Absence of Direct Cyclic Nucleotide Modulation of mEAG1 and hERG1 Channels Revealed with Fluorescence and Electrophysiological Methods

Abstract

Similar to CNG and HCN channels, EAG and ERG channels contain a cyclic nucleotide binding domain (CNBD) in their C terminus. While cyclic nucleotides have been shown to facilitate opening of CNG and HCN channels, their effect on EAG and ERG channels is less clear. Here we explored cyclic nucleotide binding and modulation of mEAG1 and hERG1 channels with fluorescence and electrophysiology. Binding of cyclic nucleotides to the isolated CNBD of mEAG1 and hERG1 channels was examined with two independent fluorescence-based methods: changes in tryptophan fluorescence and fluorescence of an analog of cAMP, 8-NBD-cAMP. As a positive control for cyclic nucleotide binding we used changes in the fluorescence of the isolated CNBD of mHCN2 channels. Our results indicated that cyclic nucleotides do not bind to the isolated CNBD domain of mEAG1 channels and bind with low affinity (K d > 51 M) to the isolated CNBD of hERG1 channels. Consistent with the results on the isolated CNBD, application of cyclic nucleotides to inside-out patches did not affect currents recorded from mEAG1 channels. Surprisingly, despite its low affinity binding to the isolated CNBD, cAMP also had no effect on currents from hERG1 channels even at high concentrations. Our results indicate that cyclic nucleotides do not directly modulate mEAG1 and hERG1 channels. Further studies are necessary to determine if the CNBD in the EAG family of K ؉ channels might harbor a binding site for a ligand yet to be uncovered.The EAG family of K ϩ channels comprises ether-à-go-go (EAG), 2 EAG-related gene (ERG), and EAG-like (ELK) K ϩ channel subfamilies (1) with diverse tissue expression patterns and physiological functions (reviewed in Ref.2). mEAG channels are overexpressed in tumor tissues (3, 4), where they are involved in regulation of tumor progression (5, 6). Inhibition of the EAG channel expression by RNAi interference (7), application of channel blockers (8, 9), and monoclonal antibody that selectively inhibits currents from EAG channels (10) decreased cell proliferation in tumor tissues. ERG channels are best known for their function in the heart. Because of their unique physiological properties, fast inactivation, and slow deactivation, ERG channels are major contributors to the repolarization phase of the cardiac action potential (11)(12)(13)(14). Mutations in the ERG channels and administration of ERG channel blockers, such as class III antiarrhythmic drugs, cause long QT syndrome, a potentially lethal cardiac arrhythmia characterized by a prolonged cardiac action potential (15)(16)(17)(18)(19). In addition to their role in cardiac excitability, ERG channels also regulate proliferation of tumor cells (20 -22). The physiological role of ELK channels is not well understood, however, early reports suggest their possible involvement in the regulation of neuronal excitability (23). K ϩ channels in the EAG family are structurally related to the cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-modulated (HCN) K ϩ channels (1, 24). All...

Structural snapshot of cyclic nucleotide binding domains from cyclic nucleotide-sensitive ion channels

“…Moreover, the mEAG1 structure supports the idea that the bound intrinsicligand conformation seems to be a conserved feature of all CNBHDs. Such an observation explains the absence of cyclic nucleotide binding to the mEAG1 CNBHD, as previously shown (Brelidze et al, 2009). Further biochemical characterization suggests that the mEAG1 CNBHD may exists in the unliganded conformation.…”

“…Not surprisingly, the overall structure of the CNBHD shows the characteristic features of previously determined CNBHDs from mEAG and zELK channels (Figure 2), including the self-liganded conformation. The C-terminal residues Y727 to M729 of β9 are bound in similar positions when compared with cyclic nucleotides in HCN CNBDs, explaining the lack of direct modulation of agERG channels by cyclic nucleotides (Brelidze et al, 2009(Brelidze et al, , 2012(Brelidze et al, , 2013.…”

Flavonoid Regulation of HCN2 Channels

“…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). The CNBHD in KCNH family channels does not bind cyclic nucleotides, and these channels are not directly regulated by cAMP or cGMP (18,21). However, we found that flavonoids such as fisetin bind to the CNBHD of EAG1 to stabilize the channel's open state (16,22).…”

“…A longer protein composed of the entire C-linker and CNBD was used for fluorescence resonance energy transfer (FRET) experiments. This protein (HCN2 C-linker/CNBD -L586W) included residues 443-645 and a tryptophan substituted for a leucine at position 586 (21). For protein expression, the DNA encoding HCN2 C-linker/CNBD -L586W was subcloned into the pETGQ vector (27).…”

Crystallization and preliminary X-ray crystallographic characterization of a cyclic nucleotide-binding homology domain from the mouse EAG potassium channel

“…In cyclic nucleotide-regulated channels there are extra helices that connect the channel pore to the CNB domain and are responsible for transmitting the cAMP-induced conformational change to the pore; this region is known as the C-linker (Craven & Zagotta, 2006). Importantly, it has been well demonstrated that KCNH channel function is not affected by cyclic nucleotides and that the CNB-homology domain from KCNH channels does not bind nucleotides (Brelidze et al, 2009). Here, we describe the protocols for purification and crystallization of the CNB-homology domain from one of the members of the KCNH family, the mouse EAG channel.…”