Activation gating in HCN2 channels.
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ABSTRACT:
SUBMITTER: Hummert S
PROVIDER: S-EPMC5863937 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are pacemaker channels whose currents contribute to rhythmic activity in the heart and brain. HCN channels open in response to hyperpolarizing voltages, and the binding of cAMP to their cyclic nucleotide-binding domain (CNBD) facilitates channel opening. Here, we report that, like cAMP, the flavonoid fisetin potentiates HCN2 channel gating. Fisetin sped HCN2 activation and shifted the conductance-voltage relationship to more depolarizing potentials with a half-maximal effective concentration (EC50) of 1.8 μM. When applied together, fisetin and cAMP regulated HCN2 gating in a nonadditive fashion. Fisetin did not potentiate HCN2 channels lacking their CNBD, and two independent fluorescence-based binding assays reported that fisetin bound to the purified CNBD. These data suggest that the CNBD mediates the fisetin potentiation of HCN2 channels. Moreover, binding assays suggest that fisetin and cAMP partially compete for binding to the CNBD. NMR experiments demonstrated that fisetin binds within the cAMP-binding pocket, interacting with some of the same residues as cAMP. Together, these data indicate that fisetin is a partial agonist for HCN2 channels.
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Project description:In a multimeric receptor protein, the binding of a ligand can modulate the binding of a succeeding ligand. This phenomenon, called cooperativity, is caused by the interaction of the receptor subunits. By using a complex Markovian model and a set of parameters determined previously, we analyzed how the successive binding of four ligands leads to a complex cooperative interaction of the subunits in homotetrameric HCN2 pacemaker channels. The individual steps in the model were characterized by Gibbs free energies for the equilibria and activation energies, specifying the affinity of the binding sites and the transition rates, respectively. Moreover, cooperative free energies were calculated for each binding step in both the closed and the open channel. We show that the cooperativity sequence positive-negative-positive determined for the binding affinity is generated by the combined effect of very different cooperativity sequences determined for the binding and unbinding rates, which are negative-negative-positive and no-negative-no, respectively. It is concluded that in the ligand-induced activation of HCN2 channels, the sequence of cooperativity based on the binding affinity is caused by two even qualitatively different sequences of cooperativity that are based on the rates of ligand binding and unbinding.
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Project description:Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dually gated channels that are operated by voltage and by neurotransmitters via the cAMP system. cAMP-dependent HCN regulation has been proposed to play a key role in regulating circuit behavior in the thalamus. By analyzing a knockin mouse model (HCN2EA), in which binding of cAMP to HCN2 was abolished by 2 amino acid exchanges (R591E, T592A), we found that cAMP gating of HCN2 is essential for regulating the transition between the burst and tonic modes of firing in thalamic dorsal-lateral geniculate (dLGN) and ventrobasal (VB) nuclei. HCN2EA mice display impaired visual learning, generalized seizures of thalamic origin, and altered NREM sleep properties. VB-specific deletion of HCN2, but not of HCN4, also induced these generalized seizures of the absence type, corroborating a key role of HCN2 in this particular nucleus for controlling consciousness. Together, our data define distinct pathological phenotypes resulting from the loss of cAMP-mediated gating of a neuronal HCN channel.
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