The comparison of the ¹H-¹⁵N HSQC spectra of TRIP8b_nano with and without CNBD bound shows that the peptide folds upon interaction with the CNBD. Thus, the ¹H-¹⁵N HSQC spectrum of TRIP8b_nano without CNBD shows a limited ¹H resonance dispersion, characteristic of unstructured proteins (Dyson and Wright, 2004), while a larger number of well-dispersed amide signals appear in the spectrum of the CNBD-bound form (Figure 1C). Importantly, we were now able to assign the backbone chemical shift resonances of TRIP8b_nano bound to the CNBD. The φ and ψ dihedral angles obtained from the NMR assignment indicate that the peptide displays two α-helices (stretch L₂₃₈-E₂₅₀ named helix N and stretch T₂₅₃-R₂₆₉ named helix C) when bound to CNBD. The helices are separated by two amino acids; three and six residues at the N- and C- termini, respectively, are unstructured (Figure 1D).

NMR-analysis of the CNBD fragment bound to TRIP8b_nano revealed that the interaction with the peptide does not affect the overall fold of the protein. Thus, the CNBD adopts the typical fold of the cAMP-free state, in line with previous evidence that this is the form bound by TRIP8b (Saponaro et al., 2014; DeBerg et al., 2015) More specifically, the secondary structure elements of the cAMP-free CNBD are all conserved in the TRIP8b_nano–bound CNBD (Figure 2). This finding generally agrees with a previous double electron-electron resonance (DEER) analysis of the CNBD - TRIP8b interaction, which showed that TRIP8b binds to a conformation largely similar to the cAMP-free state (DeBerg et al., 2015). Despite the overall agreement with the DEER study, the NMR data also reveal a new and unexpected feature of TRIP8b binding to the CNBD. Indeed, our results show that TRIP8b_nano binding to the CNBD induces a well-defined secondary structure of the distal region of the C-helix (Figure 2). This means that the distal region of the C-helix (residues 657–662), which is unstructured in the free form of the CNBD (Saponaro et al., 2014; Lee and MacKinnon, 2017), extends into a helical structure upon ligand binding irrespectively of whether the ligand is cAMP (Puljung and Zagotta, 2013; Saponaro et al., 2014; Lee and MacKinnon, 2017) or TRIP8b (Figure 2). In contrast, and very differently from cAMP, which directly contacts the P-helix in the Phosphate Binding cassette (PBC) and causes its folding (Saponaro et al., 2014; Lee and MacKinnon, 2017), the NMR data show that TRIP8b_nano binding to the CNBD does not induce P-helix formation (Figure 2).

Figure 2

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Despite the significant improvement in sample stability and NMR spectra quality achieved upon TRIP8b_nano binding, we were still unable to assign the side chains of both proteins in the complex and thus could not solve the solution structure of the complex by the canonical NMR procedure. We therefore built a model (Figure 3) of the CNBD - TRIP8b_nano complex by docking the two NMR-derived structures described above using the Haddock program (a detailed description of how the respective structures were generated is provided in Materials and methods and Figure 3—source data 1).

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Figure 3—source data 1

Acquisition parameters for NMR experiments performed on cAMP-free human HCN2 CNBD in complex with TRIP8b_nano and vice-versa.

https://doi.org/10.7554/eLife.35753.011

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Figure 3—source data 2

Docking calculation.

https://doi.org/10.7554/eLife.35753.012

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In order to define the active residues (ambiguous interaction restraints) on the CNBD we used the chemical shift perturbation values as described in Figure 3—figure supplement 1. For TRIP8b_nano, we defined as active a stretch of residues, E₂₃₉-E₂₄₃, previously identified as critical for the interaction (Santoro et al., 2011). Output clusters of this first molecular docking calculation (settings can be found in Materials and methods) were further screened for TRIP8b_nano orientations in agreement with a previous DEER analysis, which placed TRIP8b residue A₂₄₈ closer to the proximal portion and TRIP8b residue A₂₆₁ closer to the distal portion of the CNBD C-helix (DeBerg et al., 2015). Remarkably, in all clusters selected in this way, residues E₂₆₄ or E₂₆₅ in TRIP8b were found to interact with residues K₆₆₅ or K₆₆₆ of the CNBD (Figure 3—figure supplement 2). This finding was notable, because we previously identified K₆₆₅/K₆₆₆ as being critical for TRIP8b interaction in a biochemical binding assay (Saponaro et al., 2014). We thus proceeded to individually mutate each of these four positions, and test their effect on binding affinity through ITC. As expected, reverse charge mutations K₆₆₅E or K₆₆₆E (CNBD) as well as E₂₆₄K or E₂₆₅K (TRIP8b_nano) each strongly reduced the CNBD/TRIP8b_nano binding affinity (Figure 3—figure supplement 3).

Based on these observations, we performed a second molecular docking calculation, including E₂₆₄ and E₂₆₅ as additional active residues for TRIP8b_nano. This procedure resulted in the model shown in Figure 3, which represents the top-ranking cluster for energetic and scoring function (Figure 3—source data 2) and was fully validated by mutagenesis analysis as described below. Scrutiny of the model shows that TRIP8b_nano binds to both the C-helix and the N-bundle loop (Figure 3A). Binding to the C-helix is mainly guided by electrostatic interactions between the negative charges on TRIP8b_nano, and the positive charges on the CNBD (Figure 3A). As shown in Figure 3B, the model highlights a double saline bridge (K₆₆₅ and K₆₆₆ of CNBD with E₂₆₅ and E₂₆₄ of TRIP8b_nano) in line with the ITC results described above (Figure 3—figure supplement 3). Of note, the contribution of residue R₆₆₂ to the binding is also consistent with previous experiments showing residual TRIP8b interaction in a CNBD deletion mutant ending at position 663 (Saponaro et al., 2014). Our modeling data suggest that, upon folding of the distal portion of the C-helix, the side chains of residues R₆₆₂ and K₆₆₅ face to the inside when contacting cAMP, but face to the outside when binding TRIP8b (Figure 3—figure supplement 4). This indicates that cAMP and TRIP8b directly compete for the binding to the distal region of C-helix.

In addition to clarifying the role of residues in the distal portion of the CNBD C-helix, the model also highlights a second important cluster of electrostatic interactions, with R₆₅₀ in the proximal portion of the CNBD C-helix contacting E₂₄₀ and E₂₄₁ in helix N of TRIP8b_nano (Figure 3C). To confirm the contribution of these residues, we reversed charges and tested each residue mutation for binding in ITC. The results in Figure 3—figure supplement 3 show that R₆₅₀E caused a more than six-fold reduction in binding affinity for TRIP8b_nano, with smaller but significant effects seen also for E₂₄₀R and E₂₄₁R.

A third important contact highlighted by the model is the interaction between N₅₄₇ in the N-bundle loop of the CNBD and D₂₅₂ in the link between helix N and helix C of TRIP8b_nano (Figure 3D). We tested this potential interaction by disrupting the expected hydrogen bond between N₅₄₇ and the carboxyl group of the negative residue (D₂₅₂) in TRIP8b_nano. The asparagine in CNBD was mutated into aspartate (N₅₄₇D) to generate an electrostatic repulsion for D₂₅₂, and the carboxyl group in D₂₅₂ of TRIP8b_nano was removed by mutation into asparagine (D₂₅₂N). As predicted, N₅₄₇D greatly reduced binding to TRIP8b in ITC assays (Figure 3—figure supplement 3), with a smaller but significant effect observed also for D₂₅₂N (Figure 3—figure supplement 3). These results confirm and extend our previous finding that the N-bundle loop contributes in a substantial manner to the binding of TRIP8b (Saponaro et al., 2014). To understand the mechanism for the allosteric effect of TRIP8b on cAMP binding, which has been postulated on the basis of electrophysiological and structural data (Hu et al., 2013; Saponaro et al., 2014), we further tested by ITC the affinity of the N₅₄₇D CNBD mutant for cAMP. Somewhat surprisingly, the affinity of the mutant is much lower than that of the wt (N₅₄₇D K_D = 5.5 ± 0.4 µM (n = 3) vs. wt K_D = 1.4 ± 0.1 µM (n = 3)). Moreover, we measured a reduced sensitivity to cAMP also in patch clamp experiments where addition of 5 μM cAMP caused a right shift in the V_1/2 of the mutant HCN2 channel of only 5 mV while the wt channel shifted by 12 mV (Figure 3—figure supplement 5). To exclude that the N₅₄₇D mutation affects the overall structure of the CNBD, we performed the NMR (¹H-¹⁵N HSQC spectrum) analysis of the N₅₄₇D CNBD mutant. Our data show that the protein is appropriately folded (Figure 3—figure supplement 6). In conclusion, since the N-bundle loop does not directly contact any of the residues of the cAMP binding pocket, these findings underscore a previously unaddressed role of the N-bundle loop in allosterically modulating cAMP binding to the CNBD (see Discussion).

Next, we asked whether the relatively short TRIP8b_nano could be used to block cAMP-dependent modulation of HCN channels by delivering the peptide to full length channels. To this end, we dialyzed TRIP8b_nano into the cytosol of HEK 293 T cells transfected either with HCN1, HCN2, or HCN4 channels. The peptide was added (10 µM) in the recording pipette together with a non-saturating concentration of cAMP (5 µM for HCN2, 1 µM for HCN4) expected to induce a ~ 10 mV rightward shift in the half-activation potential (V_1/2) of the channels (Figure 4). No cAMP was added in the case of HCN1, because, in HEK 293 T cells, this isoform is already fully shifted to the right by the endogenous cAMP and does not respond further (Figure 4—figure supplement 1). Indeed, it is possible to induce a ~10 mV left shift in HCN1 V_1/2 by introducing the mutation R₅₄₉E that prevents cAMP binding to the CNBD (Figure 4—figure supplement 1).

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Figure 4A–C show representative current traces recorded at four given voltages, in control solution, cAMP, and cAMP +10 µM TRIP8b_nano (HCN4 and HCN2) or +10 µM TRIP8b_nano only (HCN1) in the patch pipette. Already from a visual comparison of the most positive voltage at which the current appears measurable, it is evident that TRIP8b_nano counteracts the activating effect of cAMP on the voltage-dependent gating. In the case of HCN1, the effect of TRIP8b_nano can be observed without added cAMP for the aforementioned reasons. Figure 4D–F show the mean channel activation curves obtained from the above and other experiments. Fitting the Boltzmann equation to the data (solid and dashed lines of Figure 4D–F, see Materials and methods for equation) yielded the half-activation potential values (V_1/2) plotted in Figure 4G. The addition of TRIP8b_nano prevents the cAMP-induced right shift of about 13 mV in HCN4 (V_1/2 = -102.8, –89.2, −102.1 mV, for control, cAMP and cAMP + TRIP8b_nano, respectively), and of about 11 mV in HCN2 (V_1/2 = -93.7, –83.5, −94.5 mV, for control, cAMP and cAMP + TRIP8b_nano, respectively). In HCN1, TRIP8b_nano induced a left shift in V_1/2 of about 10 mV (from – 72.8 to −82 mV) which is comparable to that induced by the R₅₄₉E mutation (from −72.7 to −80.4 mV) (Figure 4—figure supplement 1).

Figure 4G also shows the result of a control experiment performed on HCN4 where 10 µM TRIP8b_nano was added to the extracellular medium (TRIP8b_nano bath) in order to test if the peptide was able to cross the cell membrane (current traces and activation curves not shown). The V_1/2 value, which is similar to that of cAMP alone (−91.7 vs. −89.2 mV), confirmed that TRIP8b_nano peptide affects channel gating only if added to the intracellular solution presumably because it does not diffuse through the cell membrane.

It is worth noting that TRIP8b_nano prevents other related effects of cAMP activation in HCN channels, such as the acceleration of activation kinetics (Wainger et al., 2001) and, for HCN2 only, the increase in maximal current (Chen et al., 2007; Hu et al., 2013). For example, the activation kinetics (τ_on) of HCN4 measured at −120 mV was: control = 2 ± 0.2 s, 1 μM cAMP = 1.2 ± 0.1 s, 1 μM cAMP + 10 μM TRIP8b_nano = 2 ± 0.3 s (Figure 4—figure supplement 2). Moreover, Figure 4B clearly shows that TRIP8b_nano fully prevented the increase in maximal current in HCN2.

Based on these results, we reckoned the peptide may be employed as a regulatory tool for native I_f/I_h currents. As proof of principle, we tested whether TRIP8b_nano can modulate the frequency of action potential firing in SAN myocytes. In these cells, I_f is key contributor of the diastolic depolarization phase of the pacemaker action potential cycle. Moreover, the autonomic nervous system modulates the frequency of action potential firing by changing intracellular cAMP levels, which in turn acts on f-HCN channel open probability (DiFrancesco, 1993). We thus recorded the native I_f current in acutely isolated rabbit SAN myocytes with and without 10 µM TRIP8b_nano in the pipette solution (Figure 5A). Figure 5B shows that the averaged I_f activation curve measured in presence of TRIP8b_nano is significantly shifted to hyperpolarized voltages compared to the control. This indicates that the peptide is displacing the binding of endogenous cAMP to native HCN channels. Moreover, when the experiment was repeated in the presence of 1 µM cAMP, TRIP8b_nano prevented the typical cAMP-dependent potentiation of the native I_f current (Figure 5B). In light of these results, we tested whether TRIP8b_nano is also able to modulate cardiac automaticity by antagonizing basal cAMP. The data in Figure 5C show that TRIP8b_nano indeed significantly decreased the rate of action potential firing in single SAN cells. Strikingly, the observed 30% decrease in action potential rate corresponds to the effect induced by physiological concentrations of acetylcholine (DiFrancesco et al., 1989).

Figure 5

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To conclusively prove that the inhibition of the native I_f current was specifically due to TRIP8b_nano rather than caused by the dilution of the cellular content following whole cell configuration, we created a TAT version of TRIP8b_nano (hereafter TAT-TRIP8b_nano). Indeed, the TAT sequence allows the entry of biomolecules into a cell via endocytosis and/or direct translocation across the plasma membrane, thus leaving the cytosolic content unaltered (Guidotti et al., 2017).

We therefore tested whether both TRIP8b_nano and TAT-TRIP8b_nano were able to selectively inhibit the β-adrenergic stimulation of I_f current, while leaving the potentiation of L-type Ca²⁺ current (I_Ca,L) unaltered. To this end, we recorded either the native I_f or I_Ca,L current from cardiomyocytes acutely isolated from mouse sinoatrial node (SAN) in the presence and in the absence of 10 µM TRIP8b_nano or TAT-TRIP8b_nano, before and after stimulation with 100 nM isoproterenol (ISO), a β-adrenergic receptor agonist (Figure 6). Strikingly, TRIP8b_nano prevented the isoproterenol-induced increase of I_f current density, both when the peptide was added in the recording pipette solution (Figure 6A and B), and when it was used in the TAT version added to the bath (Figure 6A and C). The specificity of TRIP8b_nano for I_f current was confirmed by the absent inhibition of basal I_Ca,L (Figure 6D). In addition, we failed to record a significant difference in the isoproterenol-stimulated increase of the I_CaL current density between the control condition and 10 µM TRIP8b_nano (Figure 6E) or TAT-TRIP8b_nano (Figure 6F) conditions. To test whether the TAT-TRIP8b_nano effect described above was exclusively due to TRIP8b_nano peptide, we repeated the experiments with a scrambled version of the peptide (TAT- (SCRAMBLED) TRIP8b_nano) to exclude that the effect could be due to the TAT sequence (Figure 6—figure supplement 1). We failed to observe a significant reduction in the responsiveness of I_f to isoproterenol in the presence of TAT- (SCRAMBLED) TRIP8b_nano confirming that prevention of cAMP induced f- current stimulation was specific of the TRIP8b_nano sequence.

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In this study, we have identified the minimal binding peptide that reproduces the effects of TRIP8b on HCN channel gating. The peptide is 40 aa long and binds the HCN CNBD with high affinity (K_D = 1.4 μM). By solving the NMR structures of TRIP8b_nano and HCN CNBD in the bound form, we generated a structural model of their complex. The model provides detailed information on this protein-protein interaction at the atomic level with implications on their physiological function. The data show that the minimal binding unit of TRIP8b, TRIP8b_nano, folds in two helices upon binding and suggest that this region is intrinsically disordered when it is not bound. The model structurally validates previous indirect evidence, which suggested that TRIP8b binds to two discrete elements of the CNBD: the N-bundle loop and the C-helix (Saponaro et al., 2014). The complex forms by electrostatic interactions, which are spread throughout the contact surface. As a consequence of the interaction with TRIP8b_nano, the C-helix of CNBD increases in length, a behavior previously observed in the case of cAMP binding (Puljung and Zagotta, 2013). This portion of C-helix includes the two residues R₆₆₂ and K₆₆₅ engaged in salt bridge formation with respectively E₂₅₀/D₂₅₇ and E₂₆₄ of TRIP8b_nano. It is important to note that these two cationic residues are also involved in cAMP binding (Zagotta et al., 2003; Zhou and Siegelbaum, 2007; Lolicato et al., 2011). The finding that TRIP8b and cAMP share the same binding sites on the C-helix provides a solid molecular explanation for functional data, which imply a competition between the two regulators (Han et al., 2011; DeBerg et al., 2015; Bankston et al., 2017). Another study, however, has indicated that a direct competition model cannot fully explain the mutually antagonistic effect of the two ligands (Hu et al., 2013). Specifically, the fact that the inhibitory effect of TRIP8b on channel activity persists even at saturating cAMP concentrations advocated an allosteric component in the regulation mechanism. Our data, showing that the N₅₄₇D mutation in the N-bundle loop controls cAMP affinity in the binding pocket support the conclusion that the N-bundle loop allosterically controls cAMP binding. This is not surprising, given its crucial role of mechanically transducing to the pore the cAMP binding event within the CNBD (Saponaro et al., 2014).

The structural model also explains why a previously identified peptide selected by Lyman et al. (2017) failed to reproduce the binding affinity of the TRIP8b_core for the CNBD. This 37 aa long fragment is lacking one important contact residue, namely E_240, which, in our model, forms a salt bridge with R₆₅₀ of the CNBD. The loss of one crucial interaction is presumably the reason for the major decrease in affinity (about 20 times lower) reported for this peptide.

In functional assays, we showed that TRIP8b_nano binds the HCN channel CNBD with high affinity and fully abolishes the cAMP effect in all tested isoforms (HCN1, 2 and 4).

Given the small size of the peptide (<5 kDa), TRIP8b_nano is a good candidate for in vivo delivery into intact cells. As a proof of concept, we fused TRIP8b_nano to an internalization sequence, the TAT peptide (YGRKKRRQRRRGG). This arginine-rich Cell Penetrating Peptide (CPP) from HIV has been used in several studies as a vehicle for the delivery of large molecules across the plasma membrane (Guidotti et al., 2017). In our case, the challenge was to construct a TAT-fusion protein, which would be efficiently delivered in the cell without compromising TRIP8b_nano function. Indeed, covalent conjugation of a CPP may negatively affect both the function of the cargo, and the cell-penetrating efficacy of the CPP-peptide chimera (Kristensen et al., 2016). The design of the construct was greatly supported by the detailed knowledge of the electrostatic interactions with the target protein CNBD, provided by the NMR model structure. This structure suggested that the polycationic TAT sequence would be best linked to the N-terminus of TRIP8b_nano to avoid interference with the cationic residues of CNBD, mainly located in the distal region of C-helix, which are crucial for the binding of the peptide. From test experiments with the TAT-TRIP8b_nano peptide in SAN myocytes, we can conclude that this strategy was successful in that: (i) the peptide is efficiently delivered inside the cells; (ii) it is kept in its active conformation; (iii) the TAT sequence did not damage cell membranes and did not interfere with the basic features of I_f and I_Ca,L currents; (iv) the modification did not affect the proteolytic stability of the TRIP8b_nano peptide at least in the time frame of our experiments (30 min to 1 hr).

In conclusion, we successfully used the miniaturized TRIP8b_nano peptide to selectively control native I_f currents and the rate of spontaneous firing in SAN myocytes. Unlike channel blockers, which inhibit ionic currents, the peptide only interferes with the cAMP-based regulation of HCN channels, while leaving basal HCN functions unaltered. In addition and in contrast to even the most selective blockers, it is selective for HCN and it does not interfere with other cAMP-modulated channels present in the SAN, such as L-type Ca²⁺ channels. Collectively, this makes TRIP8b_nano a promising tool in targeted therapeutic interventions.

The cDNA fragment encoding residues 235–275 (TRIP8b_nano) of mouse TRIP8b (splice variant 1a4) was cloned into pET-52b (EMD Millipore) downstream of a Strep (II) tag sequence, while the cDNA fragment encoding residues 521–672 of human HCN2 (HCN2 CNBD) was cloned, in a previous study, into a modified pET-24b downstream of a double His₆-maltose-binding protein (MBP) (Saponaro et al., 2014). The cDNA encoding full-length human HCN1 channel and mouse TRIP8b (1a4) were cloned into the mammalian expression vector pcDNA 3.1 (Clontech Laboratories), while mouse HCN2 channel and rabbit HCN4 channel were cloned into the mammalian expression vector pCI (Promega). Mutations were generated by site-directed mutagenesis (QuikChange site-directed mutagenesis kit; Agilent Technologies) and confirmed by sequencing.

The HCN2 CNBD WT and mutant proteins, as well as the TRIP8b_core and TRIP8b_nano proteins (WT and mutants) were produced and purified following the procedure previously described (Saponaro et al., 2014).

NMR experiments were acquired on Bruker Avance III 950, 700 and 500 MHz NMR spectrometers equipped with a TXI-cryoprobe at 298 K. The acquired triple resonance NMR experiments for the assignment of backbone resonances of cAMP-free HCN2 CNBD (CNBD hereafter) in complex with TRIP8b_nano and vice versa are summarized in Figure 3-source data 1. ¹⁵N, ¹³C’, ¹³C_α, ¹³C_β, and H_α chemical shifts were used to derive ϕ and ψ dihedral angles by TALOS + program (Cornilescu et al., 1999) for both CNBD and TRIP8b_nano. For TRIP8b_nano, CYANA-2.1 structure calculation (Güntert and Buchner, 2015) was performed using 68 ϕ and ψ dihedral angles and 40 backbone hydrogen bonds as input. For CNBD, CYANA-2.1 structure calculation was performed using 108 ϕ and ψ dihedral angles, combined with the NOEs obtained in our previous determination of the cAMP-free form of the CNBD (Saponaro et al., 2014) for those regions not affected by the interaction with TRIP8b_nano. The 10 conformers of TRIP8b_nano and CNBD with the lowest residual target function values were subjected to restrained energy minimization with AMBER 12.0 (Case, 2012) (http://pyenmr.cerm.unifi.it/access/index/amps-nmr) and used as input in docking calculations.

Docking calculations were performed with HADDOCK2.2 implemented in the WeNMR/West-Life GRID-enabled web portal (www.wenmr.eu). The docking calculations are driven by ambiguous interaction restraints (AIRs) between all residues involved in the intermolecular interactions (Dominguez et al., 2003). Active residues of the CNBD were defined as the surface exposed residues (at least 50% of solvent accessibility), which show chemical shift perturbation upon TRIP8b_nano binding.

The assignment of the CNBD bound to TRIP8b_nano allowed to highlight the residues of CNBD whose backbone featured appreciable Combined Chemical Shift Perturbation (CSP) (Figure 3—figure supplement 1). The combined CSP (Δ_HN) is given by the equation Δ_HN={((H_Nfree−H_Nbound)²+((N_free−N_bound)/5)²)/2}½ (Garrett et al., 1997).

Passive residues of CNBD were defined as the residues close in space to active residues and with at least 50% solvent accessibility.

In the case of TRIP8b_nano, the conserved stretch E₂₃₉-E₂₄₃, located in helix N, was defined as active region in a first docking calculation, while all the other solvent accessible residues of the peptide were defined as passive. This docking calculation generated several clusters. A post-docking filter step allowed us to select those clusters having an orientation of TRIP8b_nano bound to CNBD in agreement with a DEER study on the CNBD - TRIP8b_nano interaction (DeBerg et al., 2015). The selected clusters grouped in two classes on the basis of the orientation of helix N of TRIP8b_nano (N) relative to CNBD (Figure 3—figure supplement 2. A second docking calculation was subsequently performed introducing also residues E₂₆₄-E_265, located in helix C of TRIP8b_nano as active residues. The active residues for CNBD were the same used for the first calculation. For this second HADDOCK calculation, 14 clusters were obtained and ranked according to their HADDOCK score. Among them only four clusters showed both an orientation of TRIP8b_nano bound to CNBD in agreement with the DEER study (DeBerg et al., 2015) and the involvement of E₂₃₉-E₂₄₃ stretch of TRIP8b_nano in the binding to CNBD. These clusters were manually analyzed and subjected to a per-cluster re-analysis following the protocol reported in http://www.bonvinlab.org/software/haddock2.2/analysis/#reanal. From this analysis, it resulted that the top-ranking cluster, i.e. the one with the best energetic and scoring functions, has a conformation in agreement with mutagenesis experiments (Figure 3—figure supplement 3). Energy parameters (van der Waals energy, electrostatic energy, desolvation energy, and the penalty energy due to violation of restraints) for this complex model are reported in Figure 3—source data 2.

Both docking calculations were performed using 10 NMR conformers of both the CNBD and the TRIP8b_nano structures calculated as described above. In the TRIP8b_nano structures the unfolded N- and C-terminal regions were removed, while in the CNBD structures only the unfolded N-terminal region was removed. This is because the C-terminal region of the CNBD is known to comprise residues involved in TRIP8b_nano binding (Saponaro et al., 2014). Flexible regions of the proteins were defined based on the active and passive residues plus two preceding and following residues. The residue solvent accessibility was calculated with the program NACCESS (Hubbard and Hornton, 1993). In the initial rigid body docking calculation phase, 5000 structures of the complex were generated, and the best 400 in terms of total intermolecular energy were further submitted to the semi-flexible simulated annealing and a final refinement in water. Random removal of the restraints was turned off. The number of flexible refinement steps was increased from the default value of 500/500/1000/1000 to 2000/2000/2000/4000. The final 400 structures were then clustered using a cutoff of 5.0 Å of RMSD to take into consideration the smaller size of protein-peptide interface.

HEK 293 T cells were cultured in Dulbecco’s modified Eagle’s medium (Euroclone) supplemented with 10% fetal bovine serum (Euroclone), 1% Pen Strep (100 U/mL of penicillin and 100 µg/ml of streptomycin), and stored in a 37°C humidified incubator with 5% CO₂. The plasmid containing cDNA of wild-type and mutant HCN1, HCN2 and HCN4 channels (1 µg) was co-transfected for transient expression into HEK 293 T cells with a plasmid containing cDNA of Green Fluorescent Protein (GFP) (1.3 µg). For co-expression with TRIP8b (1a-4), HEK 293 T cells were transiently transfected with wild-type (wt) and/or mutant human HCN1 cDNA (1 µg), wt TRIP8b (1a-4) cDNA (1 µg) and cDNA of Green Fluorescent Protein (GFP) (0.3 µg).

One day after transfection, GFP-expressing cells were selected for patch-clamp experiments in whole-cell configuration. The experiments were conducted at R.T. The pipette solution in whole cell experiments contained: 10 mM NaCl, 130 mM KCl, 1 mM egtazic acid (EGTA), 0.5 mM MgCl₂, 2 mM ATP (Mg salt) and 5 mM HEPES–KOH buffer (pH 7.4). The extracellular bath solution contained 110 mM NaCl, 30 mM KCl, 1.8 mM CaCl₂, 0.5 mM MgCl₂ and 5 mM HEPES–KOH buffer (pH 7.4).

TRIP8b_nano was added (10 µM) to the pipette solution. cAMP was added at different concentration to the pipette solution depending on the HCN isoform used: 0 µM for HCN1, 5 µM for HCN2 and 1 µM for HCN4.

Whole-cell measurements of HCN channels were performed using the following voltage clamp protocol depending on the HCN isoform measured: for HCN1, holding potential was –30 mV (1 s), with steps from –20 mV to –120 mV (10 mV interval, 3.5 s) and tail currents recorded at –40 mV (3 s); for HCN2, holding potential was –30 mV (1 s), with steps from –40 mV to –130 mV (10 mV interval, 5 s) and tail currents recorded at −40 mV (5 s); for HCN4, holding potential was –30 mV (1 s), steps from –30 mV to –165 mV (15 mV interval, 4.5 s) and tail currents were recorded at −40 mV (5 s). Current voltage relations and activation curves were obtained by the above activation and deactivation protocols and analyzed by the Boltzmann equation, see data analysis.

Animal protocols conformed to the guidelines of the care and use of laboratory animals established by Italian and European Directives (D. Lgs n° 2014/26, 2010/63/UE). New Zealand white female rabbits (0.8–1.2 kg) were anesthetized (xylazine 5 mg/Kg, i.m.), and euthanized with an overdose of sodium thiopental (i.v.); hearts were quickly removed, and the SAN region was isolated and cut in small pieces. Single SAN cardiomyocytes were isolated following an enzymatic and mechanical procedure as previously described (DiFrancesco et al., 1986). Following isolation, cells were maintained at 4°C in Tyrode solution: 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5.5 mM D-glucose, 5 mM HEPES-NaOH (pH 7.4).

For patch clamp experiments cells were placed in a chamber on an inverted microscope and experiments were performed in the whole-cell configuration at 35 ± 0.5°C. The pipette solution contained: 10 mM NaCl, 130 mM KCl, 1 mM egtazic acid (EGTA), 0.5 mM MgCl₂, and 5 mM HEPES–KOH buffer (pH 7.2). The I_f current was recorded from single cells superfused with Tyrode solution with 1 mM BaCl₂, and 2 mM MnCl₂.

I_f activation curves were obtained using a two-step protocol in which test voltage steps (from −30 to −120 mV, 15 mV interval) were applied from a holding potential of −30 mV and were followed by a step to −125 mV. Test steps had variable durations so as to reach steady –state activation at all voltages. Analysis was performed with the Boltzmann equation (see data analysis).

In current-clamp studies, spontaneous action potentials were recorded from single cells superfused with Tyrode solution, and rate was measured from the interval between successive action potential. When indicated cAMP (1 µM) and/or nanoTRIP8b (10 µM) were added to the pipette solution.

Mice were killed by cervical dislocation under general anesthesia consisting of 0.01 mg/g xylazine (2% Rompun; Bayer AG), 0.1 mg/g ketamine (Imalgène; Merial) and 0.04 mg/g of Na-pentobarbital (Euthanasol VET, Laboratoire TVM, Lempdes, France), and beating hearts were quickly removed. The SAN region was excised in warmed (35°C) Tyrode’s solution containing: 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 1 mM Hepes-NaOH (pH = 7.4), and 5.5 mM D-glucose and cut in strips. Strips were then transferred into a ‘low-Ca²⁺-low-Mg²⁺’ solution containing: 140 mM NaCl; 5.4 mM KCl, 0.5 mM MgCl₂, 0.2 mM CaCl₂, 1.2 mM KH₂PO₄, 50 mM taurine, 5.5 mM D-glucose, 1 mg/ml bovine serum albumin (BSA), 5 mM Hepes-NaOH (pH = 6.9).

Tissue was digested by adding Liberase TH (0.15 mg/ml, Roche Diagnostics GmbH, Mannheim, Germany), elastase (1.9 U/ml, Worthington, Lakewood). Digestion was carried out for a variable time of 15–18 min at 35°C. Tissue strips were then washed and transferred into a modified ‘Kraftbrühe’ (KB) medium containing: 70 mM L-glutamic acid, 20 mM KCl, 80 mM KOH, (±) 10 mM D- b-OH-butyric acid; 10 mM KH₂PO₄, 10 mM taurine, 1 mg/ml BSA and 10 mM Hepes-KOH (pH = 7.4).

Single SAN cells were isolated by manual agitation in KB solution at 35°C for 30–50 s.

Cellular automaticity was recovered by re-adapting the cells to a physiological extracellular Ca²⁺ concentration by addition of a solution containing: 10 mM NaCl, 1.8 mM CaCl₂ and normal Tyrode solution containing BSA (1 mg/ml). The final storage solution contained: 100 mM NaCl, 35 mM KCl, 1.3 mM CaCl₂, 0.7 mM MgCl₂, 14 mM L-glutamic acid, (±) 2 mM D-b-OH-butyric acid, 2 mM KH₂PO₄, 2 mM taurine, 1 mg/ml BSA, (pH = 7.4). Cells were then stored at room temperature until use. All chemicals were from SIGMA (St Quentin Fallavier, France).

For electrophysiological recording, SAN cells in the storage solution were harvested in special custom-made recording plexiglas chambers with glass bottoms for proper cell attachment and mounted on the stage of an inverted microscope (Olympus IX71) and perfused with normal Tyrode solution. The recording temperature was 36°C. We used the whole-cell variation of the patch-clamp technique to record cellular ionic currents, by employing a Multiclamp 700B (Axon Instruments Inc., Foster USA) patch clamp amplifier. Recording electrodes were fabricated from borosilicate glass, by employing a WZ DMZ-Universal microelectrode puller (Zeitz-Instruments Vertriebs GmbH, Martinsried, Germany).

I_f was recorded under standard whole-cell configuration during perfusion of standard Tyrode’s containing 2 mM BaCl₂ to block I_K1. Patch-clamp pipettes were filled with an intracellular solution containing: 130 mM KCl, 10 mM NaCl, 1 mM EGTA, 0.5 mM MgCl₂ and 5 mM HEPES (pH 7.2).

For recording of L-type Ca²⁺ currents, pipette solution contained: 125 mM CsOH, 20 mM tetraethylammonium chloride (TEA-Cl), 1.2 mM CaCl₂, 5 mM Mg-ATP, 0.1 mM Li₂-GTP, 5 mM EGTA and 10 mM HEPES (pH 7.2 with aspartate). 30 µM TTX (Latoxan, Portes lès Valence, France) to block INa was added to external solution containing: 135 mM tetraethylammonium chloride (TEA-Cl), 4 mM CaCl₂,10 mM 4-amino-pyridine, 1 mM MgCl₂, 10 mM HEPES and 1 mg/ml Glucose (pH 7.4 with TEA-OH).

Electrodes had a resistance of about 3 MΩ. Seal resistances were in the range of 2–5 GΩ. 10 µM TRIPb8_nano was added to pipette solution. 10 µM TAT-TRIPb8_nano was added in cell storage solution for at least 30 min before patch clamp recording.

TAT-TRIP8b_nano (YGRKKRRQRRRGG-NHSLEEEFERAKAAVESTEFWDKMQAEWEEMARRNWISEN, TAT sequence is shown in bold type) and TAT-(SCRAMBLED)TRIP8b_nano (YGRKKRRQRRRGG-RNEAEAAEVAQKDMINERARTHEFEWESWEMWENLSESFK, TAT sequence is shown in bold type) were purchased from CASLO ApS. TAT-peptides were dissolved in Milliq water (1.5 mM) and added to the petri dish at the final concentration (10 µM) 30 min before current recordings. During the patch clamp experiments, cells were perfused with standard Tyrode with 2 mM BaCl₂ (see above) without the peptides. Recordings from the same petri dish were performed over a time window of 10 to 60 min in peptide-free solution

Data were acquired at 1 kHz using an Axopatch 200B amplifier and pClamp10.5 or 10.7 software (Axon Instruments). Data were analyzed off-line using Clampfit 10.5 or 10.7 (Molecular Devices) and Origin 2015 or 16 (OriginLab Corp., Northampton MA). Activation curves were analyzed by the Boltzmann equation, y = 1/{1 + exp[(V−V_1/2)/s]}, where y is fractional activation, V is voltage, V_1/2 half-activation voltage, and s the inverse slope factor (mV) (DiFrancesco, 1999). Mean activation curves were obtained by fitting individual curves from each cell to the Boltzmann equation and then averaging all curves obtained. Activation time constants (τ_on) were obtained by fitting a single exponential function,

I=I₀ exp(-t/τ) to current traces recorded at the indicated voltages.

Experiments on rabbit SAN cells were performed using left-over cells obtained during experiments approved by the Animal Welfare Body of the University of Milan and by the Italian Ministry of Health (license n.1127/2015-PR). Animal procedures were conformed to the guidelines of the care and use of laboratory animals established by Italian and European Directives (D. Lgs n° 2014/26, 2010/63/UE).

Mouse primary pacemaker cells were isolated from adult C57BL/6J mice as previously described (Mangoni and Nargeot, Cardiovasc Res 2001), in accordance with the Guide for the Care and Use of Laboratory Animals (eighth edition, 2011), published by the US National Institute of Health and European directives (2010/63/EU). The protocol was approved by the ethical committee of the University of Montpellier and the French Ministry of Agriculture (protocol N°: 2017010310594939).

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

This work has been supported by Fondazione CARIPLO grant 2014–0796 to AM, BS and LB, by 2016 Schaefer Research Scholars Program of Columbia University to AM, by European Research Council (ERC) 2015 Advanced Grant 495 (AdG) n. 695078 noMAGIC to AM and GT, by National Institutes for Health Grant R01 NS036658 to BS, by Instruct-ERIC and national member subscriptions to LB, and by Accademia Nazionale dei Lincei (Giuseppe Levi foundation) to AS. We specially thank the EU ESFRI Instruct Core Centre CERM-Italy.

Animal experimentation: Experiments on rabbit SAN cells were performed using left-over cells obtained during experiments approved by the Animal Welfare Body of the University of Milan and by the Italian Ministry of Health (license n.1127/2015-PR). Animal procedures were conformed to the guidelines of the care and use of laboratory animals established by Italian and European Directives (D. Lgs no 2014/26, 2010/63/UE). Mouse primary pacemaker cells were isolated from adult C57BL/6J mice as previously described (Mangoni and Nargeot, Cardiovasc Res 2001), in accordance with the Guide for the Care and Use of Laboratory Animals (eighth edition, 2011), published by the US National Institute of Health and European directives (2010/63/EU). The protocol was approved by the ethical committee of the University of Montpellier and the French Ministry of Agriculture (protocol N°: 2017010310594939).

© 2018, Saponaro et al.

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