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Propofol rescues voltage-dependent gating of HCN1 channel epilepsy mutants

Nature volume 632, pages 451–459 (2024)Cite this article

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Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels1 are essential for pacemaking activity and neural signalling2,3. Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain4 and epileptic seizures5. The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs. 7,8). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine–phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies.

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Fig. 1: Structural resolution of the propofol–HCN1 complex.
Fig. 2: Site 1 seems to be a state-dependent pocket and mutation of residues reduced druggability.
Fig. 3: Propofol restores function of disease-causing HCN1 mutants.
Fig. 4: Homologous epilepsy-associated M305L mutant channels are voltage-independent but with intact voltage sensor movement.
Fig. 5: Propofol inhibits spHCN current without changing voltage sensor movement.
Fig. 6: The effect of propofol on WT and M305L channels.

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Data availability

The maps for HCN1 WT and M305L in nanodiscs in complex with propofol (accession codes: EMD-42116, EMD-44425) and without propofol (accession codes: EMD-42117, EMD-44426) have been deposited in the Electron Microscopy Data Bank (EMDB). Atomic coordinates for the HCN1 WT and M305L structures in nanodiscs with propofol (accession codes: 8UC7, 9BC6) and without propofol (accession codes: 8UC8, 9BC7) have been deposited in the Protein Data Bank (PDB). Figures 2–5 and Extended Data Figs. 3–5 and 7 have raw data associated with them. Raw electrophysiology and fluorescence traces are available from the corresponding authors upon request. The atomic coordinates of one replica of the free molecular dynamics simulation of DOPC:POPE:POPS lipid-solvated HCN1 channel with propofol bound at sites 1 and 2 at t = 0 and 400 ns and the topology and force field files of all system components including propofol are available at Zenodo (https://doi.org/10.5281/zenodo.11528212)84. Source data are provided with this paper.

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Acknowledgements

We thank M. Falzone and P. Schmidpeter for assistance with cryo-EM freezing and processing; M. Ferrer for help with two-electrode voltage clamp recordings; J. Wojcik for mutagenesis of human HCN1; L. Khajoueinejad for Xenopus oocyte preparation; M. Su for baculovirus guidance; and the members of C. Nimigean’s, P. Larsson’s and P. Riegelhaupt’s labs for their scientific input. Screening and data collection were performed by H. Kuang, B. Wang and W. Rice at New York University Langone Health’s Cryo-Electron Microscopy Laboratory (RRID:SCR_019202); by J. Mendez, C. Hernandez, K. Maruthi and E. Eng at the Simons Electron Microscopy Center at the New York Structural Biology Center; and by D. Acehan and E. C. Fluck at the Weill Cornell Medicine Cryo-EM Core Facility. Negative stain screening and confocal imaging were conducted at the Weill Cornell Medicine CLC Imaging Core Facility. The computational resources were provided by the Scientific Computing Unit at Weill Cornell Medicine. We thank R. Mackinnon for the pEG BacMam-HCN1EM and PET32a-eGFP nanobody constructs. The work presented here was sponsored in part by grant no. NIH GM124451 and grant no. NIH NS137561 to C.M.N., grant no. NIH GM139164 to H.P.L., grant no. NIH GM128420 to A.A., grant no. NIH R42NS129370 to P.A.G., grant no. NIH F32GM145091 to E.D.K. and a Hartwell Foundation Postdoctoral Fellowship to E.D.K. The New York University Langone Health Cryo-Electron Microscopy laboratory is partially supported by the Laura and Isaac Perlmutter Cancer Center Support Grant no. NIH NCI P30CA016087 and work at the Simons Electron Microscopy Center at the New York Structural Biology Center is supported by the Simons Foundation (grant no. SF349247). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Author notes

  1. These authors contributed equally: Elizabeth D. Kim, Xiaoan Wu

Authors and Affiliations

  1. Department of Anesthesiology, Weill Cornell Medical College, New York, NY, USA

    Elizabeth D. Kim, Sangyun Lee, Gareth R. Tibbs, Eleonora Di Zanni, Peter A. Goldstein, Alessio Accardi & Crina M. Nimigean

  2. Department of Physiology and Biophysics, University of Miami Miller School of Medicine, Miami, FL, USA

    Xiaoan Wu, Kevin P. Cunningham, Marta E. Perez & H. Peter Larsson

  3. School of Life Sciences, University of Westminster, London, UK

    Kevin P. Cunningham

  4. Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA

    Alessio Accardi & Crina M. Nimigean

  5. Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden

    H. Peter Larsson

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Contributions

E.D.K. performed mutagenesis and TEVC for human HCN1, prepared samples for and collected the cryo-EM data, and analysed the TEVC and cryo-EM data. X.W. performed mutagenesis and VCF for spHCN channels. K.P.C. performed VCF for spHCN channels. H.P.L. performed the kinetic modelling. M.E.P. performed mutagenesis for spHCN channels. S.L. and A.A. designed, performed and analysed the molecular dynamics simulations. E.D.Z. performed transfection, confocal microscopy and colocalization analysis and mutagenesis of human HCN1. G.R.T. and P.A.G. contributed to the conception of this work, established TEVC conditions and synthesized and provided the pGHE human full-length HCN1 plasmid for TEVC. H.P.L. and C.M.N. supervised, designed and acquired funding for the research. E.D.K., X.W., H.P.L. and C.M.N. interpreted the data, prepared figures and wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to H. Peter Larsson or Crina M. Nimigean.

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Competing interests

G.R.T. and P.A.G. are co-inventors on patents related to the development of novel alkylphenols for the treatment of neuropathic pain. G.R.T. and P.A.G. serve on the Scientific Advisory Board for Akelos Inc., a research-based biotechnology company that has a licensing agreement for the use of those patents.

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Extended data figures and tables

Extended Data Fig. 1 Cryo-EM data processing of HCN1 WT nanodisc in the absence and presence of 1 mM propofol.

a, SEC chromatograms and SDS-PAGE of HCN1 WT nanodisc purifications in the presence and absence of propofol. For gel source data, see Supplementary Fig. 1. The cryo-EM processing schematics are shown for b, apo HCN1 WT nanodisc and c, HCN1 WT nanodisc + pfl. Micrograph scale bar represents 50 nm. d, Backbone r.m.s.d. deviations between apo, propofol (pfl), and detergent (PDB 5U6O9) structures.

Extended Data Fig. 2 Local cryo-EM densities of propofol binding sites 1 and 2.

Shown are side and top views of the site 1 and site 2 densities from the 3D Refinement map, PostProcess map, DeepEMhancer map, and half map 1 for a, apo WT, b, WT + 1 mM propofol, c, holo M305L, and d, M305L + 1 mM propofol datasets. The HCN1 protein is in gray, tubular lipid densities in yellow, and the identified propofol densities in red. DeepEMhancer representations are used in the manuscript.

Extended Data Fig. 3 Site 2 is state-independent and does not confer propofol inhibition of HCN1 channels.

a, Overview of the propofol-HCN1 cryoEM map, from Fig. 1b, highlighting binding site 1 and site 2. b, Zoomed in view of the residues surrounding the density at site 2. c, Overlay of HCN1 + 1 mM propofol (this study, red-brown), HCN1 crosslinked (PDB 6UQF10, cyan), HCN4 open (PDB 7NMN35, light blue), and HCN1 closed (PDB 5U6O9, white). Shown are the response of HCN1 d, L218A and e, K219A in the absence (left) and presence (right) of propofol by two electrode voltage clamp. Voltage clamp ranged from +45 mV to −135 mV with tail currents measured at +50 mV. The current response at −85 mV is highlighted in red. Corresponding Boltzmann fits are shown in f, L218A (apo: V1/2 = −55.3 ± 4.0 mV, k = 6.3 ± 0.8 mV, n = 3; pfl: V1/2 = −86.2 ± 3.4 mV, k = 10.1 ± 1.2 mV, n = 4, P < 0.0001) and g, K219A (apo: V1/2 = −50.7 ± 2.8 mV, k = 6.8 ± 0.4 mV, n = 4; pfl: V1/2 = −83.0 ± 2.5 mV, k = 10.2 ± 1.6, n = 4, P < 0.0001). L218A ΔV1/2 = −31.0 ± 5.2 mV and K219A ΔV1/2 = −32.3 ± 3.7 mV, compared to that of WT ΔV1/2 = −30.9 ± 6.4 mV. P-values were determined by two-way ANOVA using a Tukey post hoc test between apo and propofol V1/2, with significance defined as P < 0.05, and n denotes biological replicates. Empty and filled symbols with error bars represent mean ± s.d. for normalized apo and propofol data, respectively.

Source Data

Extended Data Fig. 4 Propofol makes hydrophobic contacts with and exhibits longer residence in site 1.

a, HCN1 was solvated in lipids (yellow-red-blue sticks) and propofol bound at site 1 and site 2 are shown in red spheres. K+ and Cl− ions are shown in green and gray spheres, respectively. Waters are not shown for simplicity. R.m.s.d. of propofol from their originating position in a MD simulation of the HCN1 WT tetramer in a b, DOPC:POPE:POPS and c, pure POPC lipid bilayer. All 12 propofols unbound from site 2 in both lipid compositions, while 11 of 12 propofols remained bound to site 1 in the DOPC:POPE:POPS bilayer. In the POPC bilayer, 10 of 12 propofols remained at site 1. Propofol (red) at site 1 adopts multiple binding poses in both the d, DOPC:POPE:POPS and e, POPC lipid bilayers. Propofols at the center of each of the three highest populated clusters which contribute to 97% of the total frames are shown in stick model and colored by orange, red-brown, and yellow respectively. Distinct HCN1 subunits are shown in light gray and slate. Amino acid residues lining the binding pocket are in purple. f, Docking65 of propofol to HCN1 identified 5 transmembrane locations, labeled 1 through 5. Site 1 identified by cryoEM is equivalent to docking position 1. However, site 2 from the cryo-EM experiment was not identified. g, Docking of propofol to HCN1 M305L found 4 transmembrane locations. The site 1 was identified, but not site 2. Positions 2 and 4 were also the same as those found in the WT docking experiment in f. For clarity, only the transmembrane domain of the channel is shown with individual subunits colored in slate, light grey, green, and red-brown. Docked propofol molecules are in red.

Source Data

Extended Data Fig. 5 Perfusion of propofol to HCN1 M305L recovers voltage dependent gating.

a, Schematic of perfusion experiment design. Two electrode voltage clamp recordings were performed pre- and post-perfusion with 30 µM propofol for 10 min. To verify inward HCN1 currents, recording solution supplemented with 1 mM caesium chloride was perfused on and off the cell for 5 min. Shown are representative traces of n = 3 and 4 similar recordings with b, WT and c, M305L, respectively. d, For WT and M305L, the inward current is blocked by caesium while the outward depolarized tails remain intact. Corresponding Boltzmann fits are also shown for WT and M305L. Controls demonstrating inward current caesium block in the absence of 30 µM propofol are shown for e, WT and f, M305L and are representative traces of n = 3 similar recordings. Voltage clamp ranged from +45 mV to −125 mV with tail currents measured at +50 mV. The current response at −85 mV is highlighted in red. Empty and filled symbols with error bars represent mean ± s.d. for normalized apo and propofol data, respectively. n represents the number of biological replicates. HEK293S GnTI- cells transfected with HCN1 g, WT and h, M305L using Lipofectamine 2000 (Invitrogen). Nuclei are in blue, the plasma membrane in red, and HCN1 in green. Expression at the plasma membrane is demonstrated by colocalization (yellow). Shown is a representative cell of WT n = 20 and M305L n = 10 similar cells, over 3 independent transfections. Plotted to the right are intensity values across the dashed orange line. The scale bar represents 10 µm. For microscopy source data, see Supplementary Fig. 2.

Source Data

Extended Data Fig. 6 Cryo-EM data processing of HCN1 M305L nanodisc in the absence and presence of 1 mM propofol.

a, SEC chromatograms and SDS-PAGE of HCN1 M305L nanodisc purifications in the presence and absence of propofol. The nanodiscs for HCN1 M305L holo without propofol were made using MSP1E3 while the ones for HCN1 M305L with propofol were made using MSP2N2. For gel source data, see Supplementary Fig. 1. The cryoEM processing schematics are shown for b, apo HCN1 M305L nanodisc and c, HCN1 M305L nanodisc + pfl. Micrograph scalebar represents 50 nm. d, Pore diagram comparison between holo M305L nanodisc, M305L + pfl, and holo WT detergent (PDB 5U6P9) structures using HOLE63. Red indicates regions that are smaller than a single water molecule to pass, green for a single water molecule, and blue is double the radius of a single water molecule. Both holo structures contain cAMP. e, Backbone r.m.s.d. deviations of the voltage sensing domain (S1-S4) between holo M305L, holo WT (PDB 5U6P9), and M305L HCN1 propofol structures.

Extended Data Fig. 7 Voltage-independent spHCN-M375L channels are blocked by the specific HCN channel blocker ZD7288 and the Met375-Phe459 interaction is important to close spHCN channels at positive voltages.

a, Representative current traces from spHCN M375L channels before (left) and after (right) the application of 100 µM ZD7288. Dashed lines indicate no currents. Met375 and Phe459 mutants show currents at positive voltages and similar voltage sensor movement. b, GV and c, FV relations from WT (black), M375L (blue), M375F (purple), M375A (green), M375C (orange) and M375S (pink) mutant spHCN channels. d, GV and e, FV relations from WT (black), F459Y (pink), F459C (orange), F459M (purple), F459E (cyan), F459A (green), F459L (blue), F459Q (gray), F459V (magenta) and F459W (dark yellow) mutant spHCN channels. f, Representative current traces from oocytes expressing WT, M375F, F459M and M375F/F459M spHCN channels. Dashed lines indicate no currents. g, GV relations from WT (black), M375F (green), F459M (orange) and M375F/F459M (red) spHCN channels. All GV1/2, FV1/2 and n numbers are shown in Extended Data Table 2. Data are represented as mean ± s.e.m. n indicates the number of biological replicates.

Source Data

Extended Data Fig. 8 Met-aromatic interactions occur in voltage-gated HCN1 channels.

a, Local structure of HCN1 + propofol, HCN1 closed (PDB 5U6O), and HCN1 crosslinked (PDB 6UQF) around the Met305-Phe389 interaction. The homologous positions Ile307-Ile392 for the CNGA1 structure (PDB 7LFT) are also shown. Approximate distances between atoms (dashed yellow lines) are labeled between methionine, isoleucine, and the adjacent aromatic rings (purple). Propofol is colored in pink and adjacent protomers are in blue and yellow. b, Multiple sequence alignment between human HCN and CNG isoforms. Residue numbering follows the HCN1 amino acid sequence. The methionine, isoleucine, and aromatic positions labeled in panel a are highlighted in red, orange, and blue. A single aliphatic-aromatic interaction (1-bridge) exists in CNG channels which are ligand gated. In contrast, an interaction between methionine with two aromatic residues (2-bridge) occurs in HCN channels which are voltage gated.

Extended Data Table 1 CryoEM data collection, refinement, and validation statistics
Full size table
Extended Data Table 2 Fit parameters for TEVC of human HCN1 propofol binding site mutations and VCF of spHCN mutant channels expressed in Xenopus laevis oocytes
Full size table

Supplementary information

Supplementary Information

Supplementary Fig. 1. Uncropped HCN1 WT and M305L purifications. Supplementary Fig. 2. Uncropped HCN1 WT and M305L confocal images. Supplementary Appendix.

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Kim, E.D., Wu, X., Lee, S. et al. Propofol rescues voltage-dependent gating of HCN1 channel epilepsy mutants. Nature 632, 451–459 (2024). https://doi.org/10.1038/s41586-024-07743-z

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