Neurotransmitter release

Neurotransmitter release

1. Neuronal chemical axo-dendritic communcaiton

   • NT release is a highly specialised form of membrane fusion
   • Very high spatial and temporal resolution
   • Involves the release of small packets or quanta of transmitter
   • Triggered by calcium-mediated fusion of vesicles
   • Very rapid transduction of an electrical stimulus into vesicular release - less than 1ms

2. Vesicular transmitter release

   • Small synaptic vesicles (SSVs)
     • Small transmitters
     • Diameter about 50nm
   • Large dense-core Vesicles (LDCVs)
     • Peptides
     • Diameter about 250nm

3. Structure and location of synaptic vesicles

   • Large concentration of protein on post synaptic side (about 1000 different proteins involved)
   • Reserve pool of vesicles pre-synaptically are bound to actin - released from actin upon stimulation

4. Action potential

   • 1. Action potential propagation in presynaptic neuron
   • 2. Ca2+ entry into synaptic knob
   • 3. Release of neurotransmitter by exocytosis
     • 1. Docking - membrane of vesicles tightly associates with plasma membrane (otherwise vesicles are in a reserve pool)
     • 2. Priming - creating of a competent readily releasable pool of vesicles
     • 3. Fusion - active fusion of the vesicle membrane with the plasma membrane to release vesicular contents when the local calcium reaches a threshold level
     • 4. Recycling - of vesicular membrane by endocytosis to form a new vesicle of transmitter by filling
   • 4. Binding of neurotransmitter to postsynaptic receptor
   • 5. Opening of specific ion channels in subsynaptic membrane

5. Vesicle fusion regulation

   • Vesicular proteins
     • Synaptobrevin
       • Important for controlling fusion
     • Synaptotagmin
       • Calcium trigger
       • Drives membrane fusion
     • Synapsin
       • Important in holding vesicles in the reserve pool
     • Rab's
       • Important in fusion
   • Phosphorylation of synapsin 1 releases the vesicle form the reserve pool
     • CamKII is activated and phosphorylates synapsin 1 causing it to release from actin
   • Rab 3 involved in docking and fusion
     • GTP-bound Rab3 binds Rabphilin and the plasma membrane protein RIM
     • GDP-vound Rab3 dissociates from vesicles
     • C2 domain proteins are domains involved in calcium and lipid binding (common in proteins associated with the lipid membrane
   • Fusion requires N-ethlmaleimide (NEM) sensitive factors (NSF)
     • Cell-free fusion assays of golgi derived vesicles - NEM sensitive protein fraction
     • 76kDa homotrimer
     • ATPase activity
     • Required together with soluble NSF attachment proteins (SNAPs) for ER-golgi and intra-golgi membrane transport

6. Synaptic SNAREs

   • Soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor
   • All membrane fusion secretory events involve the formation of a helical coiled coil complex assembled from membrane anchored proteins known as SNAREs
   • SNARE proteins responsible for synaptic vesicle fusion are:
     • Syntaxin 1
       • Presynaptic plasma membrane (integral) tSNARE
     • SNAP 25
       • Presynaptic plasma membrane (integral) - tSNARE
     • Synaptobrevin 2
       • Vesicular membrane (integral) - vSNARE
     • Synaptic SNAREs form a complex of four parallel helices: Syntaxin and Synaptobrevin contribute one motif each and SNAP 25 two mofits
   • SNAREs squeeze target and vesicular membrane
   • Munc-13 and Munc-18 enable formation of the SNARE complex
     • Muc-18
       • Acts as a clasp
       • Binds Syntaxin 1
       • Designed to bind 4 helix bundles
   • SNARE complexes are 'clamped' by complexins
     • Complexins bind SNARES with 1:1 stochiometry
     • Complexins arrest fusion in an hemifusion state - inner but not outer leaflets fused
   • Synaptotagmin 1
     • Can displace complexin from SNAREs in the presence of Ca2+
     • Trigger for NT release
     • Integral protein on vesicles with two C-terminal C2 domains (C2A/B)
     • Calcium and phospholipid binding domains
     • Acts to put a Ca2+ sensitive inhibitory constrain on exocytosis
     • Acts upon the complexin-bound SNARE complex in response to Ca2+ and thus triggers fast diffusion