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Presynaptic Proteins, Synaptic Vesicle Docking & Membrane Fusion ¹

Active zones Vesicle trafficking & release   Steps   Botulinum toxin   Transmitter release path Presynaptic proteins   Ca++ & ATP regulation   Interactions   SNAREs   Vesicle membrane proteins   Secretion-related proteins     Other

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

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SNAREs (SNAP Receptors) ⁵

• 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)
      • Driven by: Coupled folding & assembly of 3 synaptic SNARE proteins
        • Target plasma membrane (t-SNAREs): Syntaxin-1 & SNAP-25
        • Vesicular membrane (v-SNARE): VAMP2
        • Forms a four-helix bundle
    • 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
    • Stable SNARE complexes composed of four helical bundle with one motif from each class
    • Transmembrane regions of SNAREs emerge on C-terminus
    • Specificity of SNARE complexes
      • Interaction of SNARE motifs is generally promiscuous
      • High specificity probably conferred by non-SNARE motifs
• Synaptic vesicle exocytosis: SNARE & associated proteins
  • Locations
    • Vesicles
      • Synaptobrevins (Vesicle-associated membrane proteins; VAMP): Contain R-SNARE motif
    • Presynaptic membrane
      • Syntaxin 1: Contains Qa-SNARE motif
      • SNAP-25: Contains Qb- & Qc-SNARE motifs
  • Exocytosis: Core trans-SNARE complex formed
    • Components
      • Vesicle-associated v-SNARE (synaptobrevins)
      • Target cell-associated t-SNAREs (syntaxin & SNAP-25)
  • 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 1 (Vesicle-associated membrane protein 1; VAMP1)
    • 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
    • Diseases
      • Congenital Myasthenic Syndrome 25
      • Spastic Ataxia 1, Hereditary, Dominant
  • VAMP-2 (Synaptobrevin 2)
    • High levels in brain, kidney & Langerhans islets
    • v-SNARE in skeletal muscle & adipocytes
    • Disease: NEDHAHM
  • VAMP 3 (Synaptobrevin 3; Cellubrevin)
    • Ubiquitous
    • Membrane endocytosis/exocytosis
  • VAMP7
  • GOS-28: Golgi transport (Intra & with cytoplasm)
  • 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
    • Disease: Myasthenia, Cortical hyperexcitability, Ataxia & Intellectual disability
  • Syntaxins
    • General: Cell receptors for transport vesicles
    • 1A & 1B : Neural axons & synapses
      • Syntaxin 1 cleaved by botulinum toxin C1
      • Syntaxin 1B
        • Ca⁺⁺-dependent synaptic transmission in brain
        • Binds to: UNC13A
      • Bind to molecular complex including Neurexin 1 (α-latrotoxin receptor)
    • 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
    • Also
      • Syntaxin binding protein 5-like (STXBP5L; LLGL4)
  • SNAP-23
    • Plasma membrane
    • 59% homology to SNAP-25
    • Insulin sensitive tissues
  • GOSR2
  • BET1

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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
• Syntaxin binding protein 5-like (STXBP5L; LLGL4)

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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

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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

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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
5. Front Physiol 2017;8:5

2/12/2020