SNARE

Home, Search, Index, Links, Pathology, Molecules, Syndromes,
Muscle, NMJ, Nerve, Spinal, Ataxia, Antibody & Biopsy, Patient Info

PRESYNAPTIC PROTEINS, SYNAPTIC VESICLE DOCKING & MEMBRANE FUSION ¹

Active zones
Interactions between presynaptic proteins
Transmitter release pathway
Vesicle trafficking & release: Steps
Presynaptic proteins
SNAREs
Vesicle membrane proteins
Ca⁺⁺ & ATP regulation
Other secretion-related proteins

External link: Heuser images

Presynaptic steps leading to transmitter release

Action potentials
- Invade nerve terminals
- Cause depolarization
- Other features
  - In many neurons only 10%–20% of action potentials trigger transmitter release
  - Repetitive trains of action potentials
    - At 3 Hz the number of vesicles relased per potential declines with time
    - At faster impulse rates the number of vesicles released per potential may increase for a time
Depolarization of nerve terminals
- Activates voltage-gated calcium channels (VGCC)
- Channels are located in active zones: Each active zone contains 2 parallel arrays of VGCCs
Calcium entry: Through activated VGCCs
- Ca⁺⁺ channel types: Most commonly P/Q (Ca_v2.1) or N (Ca_v2.2) -type
- Begins: At peak of the action potential
- Ends: Before terminal is fully repolarized
Vesicle pathways: Docking & Membrane fusion
- Initiated by
  - Calcium entry
  - Via Ca⁺⁺ sensor: Synaptotagmins
- Vesicle stages
  - Docking: Vesicles move toward & interact with membrane of presynaptic terminal
    - Distance of vesicles from Ca⁺⁺ channel cluster: Range 30 to 300 μM; Mean 100 μM
    - Molecular mechanisms: Interactions between secretion-related proteins
  - Priming: Vesicles become competent for fusion-pore opening
  - Fusion-pore opening
    - Ca⁺⁺-induced
    - Related to fast transmitter release
- SNARE proteins in synaptic exocytosis
  - Synaptic vesicles: Synaptobrevin (Vesicle-associated membrane protein; VAMP)
  - Presynaptic plasma membrane: Syntaxin 1 & SNAP-25
Neurotransmitter release
- Transmitter source: Synaptic vesicles
- Release types
  - Fast release: Ca⁺⁺ triggered
    - Synchronous
    - Phasic
    - Induced rapidly: Onset 50 to 500 μSec after Ca⁺⁺ transient develops
    - Ca⁺⁺ stimulation
      - Sensor: Synaptotagmin 1
      - Concentration: High
  - Slow release: Ca⁺⁺ triggered
    - Asynchronous
    - Continues for > 1 sec after Ca⁺⁺ transient develops
    - Increase in rate of spontaneous release
    - Ca⁺⁺ stimulation
      - Sensor: ? Synaptotagmins 3 or 7
      - Concentration: Low
  - Spontaneous release: Occurs at rest
- Probability of fusion-pore opening after action potential: Variable
  - Depends on tension & composition of participating membranes
- Motor nerve terminals
  - Action potentials produce the release of ACh from 10 to 200 vesicles
  - Each vesicle contains 5,000 to 20,000 ACh molecules
Vesicle recycling: Several alternative pathways

Trafficking cycle: Filling, Targeting, attachment & fusion of vesicles: Steps⁴

Neurotransmitter transport into synaptic vesicles
- Active transport
  - Driven by vacuolar protein proton pump
  - Generates electrochemical gradient that drives neurotransmitter uptake
- Proton pump structure
  - 13 subunits
  - Peripheral complex (V1): Includes ATPase activity
  - Integral membrane complex (V0): Mediates proton translocation
  - Connection between V0 & V1 complexes: 116-kDa subunit
  - Most vesicles contain only 1 proton pump molecule
  - Vacuolar
- Transporters
  - Subtypes
    - Glutamate: 3 types; Driving force is membrane potential
    - Monoamines: 2 types
    - GABA & Glycine: 1 type; Membrane potential & proton gradient contribute to uptake
    - Acetylcholine: 1 type
  - Mechanisms
    - Control of uptake amount & speed not defined
Vesicle properties
- Size: 20 nM radius
- Protein:Phospholipid ratio: 1:3
- Lipid composition
  - Phosphatidylcholine: 40%
  - Phosphatidylethanolamine: 32%,12%
  - Phosphatidylserine: 12%
  - Phosphatidylinositol: 5%
  - Cholesterol: 10%
- Protein components
  - Transport proteins
    - Functions: Involved in neurotransmitter uptake
  - Trafficking proteins
    - Functions: Participate in vesicle exo- and endocytosis & recycling
    - Functional subtypes
      - Proton pump
      - Neurotransmitter transporters: Mediate transmitter uptake
    - Structural subtypes
      - Intrinsic membrane proteins
      - Proteins associated via posttranslational lipid modifications
      - Peripherally bound proteins
Vesicle pools in presynaptic terminal
- Readily releasible: 20% of population at NMJ
- Reserve: 80% at NMJ
- Resting: Not present at NMJ
Vesicle transport: Cytoskeletal-associated proteins probably involved in process
- Vesicle transport through cytoplasm: Cluster at active zones
- Actin: Transport requires intact matrix
- Myosin systems
  - Myosin V (unconventional) : Associated with vesicles via v-snare
  - Myosin light chain kinase & Myosin ATPase: ? Enhance neurotransmitter secretion
- Microtubule-related proteins
  - rab6 : GTPase implicated in inter-Golgi trafficking
  - Kinesin motor proteins: KIF1Bβ; KIF3A
Vesicle targeting
- Formation of bridge between vesicle & membrane
- Molecular mechanism: ? Exocyst-sec6/8 complex
  - Independent of cytoskeleton
  - Present mainly in developing neurons
Vesicle tethering
- Reversible attachment of vesicles to membrane
- Molecular mechanism: ? rab complex
  - Family of low molecular weight GTPases
  - ? Function in vesicle attachment to acceptor membranes
    - rab3 required for vesicle sequestration at presynaptic terminals
    - rab3A: Deficit leads to synaptic fatigue & reduced plasticity
  - Acts via multiple effectors: Uso1p/p115 ; Rabphilin ; Rim
  - Precedes formation of SNARE complex
Vesicle docking
- Irreversible attachment of vesicles to membrane
- Molecular mechanism: SNARE complex on both fusing membranes
- Induces partial, but not complete, membrane fusion
Vesicle priming
- Conversion into state of competence for Ca⁺⁺-triggered fusion-pore opening
- Involves ATP
Vesicle fusion
- Final events in membrane fusion
- Molecular mechanisms
  - Ca⁺⁺; Calmodulin
  - Others: GTP; Phosphatase (sensitive to Microcyteine LR)
Synaptic vesicle cycling
- Alternative pathways
  - "Kiss & Stay"
    - Vesicles reacidified & refilled with neurotransmitters without undocking
    - Vesicles remain in readily releasible pool
  - "Kiss & Run"
    - Vesicles undock & recycle locally
    - Reacidify & refill with neurotransmitters
  - Endocytosis via clathrin-coated pits
    - Reacidify and refill with neurotransmitters: Directly or After passing through an endosomal intermediate
- Endocytosis features: Time course
  - After single stimulus: Rapid; Mildly slowed with increased number or vesicles released
  - Repetitive stimulation: Prominent slowing of endocytosis rate
- Recycling features
  - Most vesicles recycle directly without passing through an endosomal intermediate
- Related molecules
  - Synaptojanin : Inositol 5-phosphatase
  - Amphiphysin
  - Dynamin
  - Clathrin
    - Components
      - Heavy chain ; Light chains A & B
      - Triskelion structure: 3 noncovalently bound heavy chains & 3 light chains
    - Component of cytoplasmic face of coated vesicles & coated pits: Organelles involved in
      - Intracellular trafficking of receptors
      - Endocytosis of a variety of macromolecules
  - Rab5 : Obligatory synaptic vesicle component
    - Possible function: Role in endosome fusion during the vesicle cycle
    - Mutations: Interfere with efficient release during repetitive stimulation
  - Vti1a (β splice variant)
    - Qb-SNARE protein
    - Functions: Membrane fusion
      - Endosome and Golgi fusion reactions: No interaction with synaptobrevin
    - Present on all synaptic vesicles
Other regulatory proteins
- Synapsins 1, 2 & 3
  - Protein characteristics
    - Actin binding
    - Ca⁺⁺-regulated ATPase
  - Location
    - Peripheral membrane protein
    - Coats synaptic vesicles
    - Synapsins 1 & 2 abundant at synapses
  - Functions
    - Links vesicles to cytoskeleton: Phosphorylation dependent
    - Reduced expression causes
      - Reduced Synaptic vesicles distal to active zones
      - Synaptic fatigue
    - ? Regulates reserve pool of vesicles needed for sustained synaptic transmission
  - Knockout mouse
    - Impaired synapse formation
    - Reduced number of synaptic vesicles: Vesicles present mainly at active zones
    - Reduced transmitter release
    - Increased susceptibility to seizures with electrical stimulation
  - Disorders
    - Synapsin 1: Epilepsy, X-linked, with variable learning disabilities and behavior disorders
    - Synapsin 2: Polymorphisms associated with schizophrenia
- Synaptotagmin
- CAPS (Ca⁺⁺-dependent activator protein in secretion)
  - Binds lipids: Phosphatidylinositol 4,5-bisphosphate (PI-4,5-P₂)
  - Supports catecholamine secretion
- Rab3 proteins
  - Most abundant rab proteins in synaptic vesicles
  - Rab3 is attached to synaptic vesicles
    - State: GTP-bound
    - Via covalently linked geranylgeranyl moieties
  - Rab3 undergoes cycle of synaptic vesicle association and dissociation
    - Occurs in parallel with synaptic vesicle exo- & endocytosis
    - Dissociation: During or after synaptic vesicle fusion
      - Depends on Ca⁺⁺-triggered exocytosis of synaptic vesicles
      - GTP on Rab3 is hydrolyzed to GDP
      - Resulting GDP-bound Rab3 is dissociated from synaptic vesicles by GDI
    - Reattachment of soluble GDI/GDP-Rab3 complex to vesicles
      - Involves GDP to GTP exchange
  - Rab 3A
    - Involved in late step in exocytosis that follows transport and docking
    - Knockout: 2 phenotypes depending on synapse
      - Short-term plasticity altered without change in readily releasable vesicle pool
      - Long-term potentialtion abolished without change in short-term plasticity
  - Rab3 effectors
    - General action: Bind to GTP-Rab3 but not to GDP-Rab3
    - Rabphilin
      - Soluble protein
      - Binding to synaptic vesicles
        
        Requires Rab3
        
        Cycles on & off vesicles
      - Binds Ca⁺⁺ via C2 domains
      - Knockout: No prominent phenotype
    - RIM1α /2α
      - Insoluble
      - Active-zone protein
      - C-terminal C2 domains: No Ca++-binding sites
      - Functions: Regulates neurotransmitter release via interactions
        
        N-terminal domain with Rab3 & Munc13-1
        
        PDZ domains with ERCs
        
        C-terminal C2 domain with α-liprins & synaptotagmin 1
- SV2 proteins
  - Functions: May regulate Ca⁺⁺ levels as transporter
  - Proteins: SV2A ; SV2B ; SV2C ; SVOP
  - SV2A knockout: Reduced SNARE complexes & readily released vesicles

Interactions between secretion-related proteins

Synaptobrevin (VAMP)-SNAP-25-Syntaxin complex: 1:1:1 stoichiometry
SNAREs play important roles in membrane fusion & possibly vesicle docking
Interaction between vesicle-associated snares (v-snare) & target membrane-associated snares (t-snare)
- Forms high affinity SNARE complex
- Proteins bind in parallel & form 4 helix bundle
- Complex increases α-helix nature of constituents
- Activated by α-SNAP/NSF
SNARE complex formation
- Draws vesicles toward target membrane
- Leads to vesicle fusion with cell membrane
Interactions occur
- In fast-releasing synapses
- At the active zone
- Near voltage-dependant Ca⁺⁺ channels: Especially N-type & P/Q-type

Active Zones

General
- Biochemically insoluble
- Location: Presynaptic plasma membrane precisely opposite synaptic cleft
- Number
  - Density of active zones on terminals: 2.5/μm
  - Each bouton: ~15-20 active zones
  - Human NMJ: 200 active zones; < 0.1% of all vesicles in terminal
- Ultrastructure
  - Central: Electron-dense containing hexagonal grid; Vesicles embedded in grid depressions
  - Neuromuscular: Elongated ridge containing vesicles lined up like beads on a string
Protein components
- Munc13s : Promotes priming of synaptic vesicles; Acts through syntaxin
- RIMS
  - Protein scaffolds: Bind directly or indirectly to many other synaptic proteins
  - Help regulate vesicle exocytosis during short-term plasticity
- Piccolo : Presynaptic cytoskeletal matrix; Role in cycling of synaptic vesicles
- Bassoon : Presynaptic cytoskeletal matrix
- ERCs (ELKS/Rab3-interacting molecule/CAST) : Bind to RIMS
- α-Liprins : Bind to RIMs, ERCs & receptor protein tyrosine phosphatases
- Voltage-gated calcium channels
Linking to vesicles
- Two interactions
  - GTP-dependent binding of Rab3 to RIM1α/2α
  - Ca⁺⁺-dependent association of synaptotagmin 1 with RIM1α/2α/2β
- 2 vesicles dock at each active zone

SNAREs (SNAP Receptors)

General
- Family: Membrane-associated proteins
  - Characterized by SNARE motif: Homologous 70-residue sequence
- Function: Mediate membrane fusion
  - SNAREs are components of fusion mechanism: Function with other presynaptic proteins
  - SNARE-related events occur via protein-protein interactions
  - Vesicle-associated SNARE (v-SNARE) interact with target membrane SNARE (t-SNARE)
  - Contact drives membrane fusion
- Post-fusion: Separation & Regeneration of SNARE components
  - Involves ATPase activity associated with NSF
  - α-SNAP: NSF attachment protein
- Regulators of SNARE pairing: Munc family of syntaxin-binding proteins
SNARE Protein Properties
- Size: 15 to 40 kD
- Structure
  - Integral membrane proteins
  - Anchored by carboxy-terminus
  - Cytoplasmic domains: Amino-terminus & central region
  - Coiled coil regions: Mediate trafficking
  - SNARE motif classes: 4 types (R, Qa, Qb, and Qc); Helical structure
- Transport reactions: Stoichiometric, not enzymatic
- SNARE complexes: Multivalent
  - Contents: Four SNARE motifs
    - Located in three or four separate SNARE proteins
  - Assembly: Into parallel four-helical bundle
Synaptic vesicle exocytosis: SNARE & associated proteins
- Vesicle location
  - Synaptobrevin (Vesicle-associated membrane protein; VAMP): Contains R-SNARE motif
- Presynaptic membrane location
  - Syntaxin 1: Contains Qa-SNARE motif
  - SNAP-25: Contains Qb- & Qc-SNARE motifs
- Synaptic vesicle SNARE complex: Molecular model
  - Pulls synaptic vesicle & plasma membranes close together,
  - Creates an unstable intermediate: Does not open fusion pore
    - Can progress to full-blown fusion pore
    - Can also regress to docked state of synaptic vesicles without engaged SNAREs
- Complexins: Associated with synaptic vesicle SNARE complexes
  - Bind to assembled synaptic core complexes
  - Insert into groove formed in C-terminal half of assembled SNARE complex
  - Promotes action of synaptotagmin 1 (Ca⁺⁺ sensor)
  - Not essential for SNARE function or synaptic vesicle fusion
  - Knockout mouse: Milder version of synaptotagmin 1 knockout
- Control of SNARE complex formation at synapses & fusion reactions
  - Munc18-1: Controls & promotes synaptic vesicle fusion
    - Molecular interactions
      - Binds to a closed conformation of syntaxin
      - Blocks syntaxin SNARE motif from participating in SNARE complexes
      - Munc18-1 must dissociate from syntaxin for SNARE complexes to form
      - May couple syntaxin to cytosolic factors to organize SNARE complex assembly
    - Deletion: Completely abolishes vesicle exocytosis
  - Proteins with C-terminal R-SNARE motif: Inhibit normal SNARE assembly & exocytosis
    - Tomosyn: Lethal-giant-larvae protein family
    - Amysin
  - Synaptophysin: Vesicle membrane protein
v-SNARE proteins: Vesicle-associated
- Synaptobrevin (Vesicle-associated membrane protein; VAMP) 1 ;
  - Binds to synaptophysin: Inhibits binding to other SNARE proteins
  - High levels in brain, kidney & exocrine pancreas
  - Loss of synaptobrevin reduces vesicle exocytosis
    - Cleavage: By botulinum toxins B, D, F & tetanus
    - Deletion: Causes 90% reduction in vesicle exocytosis
    - Ca⁺⁺-triggered exocytosis more inhibited than spontaneous exocytosis
- Synaptobrevin 2 : High levels in brain, kidney & Langerhans islets
- Synaptobrevin 3 (Cellubrevin) : Ubiquitous
- VAMP-2: v-SNARE in skeletal muscle & adipocytes
- GOS-28: Golgi transport (Intra & with cytoplasm)
- Cellubrevin: Membrane endocytosis/exocytosis
- rsec22b (Yeast homolog ): Endoplasmic reticulum
- Membrin (Yeast homolog ): ER & Golgi
t-SNAREs: Target-membrane associated
- Synaptosome-associated protein of 25 kDa (SNAP-25)
  - 2 Isoforms
    - SNAP-25A: In cell body & Along axon
    - SNAP25B: At nerve terminal
  - Inhibition causes reduced Ca⁺⁺-triggered vesicle exocytosis
    - SNAP-25 cleavage by botulinum toxin A & E
    - Deletion: Causes marked reduction in vesicle exocytosis
- Syntaxins
  - 1A & 1B : Neural axons & synapses
    - Syntaxin 1 cleaved by botulinum toxin C1
  - 2 : Epithelial morphogenesis; Apical membrane of pancreatic acinar cells
  - 3 : Zymogen granule plasma membranes; Family of 5 genes
  - 4 : Basolateral membrane of pancreatic acinar cells; GLUT-4 trafficking
  - 5 : cis side of Golgi; transport from ER to Golgi
- SNAP-23
  - Plasma membrane
  - 59% homology to SNAP-25
  - Insulin sensitive tissues

Other secretion related proteins

NSF (N-ethylmaleimide-sensitive factor)
Syntaxin 6 : Trans-Golgi network
rsec22a
n-sec1
Syntaxin binding protein 1 : Retina & cerebellum; Role in transmitter release
Synaptic vesicle protein 2 (SV2) : Essential role in control of exocytosis

Vesicle membrane proteins

Synaptophysin
- Integral membrane protein
- Binds to Synaptobrevin: Forms complexes on surface of synaptic vesicles
- May inhibit binding of synaptobrevin to SNAREs: Inhibits vesicle fusion
Vesicle amine transporter 1 (VAT-1; SLC18A1)
Amphiphysin :
- Cytoplasmic surface of synaptic vesciles
- Target of serum antibodies in Stiffman syndrome with breast cancer

Ca⁺⁺ & ATP regulation

Synaptotagmin I
- Location: Synaptic vesicles; Chromaffin granules
- Functions
  - Ca⁺⁺ sensor
  - Regulates fast exocytosis at synapse
  - No clear role in slow exocytosis or membrane fusion in general
- Integral membrane protein
- 2 Cytoplasmic domains: Bind total of 5 Ca⁺⁺ ions
  - C₂A domain binds: 3 Ca⁺⁺ ions; Liposomes containing acidic phospholipids
  - C₂B domain binds: 2 Ca⁺⁺ ions
  - Affinity of C₂++ increases when domains bind to phospholipid membranes
  - Phospholipid binding is Ca⁺⁺ dependent
- Synaptotagmin also binds to SNAREs
- Binding of signaling molecules leads to change in structure
- In family of 9 genes: Most localized to neurons
- Mutants
  - Fewer synaptic vesicles near plasma membrane
  - Less ability of Ca⁺⁺ to trigger fast exocytosis
- Knockout mouse: Lethal; Selective loss of fast Ca⁺⁺-triggered exocytosis
Synaptotagmin 2
Ca⁺⁺ channels
Phosphatidylinositol transfer protein
Phosphatidylinositol-4-phosphate 5-kinase
Rabphilin : Interacts with α-actinin & β-adducin
Scinderin: Ca⁺⁺ regulated actin-severing protein
CAPS (Ca⁺⁺-dependent activator protein in secretion)
Actin cytoskeleton

Return to Neuromuscular home page
Return to Botulinum toxin

References
1. Current Opinion in Neurobiology 1997;7:310-315 & 316-322, Annu Rev Neurosci 2004;27:509–547
2. Current Opinion in Cell Biology 1997;9:505-512
3. J. Neurochem 1997;69:1781-1792
4. Curr Opin Neurobiol 1999;9:321-328

10/5/2005

PRESYNAPTIC PROTEINS, SYNAPTIC VESICLE DOCKING & MEMBRANE FUSION 1

SNAREs (SNAP Receptors)

Vesicle membrane proteins

Ca++ & ATP regulation

PRESYNAPTIC PROTEINS, SYNAPTIC VESICLE DOCKING & MEMBRANE FUSION ¹

Ca⁺⁺ & ATP regulation