SH

Synaptic Vesicle Fusion and SNARE Complexes

Overview of Synaptic Vesicle Fusion Mechanisms

Key Proteins Involved in Fusion

  • SNAP-25, Synaptobrevin, Syntaxin

    • These proteins are critical in forming the SNARE complex which is essential for synaptic vesicle fusion.

    • They facilitate the priming of vesicles, making them fusion competent.

Priming and SNARE Complex Assembly

  • Fusion Competence of Primed Vesicles

    • Primed vesicles are those that are ready to fuse with the presynaptic membrane, however, they have a challenge: syntaxin is usually in a default closed configuration, hiding the SNARE binding domain that is necessary for fusion.

  • SNARE Complex Formation

    • To achieve priming, assembly of the SNARE complex is required, involving syntaxin, synaptobrevin, and SNAP-25.

    • Closed syntaxin cannot form the SNARE complex, so understanding how to transition to an open state is crucial.

Syntaxin Configurations

  • Closed versus Open State of Syntaxin

    • Closed Syntaxin

    • Hinders SNARE complex formation.

    • Open Syntaxin

    • Allows for SNARE complex assembly with synaptobrevin and SNAP-25.

Role of UNC-13

  • UNC-13 Functionality

    • UNC-13 binds to the N-terminus of syntaxin, promoting the assembly of the SNARE complex.

    • This interaction is crucial for transitioning syntaxin from the closed to the open state, facilitating vesicle docking.

Docking of Synaptic Vesicles

  • Docking Mechanism

    • SNARE complex assembly brings the synaptic vesicle into close proximity with the presynaptic membrane, enabling docking.

    • Mutations in syntaxin or UNC-13 result in defects in synaptic vesicle docking.

    • High-Pressure Freezing Technique

    • Utilized to preserve synapses in a physiological state for examination of docked and undocked vesicles.

Imaging and Structural Insights

  • High-Resolution Imaging

    • Visual representation of the symmetrical organization of protein complexes anchoring vesicles to the plasma membrane.

    • Images include various orientations (side view, top view) with scale indicated (e.g., 50 nm).

Crystal Structure and Regulation by Calcium

  • Munc13/UNC-13 Crystal Structure

    • Discovered conformations regulated by diacylglycerol (DAG) and calcium ions (Ca2+).

    • States of Munc13

    • State 1: "Captured" with unassembled SNAREs.

    • State 2: "Pre-primed" with unassembled SNAREs.

    • State 3: "Primed" state with clamped SNARE pins.

Role of Calcium in Vesicle Fusion

  • Mechanism of Calcium Influx

    • Voltage-gated calcium channels open in response to action potentials, leading to calcium influx.

    • This calcium influx triggers vesicle fusion due to the close proximity of calcium channels to primed vesicles, typically occurring within 200 ms of influx.

Synaptotagmin's Role in Synaptic Release

  • Knockout Studies of Synaptotagmin I

    • Mice lacking synaptotagmin demonstrate a loss of synchronous release of neurotransmitters, indicating its crucial role in the process.

    • Control versus synaptotagmin knockout demonstrated the impact on release patterns.

Interaction Between Calcium and Synaptotagmin

  • Calcium Binding Interaction

    • Calcium interacts with the calcium-binding protein synaptotagmin, essential for vesicle fusion.

Synaptotagmin Structure and Mechanism

  • Ring Formation

    • Synaptotagmin forms rings around the synaptic vesicle to interact with SNARE complexes.

    • C2B Domain Role

    • Upon binding calcium, the C2B domain of synaptotagmin inserts into the plasma membrane, causing it to dimple, facilitating membrane fusion.

Process of Fusion Initiation

  • Calcium and SNARE Complex Mechanics

    • Synaptotagmin binds to the SNARE complex, holding it together until calcium presence triggers further interactions.

    • Calcium binding to the C2B domain results in C2B insertion into the target membrane, which changes membrane curvature, lowering the energy barrier for fusion.

    • Ultimately, calcium-synaptotagmin interactions drive the complete zipping of SNARE complexes to initiate fusion.

Summary of Exocytosis Process

  • Key Steps in Exocytosis

    • Munc13 promotes the docking of synaptic vesicles through SNARE complex assembly (priming).

    • Synaptotagmin clamps primed vesicles until a calcium signal is received.

    • Calcium channels open in response to action potentials, leading to synaptotagmin binding and consequent vesicle fusion.