Lecture 15- Synaptic release and presynaptic proteins

Question 1

What are the crucial things needed for transmission?

Answer 1

-an active zone, synaptic cleft (wider than normal intracellular space) then postsynaptic density (with receptors)

Question 2

What is an active zone?

Answer 2

• 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

Question 3

How many active zones are there in mammalian NMJs?

Answer 3

• 10-100
• the number depends on the size of the muscle

Question 4

How many active zones are there in most hippocampal and neocortical synapses?

Answer 4

-mostly consist of a single active zone

Question 5

What is the synaptic unit and what does it consist of?

Answer 5

-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

Question 6

How many vesicles are released?

Answer 6

• 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

Question 7

How many molecules are in a vesicle?

Answer 7

-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

Question 8

Can CNS synapses have multiple active zones?

Answer 8

-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)

Question 9

What are some characteristics of the mossy fibre synapse?

Answer 9

• 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

Question 10

Why is the mossy fiber synapse “stronger”?

Answer 10

• more current flows into the dendrite
• the synapses are located close to the cell body
• Both factors contribute to greater depolarisation of the neuron

Question 11

How many vesicles are there typically around a synapse and how many are actually available for release?

Answer 11

• 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

Question 12

What determines the probability of vesicle release?

Answer 12

• 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)

Question 13

Do biological membranes fuse spontaneously?

Answer 13

-Biological membranes do not fuse spontaneously

• Electrostaitc repulsion
• Steric hindrance from proteins many biological membranes are studded with proteins

Question 14

Why can’t membranes fuse spontaneously?

Answer 14

• 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)

Question 15

What processes are needed for the fusion of vesicles with vesicles and with other membranes?

Answer 15

• 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

Question 16

What is required for the membrane fusion event?

Answer 16

• 1. formation of a SNARE complex
• 2. 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

Question 17

What is a SNARE complex and how it it formed?

Answer 17

• SNARE- gives specificity, depending on the proteins can bind or not
• vesicles have one type of snare, and this one has intracellular part
• made up of superhelix of four alpha helical proteins, determine what it can bind to, vesicle has one snare protein protruding out (synapto brevin)
• membrane of presynaptic terminus intracellularly has two proteins, one has two alpha helices (SNAP25) the other one is called: syntaxin
• so snare complex between vesicle and presynaptic membrane, (when tethered= form SNARE complexes) so complex between presynaptic terminal and vesicle requires synpatobrevin from the vesicle and SNAP25 and syntaxin from the plasma membrane, these are all SNARE protein
• as soon as get SNARE complex it quickly proceeds to fusion, only in case of synaptic release need calcium for release
• in here need an SM protein (eg. Munc18= in neurotransmission),
• snare complex forms, spontaneously forms a superhelix that draws the vesicle almost into the presynaptic membrane (normally this would be enough for fusion but not here, need calcium and SM)

Question 18

What does the SNARE complex do as the superhelix is formed?

Answer 18

• as the superhelix is formed it steers the vesicle as close to the membrane as possible
• the quaternary SNARE complex is very stable

Question 19

What is this?

Answer 19

• Molecular mechanisms of exocytosis during neurotransmitter release
• the helices tighten up drawing the structures together, the extra protein: synaptotagmin

Question 20

What is the sequence of events with synaptic release?

Answer 20

1. Docking: forming of SNARE complex, the formation of the 10 active zones

2. Priming:calcium related

3. Fusion: leaking of the contents and activation of the receptors

4. SNARE complex dissociation (by NSF): dissociation (by proteolyses)

5. Endocytosis: the vesicle that is part of the presynaptic terminal is withdrawn, endocytosed

6. Reacidification:each vesicle has 2 ATP dependent hydrogen pumps, which pump hydrogen pumps that pump the hydrogen into the vesicle (each pump needs one ATP) related to NA+/K+ ATPase, it only pumps hydrogen one way here! only two pumps per vesicle, if you work out the volume of the vesicle and the pH (7.4 outside the cells) in the vesicles is 4.4 (acidified), the pumps work all the time, at steady state the concentration of hydrogen ion is high, the number of free hydrogen ions however is just 1 or less than 1 (that is due to buffering! lot of proteins and they bidn the hydrogen)

7. Neurotransmitter filling: the neruotransmitter pumps are passive, specific for each transmitter (ACh etc,) they utilise the hydrogen gradients as the energy source, so the hydrogen is more than 1000 more concentrated inside than outside so coupled with movement of the neurotransmitter

Question 21

What is the detail of re-acidification in synaptic release?

Answer 21

-each vesicle has 2 ATP dependent hydrogen pumps, which pump hydrogen pumps that pump the hydrogen into the vesicle (each pump needs one ATP) related to NA+/K+ ATPase, it only pumps hydrogen one way here! only two pumps per vesicle, if you work out the volume of the vesicle and the pH (7.4 outside the cells) in the vesicles is 4.4 (acidified), the pumps work all the time, at steady state the concentration of hydrogen ion is high, the number of free hydrogen ions however is just 1 or less than 1 (that is due to buffering! lot of proteins and they bind the hydrogen)

Question 22

What types of vesicle recycling are there?

Answer 22

-the fast and slow routes

Question 23

Which vesicle recycling route occurs more often and how does it work?

Answer 23

• 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

Question 24

What is the slow route of vesicle recycling?

Answer 24

• 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

Question 25

What is a fusion pore?

Answer 25

- 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

Question 26

What is the reason for SNARE complexes in neurons being unable to proceed to full fusion without the presence of calcium?

Answer 26

- 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

Question 27

What is the function of synaptotagmin?

Answer 27

- 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

Question 28

What determines the probability of neurotransmitter release?

Answer 28

- how many synaptotagmin calcium sites occupied - it is probabilistic: no site occupied= means almost zero chance of it unclamping, if 4/5 then slight chance of the confirmational change occuring, 5/5 almost 100% but not quite - synaptotagmin is a vesicular protein, the part poking out of teh vesicle is the part that inserts into SNARE complex, when calcium then gets out of the SNARE but gets into the presynaptic membrane

Question 29

Does synaptotagmin remain attached to the vesicular membrane after calcium binding occurs?

Answer 29

- 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

Question 30

What are some other proteins involved in the vesicle fusion and what are their functions?

Answer 30

- 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

Question 31

What is this?

Answer 31

- 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

Question 32

How does botulin and other toxins affect the SNARE complex?

Answer 32

-botulin toxins work via lysing or proteasing the SNARE complex proteins, and the one used in botox (botoxA), works by disabling the SNARE complex so cannot get release of neurotransmitter hence the muscle cannot contract

Question 33

What are the molecular mechanisms of endocytosis following neurotransmitter release?

Answer 33

- 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

Question 34

Is vesicle retrieval similar to endocytosis?

Answer 34

- yes - Clathrin induces curvature of the membrane - Dynamin causes “pinching off” - it recognises the vesicular membrane, it is thought thanks to synaptotagmin (as it is vesicular) and clathrin binds to the membrane and makes this network and uses ATP to curve it

Question 35

What does dynamin do?

Answer 35

- 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

Question 36

How many VAMPs (synaptobrevin) are there in a vesicle?

Answer 36

- about 70 VAMP (synaptobrevin), only one is required to form a SNARE complex as each(has one alpha helix) the 3 other helices are from the membrane, (1 with 2 helices, 1 with 1) - there may be 3 SNARE complexs or 4 or 6 at most when bound to the membrane, so max 6 will be utilised at once, but it is useful to have more since the docking is not dependent on the direction and orientation of the vesicles

Question 37

What does the inside of a vesicle look like?

Answer 37

-large part of the interior is filled with protein, the protein acts as a hydrogen buffer, that is why you can have acidic interior and not have free hydrogen ions around

Question 38

What are the three types of vesicle recycling?

Answer 38

1. The normal cycle: SNARE complex is disassembled. Vesicle to Reserve Pool 2. The rapid cycle: SNARE complex remains intact, and vesicle remains docked 3. The slow cycle: SNARE disassembly occurs, vesicle joins endosome compartment - 1: the fast route - 3: the slow route, and the rapid cycle - 2.= when the SNARE complex remains intact when the endocytosis occurs, and dynamin has done its function, only seen in cases of rapid stimulation (100Hz) leads to a present to a presence of a fully docked vesicle that is however empty! as it has emptied its contents, this happens as the machinery didn’t have time to be refilled and broken down the SNARE etc - this can happen with the high frequency stimulation, then failure of release - in any actve zone in the cerebral cortex means only 1 or 0 vesicle released!

Question 39

How are neurotransmitters synthesised and recycled?

Answer 39

* The non-peptide transmitters are synthesized at the nerve terminals * Enzymes required for their synthesis are produced in the cell body and transported along the axon * They are concentrated within vesicles by specific transporters - neurotransmitter recycling depend on if peptides or small molecule - all of the fast neurotransmission amino acid (glutamate = main exc. and aspartate, GABE inhibitory= brainstem, glycine in spinal cord) also others in metabortopic= dopamine etc. - there are many peptide neurotransmitters= all metabotropic - with ACh choline acetyltransferas is the enzyme that gets to terminal, so ACh can be made at the terminal

Question 40

How is neurotransmitter removed from the synaptic cleft?

Answer 40

– 1. Diffusion – 2. Enzymatic breakdown, esp. acetylcholine – 3. Re-uptake by: - pre-synaptic membrane - astrocytes - post-synaptic membrane (small amount) - all the peptide neurotransmitters are broken down (never reused, one shot only) - with small molecules ones it is only ACh that is broken down the rest of small mlecules are recycles, some recycled by the terminal that released them and sometimes (particularly with glutamate) is recycled by astrocytes that then transfer it to the terminal -all local with small molecules - in some cases (adrenaline and noradrenaline) they are taken up by the postsynaptic membrane sometimes (a bit of dopamine too) sometimes the receptors internalise and take the neurotransmitter with them, once internalised then broken down my monoamine oxidase (the adrenaline and noradrenaline)

Question 41

What are the differences in small molecule neurotransmitter and peptide neurotransmitter transfer?

Answer 41

- 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)

Question 42

How does the release of neurotransmitters differ depending on what type of molecule they are?

Answer 42

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