Abstract
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.
Highlights
HCN2 channels are regulated by membrane potential and by the direct binding of cyclic nucleotides to their carboxyl-terminal region
To study HCN2 kinetics, the rate of activation was quantified by fitting single exponentials to the slow component of the currents evoked by steps to Ϫ140 mV. 30 M fisetin sped the time constant of activation () at Ϫ140 mV from 1.08 Ϯ 0.08 to 0.73 Ϯ 0.05 s (n ϭ 9) (p ϭ 0.01, Student’s paired t test) (Fig. 1D)
We report that the flavonoid fisetin regulates HCN2 channels
Summary
HCN2 channels are regulated by membrane potential and by the direct binding of cyclic nucleotides to their carboxyl-terminal region. HCN channels open in response to hyperpolarizing voltages, and the binding of cAMP to their cyclic nucleotide-binding domain (CNBD) facilitates channel opening. Unlike other members of this superfamily, HCN channels are only weakly Kϩ-selective, activated by hyperpolarizing voltages, and regulated by the direct binding of cyclic nucleotides to their intracellularly located, carboxyl-terminal region. This carboxyl-terminal region includes a cyclic nucleotide-binding domain (CNBD) and a C-linker that connects the domain to the pore [7, 10]. All KCNH channels, including EAG1, contain a cyclic nucleotide-binding homology domain (CNBHD) in their carboxyl-terminal region that shares sequence and structural similarity with the CNBD of HCN channels (18 –20). These results indicate that fisetin is a partial agonist for HCN2
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