A Specific Negatively Charged Sequence Confers Intramolecular Regulation on Munc13-1 Function in Synaptic Exocytosis

Neurology Biophysics Basic Medical Sciences Cell Biology Biochemistry Life Sciences Medicine
Munc13-1 synaptic exocytosis negatively charged sequence autoinhibition calcium regulation protein structure neurotransmitter release

Unlocking a New Mechanism for the Regulation of Neurotransmitter Release: A Review of Munc13-1’s Novel Autoinhibitory Structure and Its Calcium-Modulated Role

I. Academic Background and Research Motivation

Signal transmission between neurons relies on chemical synapses, with precise neurotransmitter release from presynaptic terminals via synaptic exocytosis. The active zone (AZ) serves as the molecular foundation for this process. The protein complexes in the AZ not only determine vesicle docking, priming, fusion, and the accuracy of transmitter release but also play a central role in higher neural functions such as synaptic plasticity.

Among numerous molecules regulating synaptic exocytosis, the Munc13 family of proteins (Munc13s) is regarded as a key multiphasic regulator involved in nearly the entire exocytotic process, assuming critical roles in vesicle docking, priming, and final fusion. Particularly in mammalian brain tissue, Munc13-1, the major expressed isoform, maintains the basic function of synaptic transmission and also modulates important physiological processes such as short-term synaptic plasticity. Although the conserved C-terminal region (including C2 and MUN domains) of Munc13-1 has been thoroughly studied, the specific functions and regulatory mechanisms of its unique N-terminal low-complexity region remain largely unexplored, especially regarding how this region modulates Munc13-1 activity through intramolecular interactions at a molecular and physiological level.

Against this scientific background, researchers sought to answer: Does the N-terminal low-complexity region unique to Munc13-1 contain functional structural modules? How does this module influence autoinhibition and activation of the protein? Are regulators such as calcium ions (Ca²⁺) or phosphorylation involved in this process? Addressing these issues not only enriches our fundamental understanding of neurobiology but also provides a theoretical basis for investigating the mechanisms and therapeutic strategies of neurological disorders.

II. Paper Source and Author Information

The original research article, titled “A specific negatively charged sequence confers intramolecular regulation on Munc13-1 function in synaptic exocytosis,” was completed by Kexu Zhao, Li Zhang, Mengshi Lei, Ziqi Jin, Tianxin Du, et al., with Shen Wang and Cong Ma as corresponding authors. The primary research institutions include the Key Laboratory of Molecular Biophysics of the Ministry of Education at the College of Life Science and Technology of Huazhong University of Science and Technology, School of Artificial Intelligence and Automation, Key Laboratory of Neurogenetics and Channelopathies of the Ministry of Education at the Second Affiliated Hospital of Guangzhou Medical University, among others. The paper was published on June 9, 2025, in Proceedings of the National Academy of Sciences (PNAS) as a direct submission, peer-reviewed by invitation.

III. Research Workflow Detailing

This study, leveraging molecular and cellular approaches, integrates bioinformatics, structural prediction, biochemical assays, advanced optical techniques, and neuronal electrophysiology to comprehensively analyze the physicochemical properties, evolutionary conservation, intramolecular autoinhibitory effects, and calcium/phosphorylation regulation of the negatively charged polyE sequence in the N-terminal low-complexity region of Munc13-1.

1. Study Subjects and Sample Sources

  • Protein Materials: Mainly recombinant proteins of human and animal Munc13 family isoforms, expressed and purified for in vitro functional studies.
  • Cellular/Animal Models: Mouse primary cortical neurons were used for electrophysiological and gene knockdown-rescue experiments.
  • Sequence Evolutionary Comparison: Included genes from multiple species, ranging from nematodes to humans.

2. Bioinformatics and Sequence Conservation Analysis

a. Discovery of the polyE Sequence

By comparing the N-terminal low-complexity sequences of different Munc13 isoforms, the research team found that Munc13-1 contains a negatively charged cluster rich in glutamate (Glu) and aspartate (Asp) residues at positions 317-370, which is much higher than in other isoforms. Using a custom-developed Glu&Asp cluster score (public on GitHub), they quantitatively revealed the prominent acidic residue clustering of the polyE region, and confirmed its conserved presence in birds and higher homeotherms, indicating its functional importance through evolution.

b. Structural and Functional Annotation

Utilizing AlphaFold-Multimer structural predictions, the team identified polyE as an acidic cluster capable of transitioning into an α-helix, closely interacting with basic lysines K1494, K1495, K1500 in the D subdomain of the MUN domain, hinting at potential salt bridge formation that facilitates intramolecular autoinhibition.

3. Biochemical and Structural-Functional Experiments

a. Protein Interaction Experiments

Using GST pull-down, fluorescence anisotropy, and microscale thermophoresis (MST), the team systematically assessed the binding between polyE and the MUN domain. Results showed a binding constant (Kd) of 24.19 μM for the polyE-MUN interaction, which is charge-dependent and can be completely inhibited by high salt concentration (e.g., 1M NaCl). Mutations K1494E/K1495E/K1500E also abolish this interaction.

b. Validation of the Intramolecular Autoinhibitory Model

The researchers engineered a chimeric protein (Elm) linking polyE to MUN through a 23GS artificial linker, using binding activities of free polyE/MUN as controls. This confirmed that polyE folds back onto the MUN D subdomain, forming a semi-closed autoinhibitory conformation that impedes downstream substrate interactions. Circular dichroism (CD) spectra further revealed that free polyE is mostly unstructured, but adopts an α-helical configuration upon MUN binding, indicating significant conformational plasticity.

c. Functional Testing of SNARE Complex Assembly

Employing a FRET-based system, the team reconstituted a classical in vitro SNARE complex assembly reaction with Munc18-1, Syntaxin-1, Synaptobrevin, and SNAP-25. Results showed that Elm (polyE-MUN chimera) dramatically reduced SNARE complex assembly activity under physiological ionic strength, with even stronger autoinhibition under low ionic strength. Mutant/chimeric forms disrupting the interaction relieved inhibition and restored activity.

4. Exploration of Regulatory Mechanisms

a. Key Regulatory Site Mutagenesis and Phosphorylation Effects

Through phosphomimetic mutations (T1496E, S1501E, etc.) at serine/threonine sites on the MUN D subdomain, pull-down and FRET experiments revealed that these mutations attenuated polyE-MUN binding, abolished autoinhibition, and enhanced SNARE assembly efficiency. Protein expression and electrophysiology experiments confirmed that neurons expressing polyE-deleted or critical site-mutated Munc13-1 displayed significantly elevated miniature excitatory postsynaptic current (mEPSC) frequency, evoked EPSC amplitude, and readily releasable pool (RRP) size, supporting the role of the autoinhibitory mechanism.

b. Calcium Ion Regulation Studies

Isothermal titration calorimetry (ITC) showed a polyE-Ca²⁺ binding constant of approximately 12.5 μM, matching local [Ca²⁺] levels induced by a single neuronal action potential. Further, single-molecule FRET (smFRET) tracking of Syntaxin-1 conformational changes and SNARE complex assembly showed that adding 40 μM CaCl₂ significantly relieved the Elm autoinhibitory state and increased downstream activity, demonstrating that Ca²⁺ is a critical physiological activator of polyE regulation.

5. Neuronal-Level Functional Verification

By employing knockdown-rescue strategies in mouse cortical neurons with polyE-deleted/critical site-mutated/phosphomimetic variants of Munc13-1, systematic recordings of mEPSC, evoked EPSC, and sucrose-induced release showed that all autoinhibition-deficient forms significantly increased release probability, confirming their regulatory role in synaptic function. In contrast, wild-type and dephosphorylation-mimic (T1496A) mutants showed no significant difference from control, highlighting the importance of this regulatory mechanism for fine-tuning physiological function.

IV. Main Research Findings and Conclusions

This study systematically reveals that the polyE acidic cluster in the N-terminal low-complexity region of Munc13-1 serves as a unique autoinhibitory module, capable of stabilizing a “closed” inactive state through intramolecular electrostatic interactions. Transient local increases in Ca²⁺ influx during high-frequency neuronal activity directly bind polyE, efficiently disrupting the autoinhibitory interaction and activating Munc13-1 to trigger SNARE assembly and neurotransmitter release. At the same time, dynamic and reversible phosphorylation (e.g., at T1496) provides longer timescale regulation of the autoinhibitory conformation. Evolutionary analysis indicates that polyE is unique to Munc13-1 in higher homeothermic animals, conferring upon it the advanced capacity for synaptic plasticity modulation.

V. Scientific Significance and Application Prospects

  1. Scientific Value

    • Reveals a novel molecular mechanism by which Munc13-1 regulates neurotransmitter release, assigning a definitive autoinhibitory function to its low-complexity region.
    • Identifies the polyE sequence as an unconventional calcium sensor, expanding the molecular landscape of Ca²⁺ participation in neurotransmission regulation.

  2. Application Prospects

    • This autoinhibition/relief mechanism underpins the biological basis for synaptic short-term plasticity, activity-dependent regulation, and information encoding, suggesting that targeting the polyE-MUN interaction may be a therapeutic strategy for certain neuropsychiatric disorders, epilepsy, etc.
    • Provides a molecular target for designing novel protein regulators and finely modulating synaptic release probability.

  3. Research Highlights

    • Systematically elucidates and validates the function of Munc13-1’s N-terminal low-complexity region, addressing a longstanding question in the field.
    • Integrates protein engineering, structural prediction, a custom algorithm (Glu&Asp cluster score), high-precision biophysical techniques, and calcium regulation to achieve a full-chain, in-depth analysis from sequence and structure to neuronal function.
    • Shows that polyE is an evolutionary addition, potentially serving as a molecular basis for advanced brain regions and complex behaviors.

VI. Other Valuable Content in the Study

  • The Glu&Asp cluster score algorithm developed in this study is publicly available on GitHub (https://github.com/shenwang3333/edclusterscore), providing a technical tool for future studies linking low-complexity sequences to function.
  • The phosphorylation sites examined are associated with multiple presynaptic protein kinases, connecting second messengers, protein modification, and synaptic activity into a regulatory network.

VII. Summary and Outlook

Through a multidisciplinary perspective, this research breakthrough reveals a novel intramolecular autoinhibitory-relief mechanism for Munc13-1, providing a key molecular and regulatory axis for understanding complex information processing and plasticity in the nervous system. In the future, this line of research could be extended to studies on protein aggregation regulation, synaptic pathology, and drug screening, possessing broad fundamental and applied value.