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NOMPC ion channel hinge forms a gating spring that initiates mechanosensation

Nature Neuroscience volume 28, pages 259–267 (2025)Cite this article

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Abstract

The sensation of mechanical stimuli is initiated by elastic gating springs that pull open mechanosensory transduction channels. Searches for gating springs have focused on force-conveying protein tethers such as the amino-terminal ankyrin tether of the Drosophila mechanosensory transduction channel NOMPC. Here, by combining protein domain duplications with mechanical measurements, electrophysiology, molecular dynamics simulations and modeling, we identify the NOMPC gating-spring as the short linker between the ankyrin tether and the channel gate. This linker acts as a Hookean hinge that is ten times more elastic than the tether, with the linker hinge dictating channel gating and the intrinsic stiffness of the gating spring. Our study shows how mechanosensation is initiated molecularly; disentangles gating springs and tethers, and respective paradigms of channel gating; and puts forward gating springs as core ion channel constituents that enable efficient gating by diverse stimuli and in a wide variety of channels.

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Fig. 1: Effects of domain duplications on in vitro NOMPC function.
Fig. 2: Effects of domain duplications on JO neuron CAP responses.
Fig. 3: Effects of domain duplications on gating compliance and gating-spring stiffness.
Fig. 4: Pulling force acting on ARs deforms the LH domain.
Fig. 5: Constraining one-half of the duplicated LH domain by crosslinking cysteine pairs reverts it functionally to a single LH domain.

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Data availability

Source data can be found in a public repository74. Plasmids and flies generated in this study will be provided on request. The NOMPC.L structure used for the molecular dynamic simulations is accessible at Protein Data Bank (PDB) with accession 5VKQ. Source data are provided with this paper.

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Acknowledgements

We thank Y.-N. Jan and L. Jan for providing nompC.L and AR+AR-nompC.L plasmids, UAS-AR+AR-nompC.L and an anti-NOMPC antibody. C. Dean helped with TIRF microscopy, and S. Pauls and N. Schwedthelm-Domeyer provided technical assistance. This study was supported by the German Science Foundation (grants SFB 889 A1, SPP1680, GO 1092/1-2) to M.C.G. and the Cluster of Excellence MBExC (B.L.d.G. and M.C.G.).

Author information

Author notes

  1. Ruo-Xu Gu

    Present address: School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China

  2. Bart R. H. Geurten

    Present address: Department of Zoology, University of Otago, Dunedin, New Zealand

  3. These authors contributed equally: Philip Hehlert, Thomas Effertz.

Authors and Affiliations

  1. Department of Cellular Neurobiology, University of Göttingen, Göttingen, Germany

    Philip Hehlert, Björn Nadrowski, Bart R. H. Geurten & Martin C. Göpfert

  2. Department of Otorhinolaryngology, Head and Neck Surgery and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany

    Thomas Effertz & Dirk Beutner

  3. Computational Biomolecular Dynamics Group, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany

    Ruo-Xu Gu & Bert L. de Groot

  4. Multiscale Bioimaging Cluster of Excellence (MBExC), University of Göttingen, Göttingen, Germany

    Bert L. de Groot & Martin C. Göpfert

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Contributions

M.C.G. initiated this work. P.H. performed molecular biology and cell transfections and ran all the in vivo experiments together with T.E. T.E. performed in vitro patch-clamp recordings and analyzed experimental data together with P.H., B.N., B.R.H.G., D.B. and M.C.G. R.-X.G. and B.L.d.G. carried out and analyzed molecular dynamics simulations. M.C.G. wrote the manuscript together with P.H., T.E., R.-X.G, B.N., D.B. and B.L.d.G., and all authors commented on it and all results.

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Correspondence to Martin C. Göpfert.

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Extended data

Extended Data Fig. 1 Expression of GFP-tagged NOMPC variants in Drosophila S2 cells.

a, Protein localization. GFP fluorescence reports cell surface localization for all three variants. n ≥ 100 cells/construct. b, Currents and respective channel numbers in excised outside-out membrane patches. Left: channel number \(N\) per patch deduced by fitting \({p}_{o,\ge 1}\left(P\right)\) (Fig. 1h) with \({Y\left(P\right)=1-\left(1-{p}_{o}\left(P\right)\right)}^{N}\). Middle: respective peak current amplitudes. Right: channel number deduced by dividing the peak current (middle) by the respective single channel current amplitude (Fig. 1d) (n = (n = 7 cells per protein variant except for n = 9 (NOMPC::GFP) and n = 6 (LH+LHCys-NOMPC::GFP)). Both methods yield similar channel numbers. c, Top: respective normalized amplitudes (\(I/{I}_{\max }\)) as function of the stimulus pressure (n = 7 cells per protein variant except for n = 9 (NOMPC::GFP) and n = 6 (AR + AR-NOMPC::GFP)). Solid lines: respective Boltzmann fits. Hatched lines: \({p}_{o}\left(P\right)\) deduced from Fig. 1f by fitting \({p}_{o,\ge 1}\left(P\right)\) with \({Y\left(P\right)=1-\left(1-{p}_{o}\left(P\right)\right)}^{N}\). Bottom: superimposed Boltzmann fits from the top panels and respective offset pressure corresponding to half-maximal current amplitude (shown for \({p}_{o}\left(P\right)\) in Fig. 1g). Compared to the deduced \({p}_{o}\left(P\right)\), the normalized current amplitudes increase less steeply with the stimulus pressure (top), yet, like \({p}_{o}\left(P\right)\), they superimpose for AR + AR-NOMPC::GFP and NOMPC::GFP and are shifted to approximately twice larger pressures for LH + LH-NOMPC::GFP and LH+LHCys-NOMPC::GFP.

Source data

Extended Data Fig. 2 Localization of GFP-tagged NOMPC variants in Drosophila JO neurons.

GFP signals are enhanced with an anti-GFP antibody (cyan), and JO neurons are counterstained with anti-horseradish peroxidase (HRP), which recognizes sugar residues of glycoproteins that are secreted by JO neurons in two bands (h, see inset upper right). The proximal band (arrowhead) demarks the junction between the cilium (c) and the dendritic inner segment (d), and the distal band demarks the ciliary dilation (“cd”) that separates cilium tip and base regions. s: Cell soma. Anti-GFP staining shows that NOMPC::GFP, AR + AR-NOMPC::GFP, and LH + LH-NOMPC::GFP localize to the cilium tip regions, same as native NOMPC (lower panel, stained with the anti-NOMPC antibody αNOMPC-EC16. Scale bars: 20 µm. n = 3 flies/strain.

Extended Data Fig. 3 Elasticities of LH and AR domains.

We simulated two systems separately to calculate the spring constants of the LH+5ARs (a, TMD + LH+5ARs) and the ARs (b, LH+26ARs). Only one subunit is shown in ribbon for clarity, with the transmembrane domain (TMD), the TRP helix, the LH domain and the ARs in gray, cyan, orange and magenta. The ARs on which forces were applied are labelled by green lines. For panel b, we also show the structure of a subunit, which is coloured based on the root-mean-square-fluctuation (RMSF) of the Cα atom of each residue (simulations with a force of 30 kJ/(mol nm) applied to AR4 are used as an example). For additional information, see Methods and Supplementary Table S2.

Extended Data Fig. 4 Correlation between distances of amino acid pairs and LH domain principal components.

Correlation coefficients between the Cα-Cα distances of the three amino-acid pairs that were converted into cysteine pairs to generate LH+LHCys-NOMPC::GFP (Fig. 5 and Extended Data Fig. 5) and the principal components (PCs) of the LH domain in equilibrium (left), pushing (middle) and pulling (right) simulations. Correlation coefficients whose absolute values exceed 0.5 are highlighted in blue. The positions of the respective amino-acid pairs and their Cα-Cα distances in equilibrium (without forcing) and non-equilibrium (with pushing force) simulations are indicated in Fig. 5b.

Extended Data Fig. 5 Transient effects of crosslinking cysteine pairs on in vitro NOMPC function.

a, Representative traces of spontaneous currents recorded before and at different times after application of the crosslinking agent MTS6. b, Corresponding traces of pressure-evoked currents. MTS6 transiently increases spontaneous and pressure-evoked currents of LH+LHCys-NOMPC::GFP carrying three cysteine pairs (I1161C–C1203, K1177C–N1241C, A1212C–H1249C), but not of NOMPC::GFP, LH-LH-NOMPC::GFP, and also LH+LHCys-ctrl-NOMPC::GFP, in which two of the three cysteine pairs of LH+LHCys-NOMPC::GFP (K1177C–N1241C and A1212C–H1249C) are reverted back to single cysteines (K1177C and A1212C). c, Corresponding pressure\(\,\left({\rm{P}}\right)\)-dependent open probability \({{\rm{p}}}_{{\rm{o}}}\left({\rm{P}}\right)\) before (hatched lines) and immediately after (solid lines) MTS6 application (N = 5 cells per construct). For each construct, \({{\rm{p}}}_{{\rm{o}},\ge 1}\left({\rm{P}}\right)={1-(1-{{\rm{p}}}_{{\rm{o}}}\left({\rm{P}}\right))}^{{\rm{N}}}\) (left) and \({{\rm{p}}}_{{\rm{o}}}\left({\rm{P}}\right)\) (right) are shown. MTS6 shifts \({{\rm{p}}}_{{\rm{o}}}\left({\rm{P}}\right)\) to lower pressure amplitudes for LH+LHCys-NOMPC::GFP, but not NOMPC::GFP, LH + LH-NOMPC::GFP, and LH+LHCys-ctrl-NOMPC::GFP. This narrows down the effect to modification of the two cysteine pairs that are present only in LH+LHCys-NOMPC::GFP (K1177C–N1241C and A1212C–H1249C) and that, according to our molecular dynamics (MD) simulations, change distance in response to force (Fig. 5b). At the same time, it shows that this increase arises neither from modification of the cysteine pair that is present in both constructs (I1161C–C1203, which does not change distance in our MD simulations (Fig. 5b)), nor from modification of the two single cysteines K1177C and A1212C. In principle, the effect could arise from modification of the two single cysteines N1241C and H1249C, yet this possibility seems unlikely, not only because MTS6 crosslinks cysteine pairs, but because MTS6 does not randomly impair LH+LHCys-NOMPC::GFP, but shifts its \({p}_{o}\left(P\right)\) to that of NOMPC::GFP.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2.

Reporting Summary

Supplementary Video 1

PC1, the ‘core protein.’

Supplementary Video 2

PC1, LH domain only.

Supplementary Video 3

PC2, LH domain only.

Supplementary Video 4

PC3, LH domain only.

Source data

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Source Data Extended Data Fig. 1

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Hehlert, P., Effertz, T., Gu, RX. et al. NOMPC ion channel hinge forms a gating spring that initiates mechanosensation. Nat Neurosci 28, 259–267 (2025). https://doi.org/10.1038/s41593-024-01849-3

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