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.
Highlights
hyperpolarization-activated cyclic nucleotidegated (HCN) channels are dually gated by membrane hyperpolarization and binding of cAMP
CCMP is a partial agonist of HCN channels, displaying an efficacy of ϳ0.6. cCMP increases the frequency of pacemaker potentials from isolated sinoatrial pacemaker cells in the presence of endogenous cAMP concentrations
In agreement with the planar patch-clamp measurements, the maximal shift in V0.5 by cCMP at the saturating concentration was only ϳ60% of that by cAMP (Fig. 5B). These results suggest that cCMP is a partial agonist of HCN channels
Summary
HCN channels are dually gated by membrane hyperpolarization and binding of cAMP. Results: We identify cCMP as a novel regulator of heterologously expressed and native HCN channels. Activation of hyperpolarization-activated cyclic nucleotidegated (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. HCN channel activity is enhanced by direct binding of cAMP to a cyclic nucleotidebinding domain (CNBD) in the C terminus of the channels This type of channel regulation plays an important role in a variety of physiological settings. A specific cCMP receptor has not yet been identified, it was shown that this cyclic nucleotide cross-activates cAMP- and cGMP-dependent protein kinases [19]. The CNBDs of cGMP-dependent protein kinases reveal strong sequence homology to the CNBDs of HCN channels [1] Given this structural relationship, we wondered whether HCN channels may be regulated by interaction with cCMP. We set out to study the consequences of cCMP-dependent HCN channel regulation in native SAN cells
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