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) > or = 51 microm) 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.

FIGURE 1.

The invariant Glu and Arg residues are not conserved in the EAG family of K⁺ channels. Amino acid sequence alignment of the CNBD of the EAG family of K⁺ channels and other cyclic nucleotide-binding proteins. Identical residues are red on yellow background, conserved residues are blue on cyan background, similar residues are black on green background, and weakly similar residues are in green. The α-helices (red rectangles) and β-sheets (blue arrows) represent structural motifs from the crystal structure of the CNBD of mHCN2 channels (31). The six invariant residues are indicated by black arrows. The GenInfo identifier numbers are: mEAG1, 487740; rEAG1, 557265; dEAG, 24642070; hERG1, 103488986; mELK2, 187954689; mHCN2, 148699724; bCNGA1, 231739; MlotiK1, 81779664; bPKAIαB, 145559486; CAP, 168751822.

FIGURE 2.

Biochemical studies of the CNBD of the mHCN2, mEAG1, and hERG1 channels. A, size exclusion profiles of the isolated CNBD used in the study. Runs were performed on a Superdex 200 column. The void volume is indicated by the arrow. B–D, CD spectra of the isolated CNBD of the indicated channels in the far-ultraviolet region. 100 μm cAMP was added to the CNBD in C and D as indicated.

FIGURE 3.

cAMP and cGMP decrease Trp fluorescence of mHCN2J-L586W and have no effect on the fluorescence of mEAG1-(505–702). A, ribbon representation of the homology model of mEAG1-(505–702) obtained with SWISS-MODEL (70) based on the crystal structure of mHCN2J (31). cAMP, colored in pink, is shown to illustrate a possible binding location as seen in the crystal structure of the CNBD of mHCN2 channels. Enlarged view of the cAMP binding pocket and the Trp residue are shown on the right. B and D, emission spectra of mHCN2J-L586W and mEAG1-(505–702) recorded without and with the indicated concentrations of cAMP. The excitation wavelength was 295 nm. Protein concentration was 4 μm. C and E, plots of change in the peak fluorescence intensity versus total cyclic nucleotide concentration for mHCN2J-L586W and mEAG1-(505–702). The change in the peak fluorescence intensity (ΔF) was calculated by subtracting averaged peak emission intensities for low cyclic nucleotide concentrations (intensities at 0, 0.01, and 0.1 μm cAMP at 342 nm) from the peak emission intensities. Data in C were fitted with Equation 4. The binding affinities were 13 ± 2 μm for cAMP and 62 ± 23 μm for cGMP for mHCN2J-L586W.

FIGURE 4.

Fluorescence of 8-NBD-cAMP increases upon interaction with mHCN2J-L586W and hERG1-(666–872)/MBP but not mEAG1-(505–702). A, C, and E, emission spectra of 8-NBD-cAMP at the indicated concentrations in the presence of 1 μm mHCN2J-L586W, mEAG1-(666–872), and hERG-(666–872)/MBP, respectively. The excitation wavelength was 470 nm. B, D, and F, plots of the change in the peak fluorescence intensity versus total 8-NBD-cAMP concentration for mHCN2J-L586W, mEAG1-(666–872), hERG-(666–872)/MBP, and MBP, respectively. The change in the peak fluorescence intensity (ΔF) was calculated by subtracting the peak emission intensity at 536 nm in the absence of 8-NBD-cAMP from the peak emission intensities in the presence of 8-NBD-cAMP. The data in B and F were fitted with Equation 4. The binding affinities for 8-NBD-cAMP were 3.8 ± 0.6 μm for mHCN2J-L586W, and ≥ 51 ± 4 μm for mERG1-(666–872)/MBP.

FIGURE 5.

Cyclic nucleotides do not modulate currents from mEAG1 and hERG1 channels. Currents and conductance-voltage relations for mHCN2 (A), mEAG1 (B), and hERG1-S631A (C) channels recorded in the inside-out patch configuration with cAMP (red), cGMP (green), and without the cyclic nucleotides (black). cAMP and cGMP were applied at 1 mm to the mHCN2 channels and 10 mm to mEAG1 and hERG1-S631A channels. The conductance-voltage relations were obtained from tail currents.