L13 & 14 - Presynaptic Processes

Question 1

Synapses consist of Active Zones. What are active zones?

Answer 1

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.

Question 2

Type 1 vs Type 2 synapses – which is excit/inhib, which is thicker

Answer 2

Type 1 (excitatory) synapses have a thicker PSD than type 2 (inhibitory)

Question 3

Frog active zones vs mammalian ones

Answer 3

Frogs - 300 active zones
Mammals – 10-100 active zones
-Many hippocampal and neocortical synapses consist of a single active zone

Question 4

How many vesicles are released at an active zone?

Answer 4

One or none

Question 5

How many molecules in a vesicle?

Answer 5

3000 – 5000

Enough in one vesicle to saturate a receptor hence only one vesicle is released.

Question 6

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?

Answer 6

Mossy fibre synapse aka detonator synapses – 10 – 40 active zones
It interacts with the dendrite of CA3 pyramidal neuron

Question 7

Why is the mossy fibre synapse “stronger”?

Answer 7

-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

Question 8

Vesicle pools

Answer 8

• 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

Question 9

What are the recycling and reserve vesicles tethered to?

Answer 9

Linked by synapsin to Actin filaments

Question 10

What determines the probability of vesicle release?

Answer 10

• 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)

Question 11

Why don’t biological membranes fuse spontaneously?

Answer 11

Electrostatic repulsion and steric hindrance from proteins

Question 12

Is the fusion of vesicles with vesicles or with other membranes is a spontaneous process?

Answer 12

No – it is highly specific, tightly regulated and requires an elaborate protein machinery.

Question 13

What surface proteins can be found on vesicles?

Answer 13

• 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.

Question 14

All membrane fusion events require? Is Calcium required?

Answer 14

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

Question 15

Vesicle docking requires the fusion of 4 SNARE protein alpha helices – what are they?

Answer 15

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

Question 16

What do the fusion of the outer and inner leaflets form?

Answer 16

• 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

Question 17

How do Munc13 and 18 promote membrane bending?

Answer 17

• 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)

Question 18

What does RIM protein bind to?

Answer 18

RIM binds to Rab3, VACC, Munc 13

Question 19

Why can’t vesicles fuses with other vesicles?

Answer 19

Synaptobrevin of one vesicle cannot form SNARE complexes with synaptobrevin of another vesicle

Question 20

Calcium is not required for most non-synaptic SNARE-mediated fusion - what are the exceptions? Why does synaptic vesicle fusion require Ca2+

Answer 20

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

Question 21

What is needed for stable docking/tethering?

Answer 21

3 SNARE complexes present (each with 4 alpha helices)

Question 22

OLD MODEL: How many Ca2+ binding sites per synaptotagmin molecule? What happens when Ca2+ binds?

Answer 22

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.

Question 23

Model of Complexin function

Answer 23

• 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

Question 24

The 7 Sequence of Events

Answer 24

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

Question 25

Is vesicular release quantal?

Answer 25

Yes, only get 1, 2 or 3 release – not 1.5

-BUT Sometimes process stops at fusion pore and there’s partial emptying - only a little bit trickles out

Question 26

Diseases that affect the presynaptic terminal

Answer 26

(B) Cleavage of SNARE proteins by clostridiaol toxins,

• Tetanus toxin – protease cleaving synaptobrevin & hence you can’t release NT
• Botulinum toxins – can cleave SNARE proteins e.g. BoTX-A (cleaves syntaxin) BoTX-B (cleaves synaptobrevin)

Question 27

Molecular mechanisms of endocytosis following NT release

Answer 27

1) ATP-driven process where Clathrin forms a coat, curving the membrane and pulling it out of the membrane and into the cytosol
2) Towards the end, it is a GTP-driven process where dynamin ring forms a lasso around the vesicle and pinches it off the membrane, completing the process of endocytosis
3) Coated vesicle translocated by actin filaments
4) Hsc-70 and auxilin uncoat vesicle

Question 28

When internalization occurs, besides for vesicular retrieval, what else is internalized?

Answer 28

ECF to send to nucleus

Question 29

Under high frequency stimulation, what is a possible problem that could arise in the internalization of vesicles?

Answer 29

Vesicle internalized before vesicle is broken down – SNARE complex not disassembled -> Faulty vesicle put back into the RRP (readily releasable pool) -> not reacidified or been filled with NT -> synaptic failure

Question 30

How many SNARE complexes are need for vesicular fusion?

Answer 30

3

Question 31

How many synaptobrevin molecules per vesicle? What is the reason for this?

Answer 31

70. Allows it to dock in any orientation.

Question 32

3 Types of Vesicle recycling

Answer 32

1) The normal cycle; SNARE complex is disassemble. Vesicle to Recycling Pool
2) The rapid cycle: SNARE complex remains intact, & vesicle remains docked (in RRP)
3) The slow cycle: SNARE disassembly occurs, vesicle joins endosome compartment, then enters the Reserve pool
* Not known how vesicle is chosen for recycling, could be random

Question 33

Where are the non-peptide transmitters synthesised? What about their enzymes?

Answer 33

Nerve terminals - enzymes required for their synthesis are produced in the cell body and transported along the axon

Question 34

Peptide NT – Differences with small amino acid NT

Answer 34

Small molecule neurotransmitters (that is, non – Peptide neurotransmitters) are synthesized at the nerve terminals.
The enzymes required for their synthesis are produced in the cell body and transported along the axon to the terminal.

Peptide neurotransmitters, however, are often formed first by the formation of neurotransmitter precursor proteins (formed through the usual processes of transcription and translation of proteins).