L13 & 14 - Presynaptic Processes Flashcards
Synapses consist of Active Zones. What are active zones?
An active zone consists of:
- a pre-synaptic region aka bouton containing an accumulation of vesicles (a few hundred) but only about 10 that are primed/tethered – ready for release
- a widened, electron-dense intercellular space (synaptic cleft), typically 20-50 nm
- a post-synaptic density, rich in protein.
Type 1 vs Type 2 synapses – which is excit/inhib, which is thicker
Type 1 (excitatory) synapses have a thicker PSD than type 2 (inhibitory)
Frog active zones vs mammalian ones
Frogs - 300 active zones
Mammals – 10-100 active zones
-Many hippocampal and neocortical synapses consist of a single active zone
How many vesicles are released at an active zone?
One or none
How many molecules in a vesicle?
3000 – 5000
Enough in one vesicle to saturate a receptor hence only one vesicle is released.
What synapse in the CNS – Hippocampus is similar to NMJ (single nerve impulse is able to get to threshold on the post synaptic neuron) and what does it interact with?
Mossy fibre synapse aka detonator synapses – 10 – 40 active zones
It interacts with the dendrite of CA3 pyramidal neuron
Why is the mossy fibre synapse “stronger”?
-More current flows into the dendrite
-The synapses are located close to cell body (less current leakage)
Both lead to greater depolarisation of the neuron
Vesicle pools
- Readily releasable pool (RRP) (10): pre-docked, closely located to Ca2+ voltage gated channel, tethering sites is attached to Ca2+ voltage gated channel
- Recycling pool (20-50): occupies docking site after RRP releases one vesicle
- Reserve Pool (200) – needs to undergo protein modification before it joins the recycling pool
- Recycling and reserve pool are actually intermingled but distinguishable by different proteins on them
What are the recycling and reserve vesicles tethered to?
Linked by synapsin to Actin filaments
What determines the probability of vesicle release?
- The number of pre-docked vesicles
- The sensitivity of the release trigger (Synaptotagmin) to calcium.
- The amount of calcium entry
- The site of calcium entry (VACC are located very close to docked vesicles)
Why don’t biological membranes fuse spontaneously?
Electrostatic repulsion and steric hindrance from proteins
Is the fusion of vesicles with vesicles or with other membranes is a spontaneous process?
No – it is highly specific, tightly regulated and requires an elaborate protein machinery.
What surface proteins can be found on vesicles?
• Synaptotagmin is the calcium trigger
• Synapsin tethers “resting” vesicles to actin
-Phosphorylation of synapsin allows forward movement of vesicle
• Rab 3 targets vesicles to the docking site (by binding to RIM protein, which is bound to
VACCs)
• Rab 5 is important for re-uptake after exocytosis
• VAMP (Synaptobrevin) is a vesicular SNARE protein. Formation of the docking complex requires a SNARE complex.
All membrane fusion events require? Is Calcium required?
1) Formation of a SNARE complex
2) An SM protein e.g. Munc 18
The SNARE complex pulls the 2 membranes together
The SM protein initiates phospholipid mixing – by bending the opposite membranes towards each other
*Calcium is not required for most non-synaptic SNARE-mediated fusion
Vesicle docking requires the fusion of 4 SNARE protein alpha helices – what are they?
One supplied by the vesicle (Synaptobrevin/VAMP)
• Three supplied by the plasma membrane at the synaptic terminal
-Two helices per SNAP-25 protein
-One helix per syntaxin protein
What do the fusion of the outer and inner leaflets form?
- Bending of the membrane is essential for fusion (mere pulling is not enough)
- Fusion of the outer (proximal) leaflets produces a stalk
- Fusion of the inner leaflets produces an fusion pore
How do Munc13 and 18 promote membrane bending?
- Munc13 and Munc18 promote membrane bending when they form a complex with Syntaxin, this results in steric hindrance (Munc proteins keep them apart – resulting in bending)
What does RIM protein bind to?
RIM binds to Rab3, VACC, Munc 13
Why can’t vesicles fuses with other vesicles?
Synaptobrevin of one vesicle cannot form SNARE complexes with synaptobrevin of another vesicle
Calcium is not required for most non-synaptic SNARE-mediated fusion - what are the exceptions? Why does synaptic vesicle fusion require Ca2+
Exception for non-synaptic SNARE-mediated fusion: secretion of some peptide hormones (e.g. Insulin, glucagon) are Ca2+ regulated -necessary for fusion of vesicles to occur
Synaptotagmin-1 and Complexin is why its Ca2+ dependent, they act together to provide:
1) A clamp 2) A spanner in the works of the SNARE machinery 3) Calcium sensor
* Ca2+ is required to bind to and change conformation of Synaptotagmin-1 which then extracts Complexin (which is inserted into SNARE complex) and allows for vesicular fusion to occur
What is needed for stable docking/tethering?
3 SNARE complexes present (each with 4 alpha helices)
OLD MODEL: How many Ca2+ binding sites per synaptotagmin molecule? What happens when Ca2+ binds?
5
Ca2+ binding increases the probability of release. If sufficient Ca2+ binds, synaptotagmin then dissociates from the synaptic membrane - releasing the clamp and allows vesicle to get close enough to fuse. Synaptotagmin remains attached to the vesicular membrane. Release occurs but SNARE complex is still intact. It is unwound via NSF & SNAP proteins.
Model of Complexin function
- Inserts itself ONLY into fully formed SNARE complex
- Has loose association with synaptotagmin 1 (doesn’t bind to cell membrane as old model suggested)
- Ca2+ enters -> binds to synaptotagmin, changes conformation -> binds tightly to complexin and extracts it from SNARE complex -> So vesicular fusion can occur
The 7 Sequence of Events
1) Docking - via SNARE complexes, Rab3, RIM protein
2) Priming - contraction, SNARE complex tightens
3) Fusion
4) SNARE complex dissociation (by NSF & SNAP proteins)
5) Endocytosis
6) Reacidification (pumping protons into vesicle, this drives NT uptake)
7) NT filling
