week 6 lecture 1

Overview of Synaptic Vesicle Fusion Mechanism

SNARE Complex Formation

  • Key Proteins Involved: SNARE proteins include SNAP-25, synaptobrevin, and syntaxin.

    • SNAP-25: A cytoplasmic SNARE protein that interacts with other SNARE proteins.

    • Synaptobrevin: A vesicle-associated SNARE protein that is crucial for vesicle fusion.

    • Syntaxin: A membrane-bound SNARE protein that exists in two conformations: closed and open.

  • Primed Vesicles: Vesicles that are fusion competent; readiness for fusion is achieved through SNARE complex assembly.

  • Priming Process: Requires the assembly of the SNARE complex between syntaxin, synaptobrevin, and SNAP-25.

Syntaxin Configuration

  • Closed Configuration: Syntaxin adopts a default closed configuration that conceals the SNARE binding domain, leading to incompatibility with SNARE complex formation.

  • Problem Statement: Priming requires syntaxin to transition from a closed to an open state to enable SNARE complex assembly.

Opening Syntaxin

  • Mechanism for Opening Syntaxin:

    • UNC-13 protein is crucial as it binds to the N-terminus of syntaxin, effectively promoting the SNARE complex assembly and facilitating the opening of syntaxin.

    • Reference: Betz et al., 1997.

Role of UNC-13

  • Function of UNC-13: Enhances SNARE complex assembly by transitioning syntaxin from a closed to an open conformation, allowing for effective binding and complex formation.

Vesicle Docking

  • Importance of Syntaxin and UNC-13: Both proteins are vital for synaptic vesicle docking at the presynaptic membrane.

  • Mutant Studies: Mutant studies showcase defects in synaptic vesicle docking when either UNC-13 or syntaxin is impaired.

    • High-Pressure Freezing Technique: Used to preserve synapses in a physiological state for analysis.

Imaging Studies

  • High-resolution Images: Show symmetrical organization of protein complexes attaching vesicles to the plasma membrane.

    • Demonstrates intricate spatial arrangement at a synapse, highlighting the role of various protein complexes.

Crystal Structure of Munc13/UNC-13

  • Regulatory Conformations: Munc13 exhibits different conformations influenced by factors such as Diacylglycerol (DAG) and Calcium ions ($Ca^{2+}$).

    • States Explained:

    • State 1 (Captured): Represents unassembled SNAREs.

    • State 2: Designated as 'Pre-primed' with the presence of unassembled SNAREs.

    • State 3: 'Primed' state; clamped SNAREpins are ready for fusion.

Regulation of Vesicle Fusion by Calcium

  • Calcium Influx: Triggered by action potentials through voltage-gated calcium channels.

    • Calcium levels peak within 200 ms of influx due to close proximity of calcium channels to primed vesicles.

Synaptotagmin's Role in Fusion

  • Effects of Synaptotagmin I Knockout: Loss leads to impaired synchronous release, highlighting the protein's critical role in fusion events.

Calcium-Synaptotagmin Interactions

  • Calcium Regulation: Calcium interacts with synaptotagmin, facilitating the fusion process.

    • Synaptotagmin forms rings on synaptic vesicles (SV) that interact with SNARE complexes.

  • Mechanics of Fusion:

    • Synaptotagmin C2B domain inserts into the plasma membrane upon binding with $Ca^{2+}$, inducing membrane curvature which reduces the energy barrier for fusion.

    • The interaction between calcium and synaptotagmin causes SNARE complexes to zipper fully, initiating vesicle fusion.

Summary of Exocytosis Mechanism

  • Munc13 Rings: Dock synaptic vesicles at the membrane.

  • Priming: Munc13 promotes SNARE complex assembly.

  • Triggering Fusion: Calcium channels open in response to action potentials, causing calcium to bind synaptotagmin, thereby triggering vesicle fusion with the presynaptic membrane.