NOMPC Ion Channel Hinge Forms Gating Spring to Initiate Mechanosensation

Academic Background

Mechanosensation is the process by which organisms perceive external mechanical stimuli and convert them into electrical signals. This process plays a crucial role in touch, hearing, gravity perception, and the movement of internal organs and limbs. The initiation of mechanosensation relies on mechanosensory transduction channels (MET channels), which transmit mechanical force to the channel gate through a gating spring, thereby controlling the opening and closing of the channel. The elasticity of the gating spring allows the channel to switch between open and closed states in response to mechanical stimuli.

For a long time, the molecular identity of the gating spring has been a subject of debate in the scientific community. Most research has focused on force-transmitting proteins such as the ankyrin repeat domain, hypothesizing that they might serve as the gating spring. However, these hypotheses lacked direct experimental evidence. This study, by combining protein domain duplication, mechanical measurements, electrophysiology, molecular dynamics simulations, and modeling, revealed that the gating spring of the Drosophila mechanosensory channel NOMPC is actually the short linker helix (LH domain) connecting the ankyrin domain to the channel gate. This discovery provides new insights into the molecular mechanisms of mechanosensation.

Source of the Paper

This paper was jointly authored by Philip Hehlert, Thomas Effertz, Ruo-Xu Gu, and their team from the University of Göttingen, the Max Planck Institute for Multidisciplinary Sciences, and other institutions. It was published in February 2025 in the journal Nature Neuroscience under the title “NOMPC ion channel hinge forms a gating spring that initiates mechanosensation.”

Research Process

1. In Vitro Experiments with NOMPC Domain Duplication

The research team first used genetic engineering to duplicate the ankyrin repeat domain (AR domain) and the linker helix domain (LH domain) of NOMPC, constructing AR+AR-NOMPC and LH+LH-NOMPC variants. These variants were heterologously expressed in Drosophila S2 cells, and their localization on the cell membrane was verified using fluorescent labeling.

Subsequently, the team used patch-clamp techniques to record the spontaneous currents and mechanical stimulus-induced current responses of these variants. The results showed that AR domain duplication had almost no effect on the spontaneous activity and mechanosensitivity of NOMPC, while LH domain duplication significantly reduced the spontaneous activity frequency and mechanosensitivity of NOMPC. Specifically, the LH+LH-NOMPC variant required greater pressure to activate the channel under negative pressure stimulation.

2. In Vivo Experiments with NOMPC Domain Duplication

To validate the in vitro results, the research team expressed NOMPC variants in the Johnston’s organ (JO) neurons of Drosophila. The Johnston’s organ is the auditory organ of Drosophila, where NOMPC plays a critical role in mechanosensation. By recording the compound action potentials (CAPs) of JO neurons, the team found that the AR+AR-NOMPC variant fully restored the mechanosensitivity of NOMPC-deficient mutants, while the LH+LH-NOMPC variant only partially restored it, indicating that LH domain duplication reduced the mechanosensitivity of NOMPC.

3. Measurement of Gating Spring Stiffness

The team further assessed the impact of NOMPC variants on gating spring stiffness by measuring the mechanical properties of the Drosophila antennal sound receiver. The results showed that AR domain duplication did not significantly alter the stiffness of the gating spring, while LH domain duplication halved the stiffness. This indicates that the LH domain is the primary component of the NOMPC gating spring.

4. Molecular Dynamics Simulations

To reveal the mechanical properties of the LH domain, the research team performed molecular dynamics simulations on a portion of the NOMPC structure, applying both pushing and pulling forces to the AR domain. The results showed that the LH domain exhibited hinge-like motion under force, with much greater deformation than the AR domain. This suggests that the LH domain acts as an elastic hinge in NOMPC mechanosensation, transmitting force to the channel gate.

5. Crosslinking Experiments

To further validate the function of the LH domain, the research team introduced cysteine pairs into the LH domain and used the crosslinker MTS6 to stabilize its structure. The results showed that after crosslinking, the spontaneous activity and mechanosensitivity of the LH+LH-NOMPC variant were restored to levels similar to those of wild-type NOMPC. This indicates that the elastic hinge structure of the LH domain is crucial for its function.

Key Findings

1. The LH domain is the primary component of the NOMPC gating spring: LH domain duplication significantly reduced the mechanosensitivity and stiffness of the NOMPC gating spring, while AR domain duplication had minimal impact.
2. The LH domain functions as an elastic hinge: Molecular dynamics simulations revealed that the LH domain exhibits hinge-like motion under force, transmitting force to the channel gate.
3. The function of the LH domain can be restored through crosslinking: By stabilizing the structure of the LH domain, the function of the LH+LH-NOMPC variant was restored to levels similar to those of wild-type NOMPC.

Conclusion

This study is the first to reveal the molecular mechanism of the LH domain as the gating spring of the NOMPC ion channel. Through its elastic hinge function, the LH domain efficiently transmits mechanical stimuli to the channel gate, thereby initiating mechanosensation. This discovery not only resolves the long-standing debate over the molecular identity of the gating spring but also provides new insights into the gating mechanisms of other ion channels.

Research Highlights

1. Resolves the molecular identity of the gating spring: Through experiments and simulations, the research team confirmed that the LH domain is the primary component of the NOMPC gating spring.
2. Innovative experimental design: The team systematically validated the function of the LH domain through domain duplication and crosslinking experiments.
3. Broad application value: This discovery is not only applicable to NOMPC channels but may also provide insights for research on the gating mechanisms of other mechanosensitive channels and ion channels.

Additional Valuable Information

The molecular dynamics simulations and crosslinking experiments in this study provide new methodological tools for future ion channel research. Additionally, the detailed functional analysis of the NOMPC channel offers potential targets for developing drugs targeting mechanosensation-related diseases.