Wang ZJ et al. (2023), Molecular mechanism of EAG1 channel inhibition ...

XB-ART-61218

J Biol Chem 2023 Dec 28;29912:105391. doi: 10.1016/j.jbc.2023.105391.

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Molecular mechanism of EAG1 channel inhibition by imipramine binding to the PAS domain.

Wang ZJ , Ghorbani M , Chen X , Tiwari PB , Klauda JB , Brelidze TI .

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Ether-a-go-go (EAG) channels are key regulators of neuronal excitability and tumorigenesis. EAG channels contain an N-terminal Per-Arnt-Sim (PAS) domain that can regulate currents from EAG channels by binding small molecules. The molecular mechanism of this regulation is not clear. Using surface plasmon resonance and electrophysiology we show that a small molecule ligand imipramine can bind to the PAS domain of EAG1 channels and inhibit EAG1 currents via this binding. We further used a combination of molecular dynamics (MD) simulations, electrophysiology, and mutagenesis to investigate the molecular mechanism of EAG1 current inhibition by imipramine binding to the PAS domain. We found that Tyr71, located at the entrance to the PAS domain cavity, serves as a "gatekeeper" limiting access of imipramine to the cavity. MD simulations indicate that the hydrophobic electrostatic profile of the cavity facilitates imipramine binding and in silico mutations of hydrophobic cavity-lining residues to negatively charged glutamates decreased imipramine binding. Probing the PAS domain cavity-lining residues with site-directed mutagenesis, guided by MD simulations, identified D39 and R84 as residues essential for the EAG1 channel inhibition by imipramine binding to the PAS domain. Taken together, our study identified specific residues in the PAS domain that could increase or decrease EAG1 current inhibition by imipramine binding to the PAS domain. These findings should further the understanding of molecular mechanisms of EAG1 channel regulation by ligands and facilitate the development of therapeutic agents targeting these channels.

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	Figure 1. Imipramine binds to the PAS domain of EAG1 channels.A, topology of an EAG1 channel monomeric subunit. The PAS domain is shown in green, membrane-spanning segments in gray, C-linker in cyan, and CNBH domain in blue. The dashed two-sided arrow signifies interactions between the PAS and the CNBH domains from adjacent subunits in an EAG1 channel tetramer. B, schematic of the PAS domain immobilized on the CM5 chip using amine coupling and SPR sensorgrams recorded for the immobilized PAS domain in response to the application of imipramine at 1, 10, and 30 μM concentrations.
	Figure 2. Y71 restrictsbinding of imipramine to the PAS domain cavity. Currents from WT (A), ΔPAS (C), Y71G (F), Y71E (G), and Y71R (I) mutant EAG1 channels were recorded at +50 mV with two-electrode voltage clamp in the presence of the indicated imipramine concentrations. B, plots of the averaged percentage of steady-state current inhibition versus imipramine concentration for WT (filled circles, n = 5), ΔPAS (open squares, n = 5) and Y71G (filled squares, n = 4) mutant EAG1 channels. D, imipramine bound inside the PAS domain cavity after REST simulations. The imipramine-bound PAS domain structure is shown in the green ribbon representation with the Y71 residue in green sticks and imipramine in yellow. The ligand-free structure of the PAS domain is shown in the transparent grey color with the Y71 residue in sticks. The curved arrow indicates a possible transition of Y71 from ligand-free to ligand-bound states in order to give access to imipramine to the PAS domain cavity. E, ligand-free PAS domain structure with glycine substituted for Y71 and imipramine docked in the same pose as in D. H, plots of the averaged percentage of steady-state current inhibition versus imipramine concentration for Y71E (downward pointing triangles, n = 5) and Y71R (upward pointing triangles, n = 4) mutant EAG1 channels. The solid lines in B and H represent fits of the data with the Hill equation. The dashed line in H corresponds to the fit with the Hill equation for WT channels from B. The IC50 values and the corresponding statistical analysis can be found in Table 1. The data in B and H are presented as mean ± SD. Scale bars in C, F, G, I: 4 μA.
	Figure 3. Hydrophobic profile of the PAS domain cavity facilitates imipramine binding.A, electrostatic surface profile of the PAS domain cavity. Acidic residues are shown in red, basic residues in blue, hydrophobic residues in white and polar residues in green. B–D, a representative binding pose of imipramine (yellow) inside the WT (B), 4E (C) and 4T (D) mutant PAS domain cavity determined with 100 ns MD simulations.
	Figure 4. Cavity-flanking residues D39G and R84G are essential for EAG1 current inhibition by imipramine binding to the PAS domain.A, close-up view of imipramine bound inside the PAS domain cavity with the residues poised to interact with the ligand shown in stick representations. Currents from D39G (B), V80G (C), V83G (D), R84G (E), F87G (F), F130G (G), and D39G/R84G (H) mutant EAG1 channels recorded at +50 mV with two-electrode voltage clamp in the presence of the indicated imipramine concentrations. Scale bars in (C–H): 4 μA. I, plots of the averaged percentage of steady-state current inhibition versus imipramine concentration for D39G (red squares), V80G (blue squares), V83G (blue circles), R84G (red circles), F87G (blue diamonds), F130G (blue downward pointing triangles) and D39G/R84G (red upward pointing triangles) mutant EAG1 channels recorded at +50 mV with two-electrode voltage clamp in the presence of the indicated imipramine concentrations. (n ≥ 5 for each condition). The solid lines represent fits of the data with the Hill equation. The black dashed line corresponds to the fit with the Hill equation for WT channels from Figure 2B and red dashed line to the fit for ΔPAS channels. The IC50 values and the corresponding statistical analysis can be found in Table 1. The data in I are presented as mean ± SD.
	Figure 5. Voltage dependence and the current profile of the WT and mutant EAG1 channels used in the study.A–N, representative current traces recorded with two-electrode voltage clamp. O–Q, plots of the averaged normalized conductance versus voltage for the WT and indicated mutant channels that increased the IC50 for imipramine inhibition of EAG1 currents (O), decreased IC50 (P) or had no effect on IC50 (Q). The size of the WT data points (filled black circles) in O–Q was increased relative to other symbols to prevent a complete overlap with the data points for the mutant channels. The averaged V1/2 values, n of experiments, and statistical analysis can be found in Table 3. The data in O–Q are presented as mean ± SD. Scale bars in (B–N): 4 μA.
	Figure 6. A model of EAG current inhibition by imipramine by a dual mechanism, via binding to the PAS domain and the intracellular pore vestibule.Ribbon representation of the full-length cryo-EM structure of rat EAG channels (PDB 5K7L) viewed from the side. Only two diagonal subunits are shown for clarity. The PAS domains are green, the CNBH blue, and the transmembrane segments gray. The arrows signify imipramine binding to the PAS domain and intracellular pore vestibule.