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Structural mechanism of muscle nicotinic receptor desensitization and block by curare

Nature Structural & Molecular Biology volume 29, pages 386–394 (2022)Cite this article

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Abstract

Binding of the neurotransmitter acetylcholine to its receptors on muscle fibers depolarizes the membrane and thereby triggers muscle contraction. We sought to understand at the level of three-dimensional structure how agonists and antagonists alter nicotinic acetylcholine receptor conformation. We used the muscle-type receptor from the Torpedo ray to first define the structure of the receptor in a resting, activatable state. We then determined the receptor structure bound to the agonist carbachol, which stabilizes an asymmetric, closed channel desensitized state. We find conformational changes in a peripheral membrane helix are tied to recovery from desensitization. To probe mechanisms of antagonism, we obtained receptor structures with the active component of curare, a poison arrow toxin and precursor to modern muscle relaxants. d-Tubocurarine stabilizes the receptor in a desensitized-like state in the presence and absence of agonist. These findings define the transitions between resting and desensitized states and reveal divergent means by which antagonists block channel activity of the muscle-type nicotinic receptor.

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Fig. 1: Resting-state structure and cholesterol interactions.
Fig. 2: Asymmetric conformational transition between resting and desensitized states.
Fig. 3: Agonist binding site and ion pore profile.
Fig. 4: M4 uncoupling in the desensitized state.
Fig. 5: Structural pharmacology of d-tubo.

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Data availability

Cryo-EM maps and atomic model coordinates have been deposited in the Electron Microscopy Data Bank and PDB, respectively; apo (EMD-25202 and PDB 7SMM), apo plus cholesterol (EMD-25205 and PDB 7SMQ), carbachol-bound desensitized state (EMD-25206 and PDB 7SMR), d-tubo bound (EMD-25207 and PDB 7SMS) and d-tubo plus carbachol bound (EMD-25208 and PDB 7SMT). Source data are provided with this paper.

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Acknowledgements

We thank S. Sine for Torpedo receptor cDNAs, and C. Noviello and S. Zhu for assistance in cryo-EM sample screening. We are grateful to S. Burke, J.J. Kim, C. Noviello, S. Sine and M. Klymkowsky for critical feedback on the manuscript, D. Borek for model building discussion and A. Sobolevsky for helpful discussion related to measuring recovery from desensitization. Single-particle cryo-EM grids were screened at the University of Texas Southwestern Medical Center Cryo-Electron Microscopy Facility, which is supported by the CPRIT Core Facility Support award no. RP170644. We thank H. Scott for cryo-EM data collection at the PNCC under user proposal nos. 50839 and 51574. A portion of this research was supported by National Institutes of Health (NIH) grant no. U24GM129547 and performed at the PNCC at the Oregon Health & Science University and accessed through EMSL (grid.436923.9), a Department of Energy Office of Science User Facility sponsored by the Office of Biological and Environmental Research. M.M.R. acknowledges a postdoctoral fellowship from the American Heart Association (no. 827474). This work was supported by grants from the NIH (nos. DA042072 to R.E.H., AG061829 to M.H.B.S. and NS120496 to R.E.H. and M.H.B.S.) and the MCDB Neurodegenerative Disease Fund to M.H.B.S.

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Authors and Affiliations

  1. Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Md. Mahfuzur Rahman, Jinfeng Teng & Ryan E. Hibbs

  2. Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO, USA

    Tamara Basta, Myeongseon Lee, Brady T. Worrell & Michael H. B. Stowell

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Contributions

M.M.R. performed the sample preparation and data processing for cryo-EM, structural analysis and drafted the manuscript with R.E.H. J.T. performed the electrophysiology. B.T.W. synthesized the ATM affinity reagent. T.B. and M.L. performed the lipid quantification. M.H.B.S. and R.E.H. assisted in structural analysis and model validation and directed the project. M.M.R., R.E.H. and M.H.B.S. revised the manuscript with input from all other authors.

Corresponding authors

Correspondence to Michael H. B. Stowell or Ryan E. Hibbs.

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The authors declare no competing interests.

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Peer review information

Nature Structural and Molecular Biology thanks Michaela Jansen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Florian Ullrich was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Relion 3.1 workflow for data processing.

Representative processing approach for apo dataset.

Extended Data Fig. 2 Local and global resolution estimates for cryo-EM maps.

Local resolution is illustrated by variation in map surface color and was estimated using RELION. Global resolution was estimated at FSC = 0.143 (dotted line) from half maps.

Extended Data Fig. 3 Lipid-receptor interactions.

a, Cholesterol (red) and phospholipid (yellow) binding sites in the receptor. α subunit - green, β subunit - khaki, γ subunit - blue, δ subunit – violet. High-affinity cholesterol binding sites are near the MX helices and low-affinity binding sites are near outer membrane leaflet. Among the five subunit interfaces, the high affinity cholesterols occupy the same positions in three interfaces: α/γ, α/δ, and β/α, but not γ/αδ and δ/β. The environment of that site is more polar in γ and δ subunits, where an asparagine replaces I291/α or L297/β (in M3). b, Conservation of the residues in the cholesterol high affinity binding site, residues numbers are according to T. californica α-subunit. Accession numbers for sequences used to determine conservation are given in the Methods.

Extended Data Fig. 4 Transition between resting and desensitized states.

a, Two electrode voltage clamp (TEVC) recording show activation and desensitization of the Torpedo nicotinic receptor by carbachol; 5 mM carbachol was supplemented into the cryo-EM sample for structure determination of the desensitized state. b, Asymmetry in the TMD conformational change between resting and desensitized states; residues at the 16′ position are shown as sticks. c, Conformational changes in loop C of an α-subunit and Loop F of a complementary subunit after agonist and antagonist binding compared to resting state. Carbachol is shown as green spheres. d, Conformational differences in a representative coupling region; representative α and γ subunits.

Extended Data Fig. 5 Permeation pathway and pore profile.

a, Pore diameter comparison of Torpedo in resting and desensitized states vs. other Cys-loop receptors: resting, α7 nAChR (PDB ID: 7EKI), 5-HT3AR (PDB ID: 6BE1), GlyR (PDB ID: 6UBS); and desensitized, α7 nAChR (PDB ID: 7KOQ), α4β2 nAChR (PDB ID: 6CNJ), α3β4 nAChR (PDB ID: 6PV7), 5-HT3AR (PDB ID: 6HIQ), GlyR (PDB ID: 6UBT). b, Permeation pathway cutaway colored by electrostatic potential. c, Water molecules at the 2′ gate of the desensitized state structure. d, −1′ residue orientation in the Torpedo receptor in resting and desensitized states. Transparent surface is experimental density map. e, Two α-subunits superposition of resting state structure. f, Dose response parameters measured by TEVC of WT and mutants. Nonlinear regression was carried out using GraphPad Prism 8. Replicate measurements are from independent oocytes.

Extended Data Fig. 6 Receptor activation and antagonism by carbachol and d-tubo; d-tubo binding sites with corresponding density maps.

a, Two-electrode voltage clamp (TEVC) recording illustrates receptor antagonism by d-tubo. b, d-Tubo at α/γ interface (site 1). c, d-Tubo at α/δ interface (site 2). d, d-Tubo in the pore (site 3). e, d-Tubo at junction of M1, M3 and M4 helices of the αγ subunit (site 4). Corresponding d-tubo densities are shown as semitransparent surfaces. The d-tubo density was very clear at sites 1 and 4. Site 2 density suggested two different orientations of d-tubo and we modeled the best fitted one. The density at site 3 is poorly resolved suggesting multiple orientations of d-tubo in the pore; we used the unsharpened map to model d-tubo there. Density map was contoured at a threshold of 0.02 for site 1, 2 & 4 and 0.013 for site 3 in UCSF chimera.

Extended Data Fig. 7 Orthosteric binding-sites details and carbachol vs. d-tubo complex superposition.

a, b, Electrostatic potential of the two orthosteric ligand-binding sites in the apo form; binding pockets are indicated by asterisks. c, Residue differences between two orthosteric ligand-binding sites; αγ/γ as colored (α- green, γ- blue) and αδ/δ as gray. d, Superposition of desensitized and d-tubo bound structures; d-Tubo model is colored (α, green; β, khaki; γ, blue; δ, violet) and desensitized structure is in gray. (e) Conformational difference in M4 of two α-subunits in resting, desensitized and pure d-tubo bound structures.

Supplementary information

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Supplementary Video 1

Structural transition between resting and desensitized states. Morphing video illustrates changes from resting state to desensitized state to resting state in the top view. α subunit, green; β subunit, khaki; γ subunit, blue and δ subunit, violet.

Supplementary Video 2

Structural transition between resting and desensitized states. Morphing video illustrates changes from resting state to desensitized state to resting state in the side view. α subunit, green; β subunit, khaki; γ subunit, blue and δ subunit, violet.

Supplementary Video 3

d-Tubo interactions at the α–γ interface (site 1). d-Tubo is shown as sticks (orange) and corresponding densities are shown as semitransparent surfaces. Interacting residues are also shown as sticks and colored by subunits: α subunit, green and γ subunit, blue.

Supplementary Video 4

d-Tubo interactions at the α–δ interface (site 2). d-Tubo is shown as sticks (orange) and corresponding densities are shown as semitransparent surfaces. Interacting residues are also shown as sticks and colored by subunits: α subunit, green and δ subunit, violet.

Supplementary Video 5

d-Tubo interactions at the pore (site 3). d-Tubo is shown as sticks (orange) and corresponding densities are shown as semitransparent surfaces. Interacting residues are also shown as sticks and colored by subunits: α subunit, green; β subunit, khaki; γ subunit, blue and δ subunit, violet.

Supplementary Video 6

d-Tubo interactions at the M1, M3 and M4 helices of αγ subunit (site 4). d-Tubo is shown as sticks (orange) and corresponding densities are shown as semitransparent surfaces. Interacting residues are also shown as sticks and colored by subunit: α subunit, green.

Source data

Source Data Fig. 1

Cholesterol and phospholipid assay.

Source Data Fig. 4

Dose–response curve and desensitization recovery.

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Rahman, M.M., Basta, T., Teng, J. et al. Structural mechanism of muscle nicotinic receptor desensitization and block by curare. Nat Struct Mol Biol 29, 386–394 (2022). https://doi.org/10.1038/s41594-022-00737-3

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