XB-ART-56680J Biol Chem January 1, 2020; 295 (13): 4114-4123.
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Chlorpromazine binding to the PAS domains uncovers the effect of ligand modulation on EAG channel activity.
Ether-a-go-go (EAG) potassium selective channels are major regulators of neuronal excitability and cancer progression. EAG channels contain a Per-Arnt-Sim (PAS) domain in their intracellular N-terminal region. The PAS domain is structurally similar to the PAS domains in non-ion channel proteins, where these domains frequently function as ligand-binding domains. Despite the structural similarity, it is not known whether the PAS domain can regulate EAG channel function via ligand binding. Here, using surface plasmon resonance, tryptophan fluorescence, and analysis of EAG currents recorded in Xenopus laevis oocytes, we show that a small molecule chlorpromazine (CH), widely used as an antipsychotic medication, binds to the isolated PAS domain of EAG channels and inhibits currents from these channels. Mutant EAG channels that lack the PAS domain show significantly lower inhibition by CH, suggesting that CH affects currents from EAG channels directly through the binding to the PAS domain. Our study lends support to the hypothesis that there are previously unaccounted steps in EAG channel gating that could be activated by ligand binding to the PAS domain. This has broad implications for understanding gating mechanisms of EAG and related ERG and ELK K+ channels and places the PAS domain as a new target for drug discovery in EAG and related channels. Up-regulation of EAG channel activity is linked to cancer and neurological disorders. Our study raises the possibility of repurposing the antipsychotic drug chlorpromazine for treatment of neurological disorders and cancer.
PubMed ID: 32047112
PMC ID: PMC7105296
Article link: J Biol Chem
Species referenced: Xenopus laevis
Genes referenced: arnt erg kcnh1
GO keywords: potassium channel activity
Article Images: [+] show captions
|Figure 1. CH binding to the PAS domain of EAG channels detected with SPR. a) Ribbon representation of the full-length cryo-EM structure of rat EAG channels (PDB accession no. 5K7L) viewed from the side and down the pore of the channel. The PAS domain is green, the CNBH is blue and the transmembrane segments are gray. Ribbon representations were created using PyMol. b) Chemical structure of CH. c) Schematic of the PAS and CNBH domains immobilized on the NTA sensor chip using Ni2+-NTA coupling. d, e) SPR sensorgrams recorded for the immobilized PAS domain of mEAG channel (d) and the corresponding dose-dependence of the SPR response (e). f, g, h, SPR sensorgrams for the immobilized PAS domain of ERG (f), and CNBH domains of EAG (g) and ERG (h) channels recorded with the indicated concentrations of CH.|
|Figure 2. CH binding to the PAS domain of EAG channels detected using tryptophan fluorescence. a) Ribbon representation of the mEAG PAS domain (PDB accession no. 4HOI) (34). The two endogenous Trp residues are shown in yellow. The PAS domain cavity is shown as a grey mesh. The ribbon representation was created and the cavity was visualized using PyMol. b, c) Background subtracted emission spectra of mEAG PAS (b) and free tryptophan in solution (c), recorded in the absence and presence of the indicated CH concentrations. The excitation wavelength was 290 nm. The mEAG PAS and free tryptophan concentration was 5 μM. d) Plots of change in the peak emission intensity versus total CH concentration for mEAG (filled circles) and free tryptophan (open circles), fit with equation (2). The peak emission intensity was at 343 nm for mEAG PAS and at 353 nm for free tryptophan. The binding affinity for CH was 1 + 0.7 μM. For clarity, the data point for Trp fluorescence in the presence of 1 μM CH was moved slightly to the right to prevent overlap with the data for mEAG PAS.|
|Figure 3. Concentration dependence of EAG channel current inhibition by CH. a) Representative mEAG current traces recorded in the inside-out configuration in the absence (black) and presence (red) of 50 μM CH. b) Plots of the percent inhibition of tail currents versus the CH concentration. Tail currents were recorded at -100 mV following a voltage step to +70 mV. The lines represent fits of the data with the Hill equation with the IC50 of 3.7 + 0.7 μM, and Hill coefficient (n) of 1. c) A representative tail current recorded at – 100 mV after a voltage step to +70 mV in the absence (black) and presence (red) of 10 μM CH. The grey lines represent fits of the tail currents with a single exponential function with the time constant of deactivation of 3.3 ms in the absence and 4.1 ms in the presence of CH. d) Plots of the averaged deactivation time constants for tail currents recorded at – 100 mV after a voltage step to +70 mV versus CH concentration. n > 4 for each condition.|
|Figure 4. Voltage dependence of EAG current inhibition by CH. a) Plots of the averaged normalized tail-currents versus test voltage obtained in the absence (black symbols) and presence (red symbols) of 10 μM CH. The line represents fit with equation (3), with V1/2 of -35.8 + 1.2 mV and s of 15.6 + 1.1. b) Plots of the averaged percent current inhibition versus voltage for tail-currents in the presence of 10 μM CH. n > 4 for each condition.|
|Figure 5. Effect of the PAS domain deletion on EAG current inhibition by CH. a, b) Representative current traces recorded with TEVC in the absence (black) and presence (red) of 100 μM CH for WT (a) and ∆PAS (b) EAG channels. c) Plots of the percent inhibition of steady-state currents, recorded at the end of the voltage pulse, versus the CH concentration. The lines represent fits of the data with Hill equation with the IC50 of 29.7 + 0.7 μM for WT (filled symbols) and 53.6 + 8.9 μM for ∆PAS (open symbols) EAG channels. Hill coefficient (n) was 1.4 for WT and 0.9 for ∆PAS channels. d) Plots of the averaged normalized conductance versus voltage obtained in the absence (black symbols) and presence (red symbols) of 100 μM CH for WT EAG channels. The line represents fit with equation (3) with V1/2 of -15.7 + 0.8 mV and s of 15.9 + 0.7 e) Plots of the averaged normalized conductance versus voltage obtained in the absence (black symbols) and presence (red symbols) of 100 μM CH for ∆PAS EAG channels. f) Plots of the averaged percent steady-state current inhibition versus voltage for WT (filled symbols) and ∆PAS (open symbols) EAG channels in the presence of 100 μM CH. n > 4 for each condition.|
|Figure 6. Model for the PAS domain-dependent action of CH on EAG channels. PAS domain interacts with the VS and CNBH domains. The PAS/CNBH interaction is proposed to favor opening of the pore (symbolized by arrows). CH binding to the PAS domain could weaken the PAS/CNBH domain interaction, decreasing the opening of the pore. VS and S6 are shown in grey on the background of the rest of the transmembrane segments. PAS domain is green, CNBH domain is blue. CH is shown as a white diamond.|
References [+] :
Adaixo, Structural properties of PAS domains from the KCNH potassium channels. 2013, Pubmed