Acid-sensing ion channels (ASICs) are trimeric1, proton-gated2,3 and sodium-selective4,5 members of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily of ion channels6,7 and are expressed throughout vertebrate central and peripheral nervous systems. Gating of ASICs occurs on a millisecond time scale8 and the mechanism involves three conformational states: high pH resting, low pH open and low pH desensitized9. Existing X-ray structures of ASIC1a describe the conformations of the open10 and desensitized1,11 states, but the structure of the high pH resting state and detailed mechanisms of the activation and desensitization of the channel have remained elusive. Here we present structures of the high pH resting state of homotrimeric chicken (Gallus gallus) ASIC1a, determined by X-ray crystallography and single particle cryo-electron microscopy, and present a comprehensive molecular mechanism for proton-dependent gating in ASICs. In the resting state, the position of the thumb domain is further from the three-fold molecular axis, thereby expanding the ‘acidic pocket’ in comparison to the open and desensitized states. Activation therefore involves ‘closure’ of the thumb into the acidic pocket, expansion of the lower palm domain and an iris-like opening of the channel gate. Furthermore, we demonstrate how the β11–β12 linkers that demarcate the upper and lower palm domains serve as a molecular ‘clutch’, and undergo a simple rearrangement to permit rapid desensitization.
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We thank A. Goehring, D. Claxton and I. Baconguis for initial construct screening and advice through all aspects of the project, L. Vaskalis for help with figures, H. Owen for manuscript preparation, all members of the Gouaux laboratory for their support and the Berkeley Center for Structural Biology and the Northeastern Collaborative Access Team for help with X-ray data collection. This research was supported by the National Institute of General Medical Sciences (5T32DK007680), and the National Institute of Neurological Disorders and Stroke (5F31NS096782 to N.Y. and 5R01NS038631 to E.G.). Additional support was provided by ARCS Foundation and Tartar Trust fellowships. E.G. is an Investigator with the Howard Hughes Medical Institute.
The authors declare no competing financial interests.
Reviewer Information Nature thanks L. Rash and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, I–V relationship for ASIC1a and ∆25 constructs between −40 and 60 mV. Individual data points are displayed and are normalized to current amplitudes at −60 mV. Lines represent mean. ∆25: n = 7 cells; ASIC1a: n = 6 cells. Experiments were performed seven (∆25) and six (ASIC1a) times with similar results. b, Representative whole-cell patch-clamp recordings at stepped potentials from −60 mV to 60 mV for ∆25 and ASIC1a. Experiments were performed seven (∆25) and six (ASIC1a) times with similar results. c, Comparison of Hill slopes of activation for ∆25 and ASIC1a channels by unpaired t-test (two-sided, mean ± s.e.m., **P ≤ 0.01, P = 0.0054; 95% confidence interval = −4.949 to −1.053). ASIC1a: n = 7; ∆25: n = 10. Experiments were performed seven (ASIC1a) and ten (∆25) times with similar results. d, Control experiment demonstrating ASIC1a currents evoked by step to low pH under reducing or ambient conditions. Results are representative of seven independent experiments. Data were collected from Sf9 cells infected with BacMam virus containing ∆25 or ASIC1a DNA.
a, SDS–PAGE of purified chicken ASIC1a. b, Representative micrograph of ASIC1a channels embedded in vitreous ice. c, Angular distribution of particle projections. d, Density map coloured according to local resolution. e, Spherical-masked and solvent-corrected FSC curves for density maps and for the refined model to the final 3D reconstruction. f, Representative density for the ASIC1a reconstruction, identified by residue range or domain above.
Representative data processing steps for the ASIC1a reconstruction.
a, Omit map |Fo|–|Fc| density contoured at 2σ for a domain-swapped TM2, top view shown in inset. b, Discontinuous TM2 helix stabilized by hydrogen bonds. c, Superposition of resting and open (PDB code: 4NTW, grey) channels demonstrates relative conformations of the GAS belt.
a, b, Superposition of resting and open (PDB code: 4NTW, grey) channels highlights interactions between Arg191, Glu314 and His328 (a) and between Val353, Glu354 and Asn357 with Met211 on an adjacent subunit (b) that stabilize the expanded, high pH conformation of the acidic pocket. c, Local superposition (α1 and α2) of resting and open channels demonstrates the α5 pivot upon activation.
a–c, Resting (a), open (b; PDB code: 4NTW) and desensitized (c; PDB code: 4NYK) channel pore profiles calculated with HOLE software (pore radius: red < 1.15 Å < green < 2.3 Å < purple). d–f, Approximate fenestration sizes for resting (d), open (e) and desensitized (f) channels; approximate fenestration edge is outlined with a solid black line.
a, Resting channel pore profile calculated with HOLE software (pore radius: red < 1.15 Å < green < 2.3 Å < purple). b, Plot of pore radius for resting, open (PDB code: 4NTW) and desensitized (PDB code: 4NYK) channels along the three-fold molecular axis. c, d, Conformation of resting and open TMDs viewed from below (c) and the side (d). e, f, Resting and open gates viewed from the side (e) and above (f).
Morph depicting channel activation via transition between high pH resting and low pH open (pdb 4NTW) states. Single subunit colored by domain. (MOV 16881 kb)
Morph depicting channel desensitization via transition between low pH open (pdb 4NTW) and low pH desensitized (pdb 4NYK) states. Single subunit colored by domain. (MOV 9120 kb)
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Yoder, N., Yoshioka, C. & Gouaux, E. Gating mechanisms of acid-sensing ion channels. Nature 555, 397–401 (2018). https://doi.org/10.1038/nature25782
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