Lecture 15- Synaptic release and presynaptic proteins Flashcards
1
Q
What are the crucial things needed for transmission?
A
-an active zone, synaptic cleft (wider than normal intracellular space) then postsynaptic density (with receptors)
2
Q
What is an active zone?
A
- group of vesicles some of which tethered to the membrane -on the presynaptic membrane
- have about 300 active zones per synapse in a neuromuscular junction (NMJ)
- upon arrival of action potential, activation of voltage activate calcium channels and so get calcium increase and trigger release of vesicles from the active zone, generally one vesicle per active zone (in a frog)
- then depolarisation in the muscle
3
Q
How many active zones are there in mammalian NMJs?
A
- 10-100
- the number depends on the size of the muscle
4
Q
How many active zones are there in most hippocampal and neocortical synapses?
A
-mostly consist of a single active zone
5
Q
What is the synaptic unit and what does it consist of?
A
-The synaptic “unit” is the active zone An active zone consists of:
- a pre-synaptic region containing an accumulation of vesicles
- a widened, electron-dense intercellular space (synaptic cleft), typically 20 – 50 nm
- a post-synaptic density, rich in protein. Type I (excitatory) synapses have a thicker PSD than type II (inhibitory)
-the size of synaptic cleft is small enough to not need a transport system, will reach the other end within a milisecond just by diffusion
6
Q
How many vesicles are released?
A
- Each active zone releases one or no vesicles per action potential
- The release of a vesicle at an active zone is a probabilistic event
- In the hippocampus, different circuits have probabilities between 10% and 90%
- At the frog NMJ, up to 300 vesicles are released
- number of vesicles depends on no of active zones, one vesicle per active zone
- and often will get zero release, it is not 100% efficient
- probability of vesicle release
7
Q
How many molecules are in a vesicle?
A
-Take the example of acetylcholine vesicles at the NMJ:
- [ACh] inside vesicle is 100 mM
- Vesicle diameter is 50 nM, predicting about 10,000 ACh molecules per vesicle
- The number of ACh (and glutamate) molecules is probably half that, because the vesicle has a diminished effective volume
- number of molecules in a vesicle is around 5000, should be a bit higher given the volume (10 000 is predicted, 5000 is real)
- this is probably becaus ethe vesicle contains other things and the available volume is smaller
8
Q
Can CNS synapses have multiple active zones?
A
-yes
• Best known example is the mossy fibre synapse in the hippocampus: 10 – 40 active zones
- mossy fibre= as each synapse looks like mossy random growth, the terminal wraps around the dendritic spine (containing the postsynaptic densities)
- similar to a NMJ in that you get 1 to 1 relationship in AP to release (almost 100% probability)
9
Q
What are some characteristics of the mossy fibre synapse?
A
- the probability due to the location (very close to the cell body) so larger concentration of the signal gontain up to 40 active zones
- some 20/some 35 etc.
- under some circumstances will release only 2 vesicles, sometimes all 40, regulated!
- each active zone when activated
10
Q
Why is the mossy fiber synapse “stronger”?
A
- more current flows into the dendrite
- the synapses are located close to the cell body
- Both factors contribute to greater depolarisation of the neuron
11
Q
How many vesicles are there typically around a synapse and how many are actually available for release?
A
- typically will have a few hundred vesicles but only about 10 that are capable of releasing their contents, these are the ones that are tethered to the presynaptic terminal and ready to be released and those are called the readily releasable pool
- behind them about 20 fully competent vesicles= the reserve pool
- and the rest in the background (200) are tethered to the actin filament, the terminal has a specialised actin network, none of the vesicles are just floating ones, everything is tethered
- when release happens, one of the tehthered sites will become empty and one of the reserve pool vesicles will come in there
12
Q
What determines the probability of vesicle release?
A
- 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)
- number of vesicles: seems to not be biologically important the variability
- the sensitivity of the trigger
- contains 5 calcium sites, if all occupied then more likely release, if less occupied then less likely to be released, -snare complex= site of release
- when calcium comes in doesn’t have to fill out the terminal, calcium comes in through the voltage calcium channel, and needs to bind to synaptotagmin (that is located very close so one of the first things it sees)
13
Q
Do biological membranes fuse spontaneously?
A
-Biological membranes do not fuse spontaneously
- Electrostaitc repulsion
- Steric hindrance from proteins many biological membranes are studded with proteins
14
Q
Why can’t membranes fuse spontaneously?
A
- this is what it looks like, overall tend to be negatively charged
- they spontaneously repel each other electrostatically, the membrane is behind the proteins (so really initially a protein and protein interaction at first)
15
Q
What processes are needed for the fusion of vesicles with vesicles and with other membranes?
A
- It requires an elaborate protein machinery
- It is tightly regulated
- It is highly specific, not random
- 20-30 proteins specific for fusion
- snare complex= the basis of all vesicle organelle fusions in a cell
16
Q
What is required for the membrane fusion event?
A
- formation of a SNARE complex
- An SM protein eg Munc 18
- The SNARE complex draws the two membranous structures together
- The SM protein initiates phospholipid mixing
- Note that calcium is NOT required
18
Q
What does the SNARE complex do as the superhelix is formed?
A
- as the superhelix is formed it steers the vesicle as close to the membrane as possible
- the quaternary SNARE complex is very stable
19
Q
What is this?
A
- Molecular mechanisms of exocytosis during neurotransmitter release
- the helices tighten up drawing the structures together, the extra protein: synaptotagmin
23
Q
Which vesicle recycling route occurs more often and how does it work?
A
- the fast route is the one used most often
- mostly what happens after fusion is that endocytosis occurs, the vesicle internalizes itself, pinches off from the presynaptic terminal membrane and remains in situ, very close to the membrane where the SNAREs are and may in fact form a SNARE complex again. or go to a reserve pool or go intot he fully competent pool that is right behind the docked vesicles
24
Q
What is the slow route of vesicle recycling?
A
- about 10% of vesicles undergo the slow route
- the vesicles are transported to the back of the presynaptic terminal where they merge with an early endosome, they diffuse via SNARE complex, undergo protein re-equipment (like going in for a service)
- the slow route is available in order to refurbish the vesicles with new protein
25
Q
What is a fusion pore?
A
- there is an intermediate phase in fusion: when there is a fusion pore
1. vesicle is about to form a SNARE complex, then forms it when calcium enters (the voltage gated calcium channels are located adjacent to the SNARE proteins so when AP arises the calcium that goes in it almost immediately encounter the synpatotagmin. so it can bind) so this initiates the process of fusion and the process of fusion begins with the formation of a fusion pore
2. fusion pore: actually a protein-lined pore (not a lipid lined one) and the proteins making up the inner lining of this pore are actually the SNARE proteins, this then proceeds to full fusion, flattening the vesicles and emptying of the contents, the emtying starts as soon as the fusion pore forms -sometimes the process is arrested at the stage of fusion pore, and full fusion doesn’t happen (in some dopamine tranfer) so only small amount of neurotransmitter is leaked, and tehn go to endocytosis, it occurs, unclear how often and how important so means that not entirely quantal (can have in between
26
Q
What is the reason for SNARE complexes in neurons being unable to proceed to full fusion without the presence of calcium?
A
- synaptotagmin acts as a clamp
- the reason that SNARE complexes in neurons don’t proceed to full fusion is due to SNARE being clamped by synaptotagmin -once calcium binds to the calcium site son synaptotagmin it moves away and
27
Q
What is the function of synaptotagmin?
A
- Synaptotagmin is the calcium sensor
- Each molecule in contains 5 calcium binding sites. 
- Calcium binding increases the probability of release. If sufficient calcium binds, synaptotagmin then dissociates from the synaptic membrane
- Calcium channels are located very close to docking sites
29
Q
Does synaptotagmin remain attached to the vesicular membrane after calcium binding occurs?
A
- Synaptotagmin remains attached to the vesicular membrane
- it remains attached to the vesicular membrane, when fusion happens then synaptotagmin also inserts into the presynaptic membrane
- synaptotagmin= is a vesicular protein, 5 calcium binding sites, inserts one end into terminal end when fusion and get out of the SNARE
- Release occurs. SNARE complex is still intact
30
Q
What are some other proteins involved in the vesicle fusion and what are their functions?
A
- SNARE is the bases and the SM too but there are lot of other proteins that are needed for this to go ahead. one of the central ones is RIM= it binds to all the major players, holds them all together at the Snare complex prior to the fusion, (orange here)
- binds to the vesicle via RAB3 /Vesciualr protein), binds to the clacium voltage channels (ensure the extreemely close proximity of vesicle and channel) also binds to the SM protein, binds to the protein Munc 13
- protein RIM holds it together, RAB3, SM protein (Munc18) and a protein called Munc 13, also synaptotagmin (complexed to complexin)
- all in close proximit
y -RIM binds to vesicle, VACC and Munc 13
31
Q
What is this?
A
- molecular mechanisms of exocytosis during vesicle fusion
- the SNARE complex once it forms it tightens up and pulls like a rope the whole thing together as close as can be. all that is needed is complexin and synaptotagmin to get out of the way and for that need calcium
1. vesicle docks
2. SNARE complexes form to pull membranes together
3. Entering Ca2+ binds to synaptotagmin
4. Ca2+-bound synaptotagmin catalyzes membrane fusion by binding to SNAREs and the plasma membrane
33
Q
What are the molecular mechanisms of endocytosis following neurotransmitter release?
A
- the process of internalization works by clathrin, the clathrin recognised the vesicular membrane and makes a network and pull the membrane inwardly in a progressive fashion until you have almost fusion type neck
- clathrin coats the vesicular membrane after it’s fused and contractes curves inwardly and another protein called dynamin (uses GTP) then forms a noose and pulls it to pinch the membrane separating it
- it is an energy dependent process as the clathrin curvature requires ATP and dynamin noose pulling requires GTP
1. Adaptor proteins connect clathrin to vesicular membrane
2. Clathrin triskelia assemble into coat, curving membrane
3. Dynamin ring forms and pinches off membrane
4. Coated vesicle translocated by actin filaments
5. Hsc-70 and auxilin uncoat vesicle
35
Q
What does dynamin do?
A
- Dynamin forms a noose that pinches off the vesicle, completing the process of endocytosis
- wraps itself around the neck of the vesicle and pinches it off
41
Q
What are the differences in small molecule neurotransmitter and peptide neurotransmitter transfer?
A
- enzymes in the cell body, transported to terminus via slow anterograde axonal transport (about 1mm a day)
- with peptides they are synthesized as protein precursors and transported along microtubules via fast transport (1mm an hour) and then stored at the terminal until release (bigger vesicles as more protein)
42
Q
How does the release of neurotransmitters differ depending on what type of molecule they are?
A
one terminal may contain two types of neurotransmitters -glutamergic terminal may have lot of glutamate and then some peptides (not as clsoely associated with the membrane)
- can get co release from the same terminal
- the small molecule (glutamate) released quickly (it is primed, it has the RIM protein, only need the calcium and release!
- with the peptide vesicles, need large calcium concentration in the whole terminal, so requires prolong stimulation to achieve that concentration throughout the whole terminal and then these will be released, so less frequently released than the small molecule one, less to high frequnecy and short
- often the peptide will modulate the sensitivity of the postsynaptic cell to the glutamate, in a positive or negative way
