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Link to original content: https://pubmed.ncbi.nlm.nih.gov/22715094
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. 2012 Aug 3;287(32):26506-12.
doi: 10.1074/jbc.M112.357129. Epub 2012 Jun 19.

Regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity by cCMP

Xiangang Zong  1 , Stefanie Krause, Cheng-Chang Chen, Jens Krüger, Christian Gruner, Xiaochun Cao-Ehlker, Stefanie Fenske, Christian Wahl-Schott, Martin Biel
Affiliations

Regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity by cCMP

Xiangang Zong et al. J Biol Chem. .

Abstract

Activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated in vivo by direct binding of the second messenger cAMP. This process plays a fundamental role in the fine-tuning of HCN channel activity and is critical for the modulation of cardiac and neuronal rhythmicity. Here, we identify the pyrimidine cyclic nucleotide cCMP as another regulator of HCN channels. We demonstrate that cCMP shifts the activation curves of two members of the HCN channel family, HCN2 and HCN4, to more depolarized voltages. Moreover, cCMP speeds up activation and slows down deactivation kinetics of these channels. The two other members of the HCN channel family, HCN1 and HCN3, are not sensitive to cCMP. The modulatory effect of cCMP is reversible and requires the presence of a functional cyclic nucleotide-binding domain. We determined an EC(50) value of ∼30 μm for cCMP compared with 1 μm for cAMP. Notably, cCMP is a partial agonist of HCN channels, displaying an efficacy of ∼0.6. cCMP increases the frequency of pacemaker potentials from isolated sinoatrial pacemaker cells in the presence of endogenous cAMP concentrations. Electrophysiological recordings indicated that this increase is caused by a depolarizing shift in the activation curve of the native HCN current, which in turn leads to an enhancement of the slope of the diastolic depolarization of sinoatrial node cells. In conclusion, our findings establish cCMP as a gating regulator of HCN channels and indicate that this cyclic nucleotide has to be considered in HCN channel-regulated processes.

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Figures

FIGURE 1.
FIGURE 1.
Modulation of HCN2 current by cCMP. A, voltage step protocol and family of current traces of HEK293 cells stably expressing HCN2 without (left) and with (right) 1 mm cCMP in the pipette solution. B, normalized activation curves of HCN2 in the presence (■) and absence (●) of 1 mm cCMP. The solid lines are the fits to the Boltzmann equation with the following parameters: in the absence of cCMP (n = 15), V0.5 = −102 mV and k = 6.34 mV; and in the presence of cCMP (n = 14), V0.5 = −93.8 mV and k = 6.53 mV. C, normalized current traces of HCN2 in the absence and presence of 1 mm cCMP. Currents were evoked by a hyperpolarizing step to −140 mV from a holding potential of −40 mV, followed by a voltage pulse to −40 mV. Maximal currents at −140 mV were normalized. D, display of current traces during HCN2 channel deactivation at −40 mV (area within the dotted box in C).
FIGURE 2.
FIGURE 2.
Effect of cCMP on activation of HCN channels. A–C, activation curves of HCN1 (A), HCN3 (B) and HCN4 (C) in the presence (■) and absence (●) of 1 mm cCMP. The solid lines are the fits to the Boltzmann equation with the following parameters: for HCN1 in the absence of cCMP (n = 14), V0.5 = −70.2 mV and k = 6.57 mV; for HCN1 in the presence of cCMP (n = 15), V0.5 = −69.5 mV and k = 7.09 mV; for HCN3 in the absence of cCMP (n = 12), V0.5 = −95.8 mV and k = 9.30 mV; for HCN3 in the presence of cCMP (n = 15), V0.5 = −97.5 mV and k = 11.2 mV; for HCN4 in the absence of cCMP (n = 16), V0.5 = −101 mV and k = 9.71 mV; and for HCN4 in the presence of cCMP (n = 15), V0.5 = −91.1 mV and k = 12.3 mV. D, summarized V0.5 data of the four HCN channels in the absence (white bars) and presence (black bars) of 1 mm cCMP. The V0.5 shift induced by cCMP in HCN2 and HCN4 is highly significant (p < 0.001).
FIGURE 3.
FIGURE 3.
Effects of cCMP and cAMP on HCN4 currents determined upon internal perfusion with planar patch-clamp system. A, time course of HCN4 currents from five cells recorded during a series of 6-s hyperpolarizing pulses from −40 to −100 mV. Currents were measured 3 s after the beginning of individual hyperpolarizing pulses and normalized to the current before application of 1 μm cAMP (■). Application and washout of cAMP are indicated by closed and open bars, respectively. HCN channel rundown without application of cAMP was monitored (□). Inset, current traces recorded at the indicated time points. B, time course of HCN4 currents recorded from six cells with application of 100 μm cCMP (●). The protocol was as described for A. HCN channel rundown was monitored without application of cCMP (○). C, activation curves of HCN4 before and 15 s after internal application of 100 μm cAMP. The solid lines are the fits of a Boltzmann function to the data with the following values: V0.5 = −112 mV and k = 11.7 mV for the control and V0.5 = −100 mV and k = 12.6 for cAMP (n = 7). D, activation curves of HCN4 before and 15 s after internal application of 100 μm cCMP. The solid lines are fits of a Boltzmann function to the data with the following values: V0.5 = −111 mV and k = 9.55 mV for the control and V0.5 = −105 mV and k = 9.69 mV for cCMP (n = 8).
FIGURE 4.
FIGURE 4.
cCMP binds to CNBD of HCN2 channels. Structural models of a portion of the CNBD of HCN2 with bound cAMP (A) and cCMP (B) are shown (upper). The pocket is visualized by a molecular surface (hydrophobic (green) and hydrogen bonding (magenta)). Interactions between the protein and cyclic nucleotide are shown schematically (lower). Dashed lines indicate π stacking (red) and hydrogen bonding (blue). The border of the binding pocket is marked by dashed green lines.
FIGURE 5.
FIGURE 5.
cAMP and cCMP display different gating efficacies in HCN2. A, activation curves of HCN2 for different cCMP concentrations as indicated. B, shift in V0.5 versus concentrations of cAMP (black circles) and cCMP (red circles) for HCN2. Data represent means ± S.E. of 7–19 cells. C, activation curves of HCN2 in the absence of cyclic nucleotides (black circles), in the presence of 10 μm cAMP (red circles), and in the presence of 10 μm cAMP + 1 mm cCMP (green circles). D, shift in the activation curves from the experiments shown in A and C. E, activation curves of HCN2 in the absence of cyclic nucleotides (black circles), in the presence of 1 μm cAMP (red circles), in the presence of 50 μm cCMP (green circles), and in the presence of 1 μm cAMP + 50 μm cCMP (blue circles). F, shift in the activation curves from the experiments shown in E. G, activation curve of an HCN2 mutant with an impaired CNBD (HCN2(R591E/T592A) (HCN2EA)) in the absence (green circles) and presence (red circles) of 1000 μm cCMP. H, V0.5 values for the experiments shown in G. ***, p < 0.001.
FIGURE 6.
FIGURE 6.
Regulation of sinoatrial pacemaking by cCMP. A, representative spontaneous action potentials of murine SAN cells recorded with the control intracellular solution or the intracellular solution containing cCMP or cAMP. B, firing rate with the control intracellular solution or the intracellular solution containing cAMP and/or cCMP at the concentrations indicated. C, increase in the SDD induced by cAMP and cCMP. D, quantification of the effect of cAMP and cCMP on the SDD. E and F, steady-state activation curves (E) and V0.5 values (F) of SAN cell If measured in the absence of cyclic nucleotides (■) or in presence of either cCMP (▿) or cAMP (○). A typical family of If traces obtained from murine SAN cells is shown in the inset in E. The numbers of tested cells are given in parentheses in B, D, and F. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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