Fisetin Regulation of HCN2 and Eag1 Channels Reveals Conserved Gating Mechanisms

Abstract

Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels are pace-making channels that contribute to rhythmic activity in the heart and brain. All HCN-family channels contain a large intracellular domain in their carboxy terminal region which includes a cyclic nucleotide binding domain (CNBD) and a C-linker that connects it to the pore. We recently reported that the flavonoids luteolin and myricetin regulate Eag1 channels by binding a domain in their carboxy-terminal region that shares sequence and structural similarity with the CNBD of HCN2. In a screen of flavonoids, we identified fisetin as another potent regulator of Eag1. Fisetin shifted the voltage-dependence of Eag1 channels toward more hyperpolarizing potentials and slowed channel deactivation. Here we demonstrate that fisetin also regulates the kinetic and steady state properties of HCN2 channels. We found that 30 μM fisetin sped activation at −140 mV, decreasing the time constant from 1.17 ± 0.10 s to 0.80 ± 0.8 s (N=10). Additionally, 30 μM fisetin shifted the Vhalf of the conductance-voltage relationship from −127.8 ± 0.7 mV, to −120.4 ± 0.7 mV (N=10). Fisetin shifted the voltage-dependence of Eag and HCN channels in opposite directions, indicating that it is not simply interacting with the voltage sensor. Furthermore we found that fisetin and cAMP had non-additive effects on HCN2 channel gating. Using a FRET-based binding assay, we showed that fisetin bound to the purified CNBD of HCN2. Additionally, fisetin did not speed activation or shift the voltage-dependence of HCN2 channels lacking their CNBD. Together these data indicate that the CNBD mediates fisetin regulation of HCN2 channels. Based on the similar regulation of Eag and HCN channels by fisetin we propose that the mechanisms underlying ligand-mediated gating is well-conserved amongst these channels.