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Synaptic Transmission - Coggle Diagram

- - - - Detergent solutions of liposomes with only t- (syntaxin & SNAP-25) or v-SNAREs (synaptobrevin) were made
      - 1.5-2% of the membrane of one was made of fluorescent phospholipids (rhodamine-PE and NBD-PE) which results in quenching of fluorescence so when fusion occurs these are diluted adn de-quenching occurs
      - It was shown that de-quenching/increased fluorescence only occurs when one vesicle has t-SNAREs and the other v-SNAREs
      - Issues:
        Fluorescence de-quenching is very slow, over a time course of hours compared to in vivo observations of ms - Rothman argued this was because there are accessory proteins in vivo which speed up the process
        [Synaptobrevin] is ~10x higher than in vivo which coudl be resulting in membrane destabilisation and anomalous results
        Dennsion's group reported that at physiological [SNAREs] fusion only occurs in the presence of polyethylene glycol (which removes water)
      - Conclusion = yes
  - - - There is probably a missing component the prevents a futile cycle forming
      - NSF has a split-washer structure which --> open washer upon complex disassembly (Alex Brunger), this movement is driven by ATP
      - This is not required at the point of fusion but is required for repeated events to occur
  - - - There is strong evidence that V-ATPases are involved somehow in some situations but it is unclear exactly how and what the importance is (could they be involved in kiss-and-run)
    - - Furthermore it is unclear how these proteins would then dissociate to allow lipid mixing
- - - - Asp residues are key to chelation of Ca2+
      - C2A binds 3 Ca2+ and C2B binds 2
        Both are predominantly made of beta sheets
      - Ca2+ binding makes the C2A domain less electronegative, this speeds up the transfer of information (compared to a conformational change)
        
        Chapman's group showed using protein pull-down that synaptotagmin binds t-SNAREs in a Ca2+ dependent manner, mirroring the correlation between neurotransmitter release and Ca2+
        
        Phosphatidylserine is required for this and C2A is the critical domain, which rapidly binds lipids (same order of kinetics as vesicle response to Ca2+)
        
        Synaptotagmin has also been shown to bind PIP2 in a Ca2+-dependent manner and C2B is the critical domain for this
        
        Synaptotagmin also increases the favourability for PIP2 in the membrane which then increases the amount that can bind
        
        Synaptotagmin is necessary for Ca2+ stimulation of neurotransmitter release (Tucker's experiments show without Syt Ca2+ induces a response similar to EGTA)
        
        These experiments also showed that Syt had an active role and wasn't just/only increasing aggregation by using yeast SNAREs and human Syt (no fusion) and then replacing the SNAREs with human versions (fusion occurs)
        
        Using [Syb] of 90/vesicle showed that fusion was highly dependent on Ca2+ in physiological scenarios
        
        Adding Syt also decreased spontaneous release (as well as greatly increasing Ca2+ induced release)
        
        Both C2A and C2B penetrate the lipid bilayer (shown using lipid-anchored nitroxide spin labels to quench Trp fluorescence - AFM also shows cavities upon Syt removal)
        
        The flexible linker is integral to this function - mutating it to an 18 Pro chain impairs neurotransmitter release and ablates force on AFM measurements
        
        Both domains interact with one another to give Syt its action
        
        C2AB is capable of sensing membrane curvature
        Liposomes of different sizes added to mixture of free syt and using immunoblotting to determine concentration of syt in the supernatant
        Lower the diameter, the less lipid is required to remove syt from the supernatant
        
        C2AB and C2B (but not C2A) are able to sense and induce curvature in liposomes - this may be why the cell membrane curves towards vesicles in the pre-synapse
        
        AFM can also be used to measure the force of interaction of C2AB with lipid bilayers - these experiments show that Ca2+ increases the force required and also creates a second higher strength interaction (no Ca2+ = 44pN peak, +Ca2+ = 73 & 122pN peaks)
        
        Reinhard Jahn was the first to use full length synaptotagmin and used it to show that Syt acts in trans to phosphatidylserine (if PS is on the same vesicle Ca2+ is inhibitory, if it is not present nothing happens and if it is only on the other vesicle then it is stimulatory)
- - - - Group identified that steps were ~the same size (25-30fF) which was thought to be vesicle fusion events - the value was consistent with known capacitance/area and size of vesicles
      - They identified that on occasion the steps upwards were followed by an immediate step down - this was termed 'flickering fusion' and agreed with predictions by Ceccarelli of 'kiss-and-run' fusion events
    - - Found three types of fusion event
        
        Instant fusion shown by rapid increase in current (no foot) and irreversible capacitance increase
        
        Small increase in current reading but reversible capacitance increase (kiss-and-run)
        
        Slow increase in current reading (foot present) and irreversible capacitance increase
    - - Both were shown to undergo reversible fusion and in microvesicles conductance measurements of 20pS were observed (thanks to Klyachko)
      - The noise in the microvesicle readings was very high thought so there were some doubts about the validity of them
  - - - Fluorescence is quenched at low pH so that it is dequenched only when there is a membrane fusion event
      - Claimed to have identified kiss-and-run events (transient fluorescence) but resolution was very poor so required faith
    - - Fluorescence with this declined with a single, slow time constant and it was blocked by inhibiting clathrin-mediated exocytosis
      - Seemed to disprove kiss-and-run hypothesis
        However given later work (Tsien) it could be argued that the findings were the result of SypHy's physiological properties
  - - - Theory was that if kiss-and-run occurred then FM4-64 could escape through the pore and Qdots couldn't
      - Upon each stimulation Qdots were depleted slower than FM4-64
      - Lagnado argues that Qdots may be hindering full fusion but this is often accepted as highly convincing proof of kiss-and-run
  - - - Theory is that SNAREs bring membranes together and destabilise them so that they mix and then separate to allow content mixing
    - - Theory is that SNAREs form complementary barrel-shaped complexes to allow mixing of vesicle contents first and then dissociate to allow lipid mixing
    - - These results (and those from similar experiments on synaptobrevin) suggest that I269, G276 and I283 sit on an alpha helix and line the pore
      - This provided evidence for a protein-lined pore
    - - Nanodiscs are too small to allow full fusion pore collapse, enabling a way of mimicking kiss-and-run events
      - Chapman's group took liposomes with t-SNAREs and nanodiscs with v-SNAREs
        
        Fusion was possible with only 2xSyb, too few for a barrel to form
        
        Maximal fusion occurred with 3xSyb, which is again too small for a barrel to be form it is thought
        
        More than 4xSyb results in a decrease in lipid mixing and glutamate release
        
        Other Chapman experiment (Das, 2020) looked extensively at lower SNARE copy numbers and pore dynamics
        
        Inhibiting (reducing availability) v-SNAREs and t-SNAREs slows the rate of release, but not the overall amount - resulting in a change in the shape of mEPSCs
        
        Increasing the number of synaptobrevin molecules in the nanodiscs increased the cargo efflux rates
        Depending on the size of the cargo these rates were changed in slightly different ways (cyclodextrin had low levels throughout, maltotriose increased steadily and maltotetraose increased greatly between 5 and 6 Syb molecules)
        
        Single bilayer electrophysiology was used to measure properties of the single fusion pores - 3xSyb resulted in transient pores forming (favours kiss-and-run) whereas 7xSyb resulted in highly stable pores forming
        Hence number of Syb determines size and stability of the pore
        
        Increasing the number of cytoplasmic syb domains drove the pore to be more often closed because they form an irreversible reaction
        More recent nature communications show that alpha-SNAP and NSF are required to close the pores
      - Mutating Syx and Syb residues to Cys to label them with MTSES identifies many of them as being exposed to water during the fusion process
      - Lindau's molecular dynamics simulation suggests that SNAREs perturb the bilayer to form a lipid-lined pore and expose SNARE protein to water (when using a small number of SNAREs)
- - - - The identification, using patch clamping of rat hippocampal neurons by Chapman and others, of membrane opening events when toxins are added at low pH suggests that pH could be a driver for light chain entry into the membrane
      - Atomic force microscopy showed that breaking disulphide bonds (with DTT) results in a doughnut shape of BoNT/B providing evidence for the heavy chain forming a pore (disulphide bond holds light and heavy chains together)
        
        This disulphide bond is broken in the higher pH of the cytosol (Mauricio Montal) allowing the light chain to detach from the heavy chain once it has translocated the membrane
- - - - Complexin I KO mice suffer ataxia and seizures
        Complexin II KO mice suffer reduced hippocampal LTP and cognitive deficits (of the two this has less physical/more cognitive symptoms)
        DOuble KO = death within hours of birth
      - Two choice swim tank experiment shows that acquisition is the same but CpxII KO has decreased ability to adapt to a reversal (Jenny Morton)
    - - Complexin II is specifically and progressively depleted in the brains of murine HD models and show similar cognitive and motor defects that exacerbate over time
      - By 9 weeks there is a large difference in [Cpx II mRNA], especially in the striatum (as determined by in-situ hybridisation), with similar results found in human post-mortem samples (humans also have a degree of [synaptobrevin-2] decrease)
        Issue with the human findings though is that there are differences in how samples are taken
      - Expression of HD mutation causes a progressive reduction in neurotransmitter release (nt release is inversely proportional to [Q72]) and a specific depletion of complexin II in PC12 cells
        
        Although in some cases [complexin II] increases over the first 24 hours and then decreases - but overall protein concentration is unaffected
        
        Complexin II becomes partially localised in inclusions (fluorescence microscopy)
        
        Using carbon fibre amperometry it was shown that transfecting these cells with Cpx II (but not Cpx I) restored the phenotype
        
        Adding Cpx II (but not Cpx I) to normal cells though reduces neurotransmitter release
    - - Interpretation of this led to the proposal of a mechanism by which complexin locks the complex in a primed half-zipper state and Ca2+ binding to Syt causes it to destabilise the two membranes and free complexin and allow progression to the full-zipper state
  - - - Weis' crystal structure shows that nSec1 binds the closed conformation of syntaxin (seemingly at odds with it positive regulatory role)
      - Newer cryo-EM structures seem to show that SM proteins act to assemble the trans-SNARE complex
  - - - There are three families of tethering factors (CATCHR, TRAPIII and HOPS) and all are quite large
      - Is thought that they prime the interaction of the PM with the vesicle by bringing them closer together
      - There is an additional proposal that these inhibit reassembly of the cis-SNARE complex after NSF-ATP action
- - - - 23 genes were identified that when mutated resulted in abnormally high numbers of vesicles (density gradient centrifugation was used to differentiate them)
    - - Addition of GlcNAc occurs in the medial Golgi, prior to this VSV-G is in the cis-Golgi and so this is a way to measure whether/to what extent transport occurs
      - Used two types of cells:
        Uninfected wt cell
        VSV-infected 15B mutant (missing GlcNAc Transferase)
        
        The Golgi fractions of each were mixed and added with tritium-GlcNAc to allow radioactivity counts of VSV-G immunoprecipitates
        
        Was found that in vitro transport required the cytosol, ATP and Mg2+
        It was additionally found that N-ethylmaleimide (NEM) and GTPγS (an unhydrolysable GTP analogue) inhibitted transport
        
        This was used to identify a key protein (NSF) which was homologous to Schekman's Sec18p analogue
        This was sensitive to NEM and had ATPase activity
        
        NSF was found to not bind directly to membranes, this role was carried out by SNAP (homologous to Schekman's Sec17p) which exists in alpha, beta and gamma isoforms
        
        SNAP binding was saturable (binds receptor = SNAREs)