only and in the presence of 7-CH-cAMP (sticks). In the HCN4 CNBD:7-CH-cAMP structure the C-linker appears to fold upon the CNBD in a quite compact conformation. Root mean square deviation of the entire chain of HCN4 CNBD:7-CH-cAMP with 3OTF, 3U11_B, and 3U0Z are 0.51, 0.90, 0.67, respectively. (b) Close-up view of the cyclic nucleotide binding pocket in the HCN4 CNBD:7-CH-cAMP complex. The protein is shown as orange cartoon with 7-CH-cAMP as yellow sticks and C7 highlighted as a sphere. The main side chains giving apolar contribution to the cofactor binding pocket are represented as sticks surrounded by dotted spheres. The structure of chain B of HCN4 CNBD:cAMP (PDB code 3U11_B18) is superposed as in panel a for comparison and colored in cyan.

hydrogen bonds, none are observed. N7 is surrounded by a rather hydrophobic environment and engaged in van der Waals interactions. The binding pocket tolerates the replacement of

the nitrogen with a CH-moiety in 7-CH-cAMP, as no displacements of surrounding protein atoms are observed. The N7 in cAMP is electron richer compared to C7 in 7-CH-cAMP. 1133

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Table 2. Comparison of Affinity Values and Activation Constants for All Analyzed Proteinsa Kact

EC50 cAMP ratio 7-CH-cAMP 7-CH-cGMP Sp-8-Br-7-CH-cAMPS a

MBP-HCN1

MBP-HCN2

MBP-HCN4

hRIα

hRIβ

hRIIα

hRIIβ

Epac1

Epac2

hPKGIβ

2.4 μM 0.0097 24 nM 453 nM 52 nM

2.0 μM 0.0157 31 nM 409 nM 53 nM

3.7 μM 0.0093 34 nM 486 nM 120 nM

88 nM 2.2 190 nM 3 μM 325 nM

41 nM 1.9 78 nM 526 nMb 72 nMb

136 nM 1.6 222 nM 4 μM 341 nM

287 nM 1.0 290 nM 12 μM 618 nM

45 μM 0.289 13 μM x 8 μM

15 μM 0.5 8 μM x x

8.2 μM 0.088 719 nM 335 nM 2.3 μM

7-CH-cAMP shows selectivity for HCN proteins. bValues are determined with a R270A mutant. x, not determined.

Therefore N7 may enhance the polarization and consequently the strength of the van der Waals interactions. Such an effect may explain the higher binding enthalpy of cAMP compared to 7-CHcAMP. The polar properties of N7 make cAMP more hydrophilic than 7-CH-cAMP, which is in agreement with its phasedistribution properties as analyzed by gradient elution HPLC. As consequence of the N7- to CH-exchange, 7-CH-cAMP has a log K′w of 1.13 compared to a log K′w of 1.09 for cAMP,40 translating to a ∼10% increased relative lipophilicity.41 While N7 in cAMP is able to accept hydrogen bonds from water in solution, the 7-CH-moiety is not and therefore 7-CH-cAMP should display a stronger preference than cAMP for the hydrophobic environment in the binding pocket of HCN4 (Figure 5b). Indeed, as shown by the ITC data, 7-CH-cAMP binding is entropically favored, whereas cAMP binding is not. 7-CH-cAMP is a High Affinity Binder Specific for HCN Proteins. CNBDs are highly conserved not only between the HCN isoforms but also in the protein kinases PKA and PKG and the guanine nucleotide exchange factors Epac1 and Epac2.24 To determine whether high affinity binding of 7-CH-cAMP is specific for HCN channel proteins, this analog was tested with PKA type Iα, Iβ, IIα, IIβ, PKG Iβ and Epac 1 and 2. In this line, activation assays were performed with PKA and PKG with different cyclic nucleotide analogs. 7-CH-cAMP and Sp-8-Br-7-CH-cAMPS activate PKA similar or slightly reduced efficiently compared to cAMP (Table 2). 7-CH-cAMP activates hPKGIβ by a factor of 11 lower compared to cAMP. Interestingly, 7-CH-cGMP increases the activation constants compared to cGMP by a factor of 2 (Table 2). Epac1 and Epac2 are activated by cAMP in the micromolar range (Table 2). Here, 7-CH-cAMP activates better than cAMP (factor 2−3.5). Still the EC50 values of 7-CH-cAMP for EPAC are higher compared to those obtained for the HCN channels. The interactions of cyclic nucleotides with CNB domains of PKA42−44 and Epac43,45−48 and the prokaryotic cAMP receptor CAP49,50 have been thoroughly studied by NMR in order to gain insight into the dynamics of the protein−nucleotide interaction. Similar studies with HCN would be highly interesting, in particular as the dynamic properties may contribute to describe the entropy term of cyclic nucleotide binding obtained from our thermodynamic studies. 7-CH-cAMP Acts As a Full Agonist on HCN4 In Vivo. To confirm whether 7-CH-cAMP can activate HCN channels under physiological conditions, the effect of cAMP and 7-CH-cAMP on HCN4 channels, transiently expressed in HEK293 cells, was tested. Voltage-dependent HCN4 currents were recorded by patch clamp in whole cell configuration and the agonist applied via pipet perfusion. Both agonists caused an acceleration of current activation kinetics and a shift in the activation curve toward more positive voltages (cAMP by 15.7 mV and 7-CHcAMP by 16.6 mV) (Figure 6a,b). At the intermediate concentration of 300 nM, 7-CH-cAMP accelerated activation

Figure 6. Effect of 7-CH-cAMP and cAMP on HCN4 currents. (a) Family of current traces recorded in whole cell without (left, control) and with (right, cyclic nucleotide) 15 μM in the pipet solution (top, cAMP; bottom, 7-CH-cAMP). (b) Normalized activation curves of the HCN4 currents shown in panel a, in the absence (squares) and the presence (circles) of 15 μM agonist. (c) Normalized current traces in the presence of 300 nM cAMP or 7-CH-cAMP. (d) Dose−response curves evaluated as shift in V1/2 (mV) versus concentration of agonist cAMP (black circles) and 7-CH-cAMP (gray circles).

kinetics and, as expected from a gating activator, reduced channel deactivation kinetics, more efficiently than cAMP, indicating that the channel has a higher affinity for 7-CH-cAMP than for cAMP (Figure 6c,d). The plot shows the same maximal shift in V1/2 (about 17 mV) for both agonists at saturating concentration which means that 7-CH-cAMP acts as a full agonist on the whole channel. Moreover, 7-CH-cAMP has a 4-fold higher affinity than cAMP for HCN4 channels. Fitting the Hill equation to the mean data points (Figure 6d) yielded k1/2 = 0.82 ± 0.3 μM and 0.22 ± 0.4 μM for cAMP and 7-CH-cAMP, respectively. Concluding Remarks. We have performed a comprehensive cyclic nucleotide screen on the three isoforms HCN1, HCN2, and HCN4 and identified a HCN channel protein specific cyclic nucleotide analog, 7-CH-cAMP in which the nitrogen at position 7 is replaced by a CH-moiety. 7-CH-cAMP binds HCN channels in the low nanomolar range with an affinity 100-fold higher than cAMP and is selective for all HCN channels compared to PKA, PKG, and Epac. A new crystal structure of a HCN4:7-CH-cAMP complex explains the measured entropically favored binding mode of 7-CH-cAMP within the CNBD compared to cAMP resulting in the high affinity. 7-CH-cAMP acts as a full agonist of 1134

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Electrophysiology of HEK293 Cells. cDNA of human HCN4 was cotransfected for transient expression into HEK293 cells with a plasmid containing green fluorescent protein (GFP). One to five days after transfection, GFP-expressing cells were selected for patch-clamp experiments in whole-cell configuration at RT (25 to 26 °C). The pipettes used in whole-cell experiments contained (mM): 10 NaCl, 130 KCl, 1 EGTA, 0.5 MgCl2, 2 ATP, and 5 HEPES−KOH buffer (pH 7.2). The extracellular bath’s solution in whole-cell experiments contained (mM): 110 NaCl, 30 KCl, 1.8 CaCl2, 0.5 MgCl2, 1 BaCl2, 2 MnCl2, and 5 HEPES− NaOH buffer (pH 7.4). cAMP (Sigma) and 7-CH-cAMP (BioLog Life Science Institute, Bremen, Germany) were added at the indicated concentrations to the pipet’s solution in whole-cell or to the bath solution in inside-out. The activation curves were obtained by standard activation and deactivation protocols and analyzed by the Boltzmann equation, y = 1/{1 + exp[(V − V1/2)/s]}, where y is fractional activation, V is voltage, V1/2 half activation voltage, and s the inverse slope factor (mV). The dose−response curves were analyzed by the Hill equation, as follows: S/Smax = 1/[1+(k1/2/ [ligand])n], where S is the shift in V1/2, k1/2 is the half-maximal concentration, and n is the Hill factor. Values are given as mean ± SEM. Protein Crystallization. Crystallization trials by vapor diffusion technique were performed with an Oryx 8.0 robot (Douglas Instruments) and manually refined in hanging drop experiments, at 4 °C. Crystals of the complex were obtained in 4−6 days by mixing human HCN4 521−723 at 10 mg mL−1 (in the presence of 7-CH-cAMP 0.5 mM) in a 2:1 ratio with 100 mM TrisCl pH 8.0, 20% PEG4000 (w/v), then cryoprotected with the well solution supplemented with 30% glycerol and flashfrozen in liquid nitrogen. A full data set at 2.5 Å resolution was collected at BM14 beamline (ESRF, Grenoble, France) and processed with the CCP4 package54 (SI Table 1). Phases were extracted by molecular replacement with the program AMoRe55 using 3U11 PDB as search model.18 Structure files for 7-CHcAMP were generated using the PRODRG server,56 and the final structure was visualized by PyMOL.57 Accession Code. The atomic coordinates and structure factors have been deposited in the Protein Data Bank. The PDB code for the hHCN4 (aa 521−723):7-CH-cAMP structure is 4NVP.

HCN channels in vivo as shown by electrophysiological measurements. Therefore, 7-CH-cAMP provides a tool to the specifically target HCN channels.



METHODS Protein Expression and Purification of HCN Channels. Following plasmids were transformed into competent cells: pET24b murine HCN1 (aa 390−592), pET-24b human HCN2 (aa 470−672), pET-24b human HCN4 (aa 521−723), and pET-28 HRV3C. All HCN isoforms comprised the C-linker and the CNBD and were overexpressed and purified following the procedure described by Lolicato et al., 2011.18 For cleavage of the H6-MBP-tag the purified proteins were incubated with HRV3C protease overnight at 4 °C. The protein digest was loaded on a column prepacked with an amylose resin (New England Biolabs) to remove the free H6-MBP-tag. The flow-through was concentrated and loaded onto a Superdex 200 prep grade column and the corresponding HCN protein fractions were collected. The production of human HCN4 (aa 521−723) for structure determination followed the protocol of Lolicato et al., 2011.18 The final buffer contained 20 mM HEPES, pH 7.4, 150 mM NaCl, and 10% glycerol. Fluorescence Polarization. Direct FP-measurements were performed as described in Moll et al., 2006.34 All cyclic nucleotide analogs were purchased from Biolog Life Science Institute (Bremen, Germany) and were dissolved in 150 mM NaCl, 20 mM MOPS, pH 7, 10% DMSO (v/v). If necessary, analogs were treated by ultrasonic bath and heating (70 °C). The concentrations of the analogs were determined via their respective extinction coefficient. FP-measurements were performed in a Fusion α-FP microtiter reader at RT in a 384-well microtiter (PerkinElmer, Optiplate, black). At a fixed concentration of 1 nM 8-Fluo-cAMP and 8-Fluo-cGMP, the protein concentration was varied over a broad range. The FP signal was detected for 2 s at excitation of 485 nm and emission of 535 nm. Data were analyzed with GraphPad Prism.51 In competition experiments, the concentration of a given cyclic nucleotide analog was varied between pM and mM whereas the protein concentration was fixed corresponding to 80% of the maximum signal of a direct binding experiment. 8Fluo-cAMP was also fixed at 1 nM. EC50 values were determined by plotting the mPol signal against the logarithm of competitor concentration and fit the data with a sigmoidal dose−response curve. Measurements were repeated with different protein preparations and at least in duplicate for each cyclic nucleotide analog. All EC50 values were analyzed statistically and visualized in a radarplot. Isothermal Titration Calorimetry. The interaction between the MBP-HCN4 monomer and the cyclic nucleotides cAMP and 7-CH-cAMP was analyzed in storage buffer using a VP-ITC microcalorimeter (MicroCal, GE Healthcare). Protein (14−17 μM) was loaded into a 1.4 mL sample cell at 20 °C. The reference cell contained water. Nucleotide concentrations in the syringe were 70 μM (cAMP) and 120 μM (7-CH-cAMP). Two injections of 1 μL were performed before the titration experiment to ensure that the nucleotide concentration was at its loading state.52 Injections (10 μL) of the nucleotides were then carried out and heat changes were recorded. After returning to baseline level (about 7 min), a new injection was started. All nucleotides were solved in storage buffer. Calorimetric data were analyzed with the software Origin for MicroCal53 using a singlesite binding model.



ASSOCIATED CONTENT

S Supporting Information *

Protocols for SPR measurements and activation assays of PKA, PKG, and Epac; raw data of SPR and ITC analysis. Data collection and refinement statistics as well as protein−nucleotide interactions of the HCN4:7-CH-cAMP complex. This material is available free of charge via the Internet at http://pubs.acs.org



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

S.M., A.A., D.B., M.A., F.S., M.L., H.R., A.M., and F.W.H. designed the experiments; S.M., A.A., D.B., M.A., and H.R. performed the experiments; S.M., A.A., D.B., H.R., A.M., and F.W.H. wrote the paper. Notes

The authors declare the following competing financial interest(s): S.M., A.A., D.B., M.A., M.L., H.R., A.M., and 1135

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F.W.H. declare no competing financial interest. F.S. is head of R&D of BIOLOG Life Science Institute, that sells the cyclic nucleotide analogs used in this study.

guanosine-3′,5′-cyclic monophosphorothioate, Rp-isomer; cCMP, cytidine-3′,5′-cyclic monophosphate; cUMP, uridine3′,5′-cyclic monophosphate; cTMP, thymidine-3′,5′-cyclic monophosphate; appr, approximately; FP, fluorescence polarization; SPR, surface plasmon resonance; ITC, isothermal titration calorimetry; MSA, mobility shift assay



ACKNOWLEDGMENTS We thank D. Minor (University of California, San Francisco) and B. Santoro (Columbia University, New York) for the MBP and mHCN1 clones and Xention (Cambridge, U.K.) for the human HCN2 and HCN4 clones. Plasmids for expression of PKA subunits were a kind gift of S.S. Taylor (University of California at San Diego, CA). Special thanks go to C. Kim (Baylor College of Medicine, Houston, TX) for the provision of the hPKGIβ protein. We thank the EMBL staff H. Belrhali and B.A. Manjasetty for providing support on the beamline and EMBLDBT for providing access to the BM14 beamline at the ESRF. Also we thank E. Franz, M. Meinold, and O. Bertinetti (University of Kassel) for excellent technical assistance. F.W. Herberg acknowledges the support of the Federal Ministry of Education and Research Project 0316177FF ‘NoPainThe Nociceptor Pain Model’ and the European Union (EU) FP7 collaborative project Affinomics (Contract No. 241481).



REFERENCES

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ABBREVIATIONS HCN, hyperpolarization-activated cyclic nucleotide-gated; cAMP, adenosine-3′,5′-cyclic monophosphate; cGMP, guanosine-3′,5′-cyclic monophosphate; 7-CH-cAMP, 7-deaza-cyclic AMP; CNBD, cyclic nucleotide binding domain; PKA, cAMPdependent protein kinase; PKG, cGMP-dependent protein kinase; Epac, guanine nucleotide exchange factor; PBC, phosphate binding cassette; 8-Fluo-cAMP, 8-(2-[fluoresceinyl]aminoethylthio)-cyclic AMP; 8-Fluo-cGMP, 8-(2[fluoresceinyl]aminoethylthio)-cyclic GMP; 1-NO-cAMP, 1-Noxid-cyclic AMP; 2-DMA-cAMP, 2-dimethylamino-cyclic AMP; 2-AHA-cAMP, 2-(6-aminohexylamino)-cyclic AMP; 3′-NHcAMP, 3′-amino-cyclic AMP; 5′-NH-cAMP, 5′-amino-cyclic AMP; 6-MB-cAMP, N6-monobutyryladenosine-cyclic AMP; 6AH-cAMP, N6-(6-aminohexyl)-cyclic AMP; 8-Br-cAMP, 8Bromo-cyclic AMP; 8-N-cAMP, 8-aza-cyclic AMP; 8-N-7-CHcAMP, 8-aza-7-deaza-cyclic AMP; 8-Cl-7-CH-cAMP, 8-chloro7-deaza-cyclic AMP; 8-CPT-cAMP, 8-(4-chlorophenylthio)cyclic AMP; 8-AHA-cAMP, 8-(6-aminohexylamino)-cyclic AMP; 8-AHT-cAMP, 8-(6-aminohexylthio)-cyclic AMP; 2′-OMe-cAMP, 2′-O-methyl-cyclic AMP; 8-pCPT-2′-O-Me-cAMP, 8-(4-chlorophenylthio)-2′-O-methyl-cyclic AMP; Sp-cAMPS, adenosine-3′,5′-cyclic monophosphorothioate, Sp-isomer; Sp8-Br-cAMPS, 8-brom-Sp-cAMPS; Sp-8-Br-7-CH-cAMPS, 8bromo-7-deaza-Sp-cAMPS; Sp-7-Br-7-CH-cAMPS, 7-bromo-7deaza-Sp-cAMPS; Rp-cAMPS, adenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer; Rp-8-Br-7-CH-cAMPS, 8bromo-7-deaza-Rp-cAMPS; Rp-8-Br-cAMPB, 8-bromo-adenosine-3′,5′-cyclic monophosphoroborat, Rp-isomer; Rp-7-CHcAMPS, 7-deaza-Rp-cAMPS; Rp-7-CH-7-Br-cAMPS, 7-deaza-7bromo-Rp-cAMPS; cPuMP, purine riboside-3′,5′-cyclic monophosphate; 6-DMA-cPuMP, 6-dimethylamino cPuMP; 2-NH2cPuMP, 2-amino cPuMP; cIMP, inosine-3′,5′-cyclic monophosphate; 1-Me-cGMP, N1-methyl-cyclic GMP; 2-DM-cGMP, 2-dimethyl-cyclic GMP; 7-CH-cGMP, 7-deaza-cyclic GMP; 8Br-cGMP, 8-bromo-cyclic GMP; 8-pCPT-cGMP, 8-(4-chlorophenylthio)-cyclic GMP; 8-AHT-cGMP, 8-(6-aminohexylthio)-cyclic GMP; 2′-O-Me-cGMP, 2′-O-methyl-cyclic GMP; SpcGMPS, guanosine-3′,5′-cyclic monophosphorothioate, Spisomer; Sp-8-Br-cGMPS, 8-bromo-Sp-cGMPS; Rp-cGMPS, 1136

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