SNARES Flashcards
Neurotransmitter release mechanism shown in an electron micrograph
Presynaptic terminal showing a population of uniform presynaptic vesicles - some are docked at the presynaptic PM
Where do neurotransmitters cluster
Around the active zone
area where synaptic transmission occurs
What are synaptic vesicles?
Specialised endosomes undergoing regulated endocytosis
Constitutive secretion
Pathway by which proteins are secreted or transferred out of the cell
6 steps to membrane fusion
- tethering
- docking/ priming- pull closer together
- fusion initiation
- hemifusion
(3/4 need fuse without bursting and losing organelles- membrane needs to bend ) - fusion pore opening- inner leaflets diffuse, let molecules out
- full colapse
Fusion pore opening
- needs to be wide and expand
- small glutamate and Act only need small pore
What are the 2 types of pores
- full fusion- stays open then disengages from each other
2. kiss and run- opens and closes
What recent evidence supports the pore model
- Cryo-Em
Found SNARE proteins not required for docking but fusion
What did slices taken from the hippocampus show?
- vesicles crowded round the active zone
- capture vesicles undergoing fusion- was space between pre and post synaptic
- GI vesicle fully collapsed into membrane
What do SNARE mediate?
Vesicular fusion
Snare hypothesis
Each class of transport vesicle contains a specific targeting protein
Types of SNARE proteins
V(Cis)- associating only with a receptor protein, complexes fused together
T (trans)- specific to appropriate acceptor membrane , fusion of snares
How does a transport choose its target?
NSF= Sec18 cytosolic protein (ATPase) required for transport vesicle fusion
SNAP
Soluable NSF attachment proteins
- 3 isoforms isolated from brain
- A,B,Y required for ER to Golgi transport
What are SNAP receptors
SNARE-distinct SNAP binding sites on membranes SNAP must be bound to SNARE before NSF can bind
What is the NSF-SNAP-SNARE complex
form a stable 20s complex which requires ATP hydrolysis to dissociate
Who identified and isolated the first mammalian SNARE?
J rothmans 1993
Snap R implicated in vesicle targeting and fusion
Detergent + Ant-my epitome IgE linked to beads
- add NSF-MYC A+ Y SNAP
- add ATPy-5 EDTA
Gives you a complex with SNAP receptors, 20s fusion particle
- lose non specific and specific elucidate
Made membrane fraction form in the brain
What is ATPy-5?
Like ATP (3p) but instead of the oxygen between the final Ps it has a sulphate so it cannot be cleaved
What do you do to NSF before action
Freeze
interacts through mystery receptors
First identified SNAREs were….
Name them
Brain specific synaptic proteins - syntaxin A and B - SNAP-25-synaptosome associated protein of 25k - Vamp/synaptobrevin 7s complex
V-SNARE
Synaptobrevin/ Vamp
T-SNARE
Syntaxin
SNAP-25
Both V snare and T snare form the…
7s complex
Docking and fusion of vesicles at presynaptic membrane occur through a series of protein protein interactions involving formation of the 7s complex
NSF + SNAP + 7S (syntaxin +vamp +SNAP25) =
20s complex
How does SNARE mediate membrane fusion?
cis and trans SNARE complex come together for docking and fusion
What is needed for SNARE dissociation
Adaptor proteins
NSF
ATP
-releases ADP+pi
Why is SNARE dissociation important?
Regenerate free SNAREs for the next round of vesicle docking and fusion
What is the conserved structure in SNARE proteins
Was Q and T, now Q and R
common motifs conserved- important for function
protein structure helps understand how they work
What are Q and R?
R- centre of SNARE motif is an R residue
Q- has glutamine at the centre
What are rationally designed mutants made for?
To hypothesise how they work
Eg. SNARE motif hidden - stops forming inappropriately= closed
What is the current model for SNAREs and membrane fusion?
Trans-SNARE complex formation helps ‘dock’ (as opposed to tether) vesicle to plasma membrane
- Full zipper promotes fusion
- Snap25 and Syntaxin dimer create acceptor complex for vesicular synaptobrevin
Why does full zipper promote fusion?
Vesicles held in place but not close to membrane
Image of trans snare complex
HSBC domain (synaptobrevin, SNAP25 and syntaxin) pushed out of way springs around each other pull membranes together
What is SNAP-25
anchored into membrane- not TM
Contains 2 snare motif glutamine containing Q( B + C snare motifs)
What is a motif
Like a kids slinky toy, coils and wraps and folds back
- 2 folded around each other then its a nightmare to pull apart
What if 4 motifs come together?
4 coil-coil motif
What is support for snare mediated membrane fusion in secretion?
- clostridial neurotoxin- paralysis
- protein protein interactions produced in vitro- force fusion
- ko mouse/c.elegans/ drosphila
- mouse= embryonic lethal die from respiratory failure
- c.elegans= feeding is passive, not lethal ko - experiments- patch clamp and high resolution imaging in chromaffinn cells, pancreatic beta and model secretory cells
Tetnus (1 type) and botulinal toxins (7 types) show the role of SNARE proteins in the regulated secretory pathway
- anaerobic clostridial bacteria found in soil
- most powerful toxin known- LETHAL 10-10/kg mouse
(PIN PIRICK OF TETNUS KILLS) - 150kDa peptide released by bacteria
What was the toxin composed of?
Heavy chain- 100kDa= contains receptor domain required for internalisation (receptor binding domain to get inside neurones to e
light chain- 50kDa= contains metal-endopeptidase domain (enzyme domain cleavage of proteins)
What happens following poisoning?
Synaptic transmission is abolished
What is R is toxin
Where in the sequence toxin works, outside amino terminus
What are botulinum neurotoxins?
Active toxin in bottom causing muscles to relax- in medicine
Different types of botulinum neurotoxin
- Bot E= SNAP25
- Bot C=SNAP25 + Syntaxin
What happens when Bot C is added
Most lethal
- the syntaxin is lethal for neurotoxins as the axons retract and die
How does botox work?
- binds with high selectivity to receptor on pre synaptic terminal
- toxin become internalised into acidic endosomal environments
- Hn domain forms proteins allowing LC to pass to cytosol
retarget for motor neurons to target pain
Why is toxin internalised into acidic endosomal environment?
separates translocation domain- inserts into membrane
mammalian snares- how many are there and what do they share?
38
all share common structural features required for function
Examples of mammalian snares
Vamp 7 - secretory lysosome in immune cells
Vamp 1,2,3- found in neurons
SNAP 27- insert into membrane, long term potentiation
What are the different snares found in different intracellular compartments?
Er- Syntaxin 18, Sec20, Sec22b, USE1
Lysosome- Syntaxin 7, VT11B, Syntaxin8, Vamp
Golgi- syntaxin-5, BET1, SEC22b, GS27
What is different about Sec22b?
Find R snare bind with 3Q motif
What are the knockdown experiment used for SNARE?
Deletion or silencing in simple model cells and whole animals - used to see snare interaction
Can snares substitute for each other?
Yes but some functionality can be lost
What does patch clamp measure?
capacitance- ability to sort charge of cell
- pm stores negative charge
What is capacitance equivalent to?
SA- fuse vesicle - increase SA= increase capacitance
- endocytosis decreases capcitance
What are caged compounds and what are they sensitive to?
(Ca2+) inside and infuse into the cell through patch electrode.
They are sensitive to UV light- flash breaks cage open so ca2+ is free to bind to ca2+ binding protein
What does SNAP 23 do?
rescue regulated exocytosis in SNAP 25 KO mice however rate of vesicle diffusion is slow
- forms snare complex with snares to support
What happens in SNAP 23/24 -/- MICE
23= low response, 10 vesicles fused (20 x10-5) 25= expressed snap23 when no 25 and secretion went up
What are the essential regulators of SNARE catalysed membrane fusion?
- cis SNARE complex
- SM protein (sec1/mun 18)
- complexion
- synaptoagmin
what do sm proteins interact with for specific functions?
- syntaxin and SNARE pins = membrane fusion
2. Complexin and synaptotagmin = ca2+ regulated endocytosis
What is the first and proposed function of Mun18/sec proteins?
- first identified as binding partner of closed conformation syntaxin
- proposed=negative regulator of secretion
nucleation - zipping - membrane fusion - release and collapse
What happens when you interfere with the transmission in C.elegans?
Uncoordinated transmission (unc)
What happens to sm proteins cytosolic?
binding of syntaxin (sec+ syntaxin), they bind in close configuration and syntaxin enters snare complex
What needs to happen to sec1?
It needs to be displayed to add syntaxin to snare complex
the Drosophila version of Sec1 and what happens if you overexpress this gene?
Roc
inhibit neurotransmission
what are the 2 independent experiments that indicate munc18 is required for and speeds uo SNARE dependent fusion
- model over simplistic
2. in vitro assays- regulation of vesicle function
Making artificial lipososomes and how is this seen
Fusion of vesicles @ 37 ^oC
Known quantity of T + V, then add munc18 with no incubation or pre 3 hours incubation at 4 degrees
- seen using fluorescent molecules- FRET pair in lysosomes
Difference in fusion for no munc18, munc18 and munc18 no incubation
absence of munc= rate of fusion slow, additional regulatory proteins used
Preincubation= fusion goes up a lot, accelerates (no expected of neg control)
Why does preincubation increase munc18 fusion
has second binding site on second form of syntaxin
What does deletion of munc18 or syntaxin produce and how do we know this?
docking defects
no fusion when no munc18
- reexpress in mice- restore secretion to control level
What is a reconstitution experiment?
Shows that a defect we see is only due munc18
What kind of regulator is munc18
positive regulator
snare by themeselves is not enough
What indicates vesicles are tethered
non random distribution of vesicles and accumulation at the membrane
What does analysis of secretory cells from munc18 ko mice show?
total number of vesicles is normal but the number docked is reduced
- 2 chromaffin cells in culture from ko, infected with wt m18 and fused with EGFP- decrease in ko and increase when reintroduce
What do colocalization studies sow about vesicles docking?
They dock to munc18 microdomains (enriched hot spots) by creating a docking site
GTP shows different colours - m18 not distributed evenly throughout cell but clusters
What does overexpression of snap 25 show?
In munc18 rescues it shows docking defect
What does snap 25 rescue
docking but NOT fusion in munc18 null chromaffin cells
snap25 + syntaxin =
docking site , need ti add snap25 to see where vesicle will go
munc18 controls syntaxin
What is the purpose of munc18
prevents formation of dead end snare
As once syntaxin is open 2 snares bind to snap25 so nowhere is left for vamp to bind,
-no munc= open syntaxin forms dead ends
What happens once syntaxin is open?
2 snare molecules bind to snap25 so nowhere is left for vamp
no munc= open syntaxin forms dead end
If you overexpress SNAP25
there is still a portion of snap25
recapitulate snap25 it doesn’t come bacl
What are the 2 roles of munc18?
- negative regulator- control docking site and prevent dead end snare
- positive regulator- control fusion
what do SNARE proteins encode?
functional selectivity
- conserved sequences buried within snare pin dictate snare pairing
- outer surface of helical snare motif, cooperate to form binding sites for specific sm proteins and provide additional evidence to facilitate fusion
What does munc12-1 binding to closed syntaxin regulate
ability of snare to form complexes and consequently vesicles to dock to pm and undergo fusion
Muncr 13 ko in c.elegans and mice
c.elegans = disruit transmission mouse = embryonic lethal
Experiment to show if Mun13 is essential for fusion of glutaminergic synaptic vesicles- steps
- Isolate brain of embryo
- put in culture
- put electrode in neuron- stimulate to fire AP activates receptor on itself
- make autopsies and make synapses with themselves
- record glutamate mediated currents
What happens when there is an absence of m13?
No generation of AP no Ca2+ channels open
What if you apply artificial ca2+ into cell and ko m13 ca2+ down?
Abolish evoked excitatory mechanism
This is because the function of munc13 is down from ca2+ signalling as if you raise ca2+ artificial mutant still doesn’t increase
Sucrose experiments where there are high sucrose levels on outside of cells what happens?
Sucks water out of cell through osmotic pressure
- water leaves, it shrinks and membrane become closer together so fuse
Difference when EPSC is -/- compared with +/- ?
\+/- = current appears as they spontaneously fuse -/- = impaired- defect as snare machinery isn't functioning
synapses show plenty of vesicles.
What is the difference in ko and wt in vesicle pool
Decrease pool in mutant
Thought vesicles were docked but actually tethered
What does m13 enable to happen
vesicles to undergo fusion
What is it called when vesicles undergo fusion after they dock?
priming reaction
What are prime vesicles about?
Rate limiting step in neurotransmission
little prime= decrease in output
How does expression of munc13 control expression of neurotransmission?
Binds syntaxin and munc18
Genetic studies on yeast 2 hybrid screen show?
1- N syntaxin bind to C mun13
2- munc13 and syntaxin GST fusion protein bind in vitro
3- munc13-1 binds to 7s core complex= regulate formation
What does C.elegans studies show about munc13?
- how munc13 promotes snare complex formation
- unc-13 mutant has different shape (uncoordinated movement)
- records post synaptic response in muscles from stimulating neuron as ion channels are opening
Recording post synaptic response in muscles from stimulating neuron as ion channels are opening. What happens when ko m13 and syntaxin
m13= no movement
syntaxin ko = no movement
Crossed syntaxin and m13 ko and reexpressed mutant syntaxin = restore function despite m13 not present
Take home message from C.elegans study?
munc13 is needed for opening syntaxin
as they spontaneously remain open once syntaxin readded
reconstruction of m13 and m18 in neurotransmitter release
donor and acceptor vesicles
synatobrevin + (munc18+ syntaxin ) add ca2+, m13, snap25
full fusion occurred
Model for muncs syntaxin freed by NSF
- NSF breaks apart snare complex (SNAP25 and syntaxin)
- munc18 comes quickly and keeps syntaxin out of a non-productive complex
- munc13 binds= facilitates opening of syntaxin
- ca2+ final fusion step
What did studies in rewards lab show?
- munc13 is a target for modulation of GqPCRs
- GPCR regulate exocytosis in response to an AP
- act independent of ion channels and ca2+ signalling - agonist dependent plasticity
seward lab 1 - histamine and Ang11 potentiate exocytosis despite inhibiting ca entry through vgcc, How?
depolarise cell to cause rise in ca2+
apply agonist histamine- size of capacitance response increases
- no change in ca2+
- increase fusion - not to do with ca2+
Seward lab 2- get more secretion of vesicles coming to membrane
Load with fluoresence
- black dots = non seen
- increase secretion but no vesicles at membrane
Monitor GqPCRs effects on vesicles docking and fusion with TIRFM
Seward lab 3- formation of munc13 clusters at PM following activation of GqPCR
Gathering a specific location
- mutation in cl regulatory domain abolishes GPCR induced translocation
Seward lab 4 - overexpress mutant munc13
potentiation of agonist blocked
What does munc18 reduce the availability of and regulate?
Syntaxin and formation of syntaxin-snap25 acceptor site and regulates opening of syntaxin and vesicle priming
What leads to formation of loose SNARE pin which docks vesicles?
Engagement of syntaxin-snap25 acceptor complex by snapotagmin and synaptobrevin
Synaptic vesicle cycle
loading, mobilisation to active zone, docking, priming, ca2+ sensing, fusion, translocation, sorting and repeat
What needs to happen at priming stage?
Need to be in a state where as soon as calcium is sensed they undergo fusion
What is the protein composition of synaptic vesicles
proteomic study- isolated synaptic vesicles from mammalian brain and quantified proteins in vesicle
Loaded with glue as in brain
created model structure to show proteins with densities
What is Rab3A
Attached to vesicles dressing, distinguishes vesicle from endosome
What is synaptotagmin the only known one for?
Regulating ca2+
How many isotopes of synaptotagmin are there and what do they have?
16
- different ca2+ affinities and membrane/vesicle localisations
- all have tandem “c2 domains” (calcium binding)
- ca2+ dependent binding with phospholipids via C2B domain could trigger fusion
what happens when you purify out snare complexes using detergent?
Synaptotagmin comes out with complex
What is Ca2+ independent binding with SNARE complex important for?
docking and together with complexin acts as fusion clamp
Constituative and regulated fusion
C- vesicles to membrane and fuse
R- vesicles come to membrane and bind (clamp)- doesn’t undergo final fusion
What are synatotagmin 1,2,9 required for?
fast synaptic transmission
bind to neuronal voltage gated calcium channels
What are the other isosforms?
Synt7- peptide secretion
Synaptotagmin 1- major ca2+ sensor for transmitter release at a central synapse
Snap 1 KO proteins
Main ca2+ sensor- get dead mice
syt 7 = not lethal
What happened when ko Synaptotagmin?
- Evoked synchronised synaptic transmission depressed in ko
- asynchronous release persists
- hypertonic sucrose evoked release is normal
Difference in WT and ko when you stimulate neuron to fire AP and record activation of glutamate receptor
See post synaptic current
KO- no fast phase of synchronous release but still some mini events (vesicles still loaded with NT and fuse )
What is the fast phase?
Response on off in 50ms
Knock-in mouse expressing mutant synaptotagmin 1 = evidence for its function in regulating ca2+ sensitivity of synaptic transmission, how?
Mutant = asp133- arg133 (neg to pis aa charge change)
- c2b not c2a
- synaptic transmission = AP fired down neurons at 20 per second speed
- depletion of available residues of release, lots of extra vesicles
What are low and high release probability synapses?
Low= release at little( small response) as few primed vesicle. Ca2+ come in helps to prime more and increase pool size High= release a lot of available pool so next smaller
Ca2+ sensing function
Key role in vesicle ability to respond to a ca2+ signal
What happens when ca2+ changes on outside?
Change conc gradient across synapse
Alter affinity of C2AB= alter sensitivity of synaptic function
What do addition of calcium and C2AB domains of synaptotagmin do?
Increase speed of fusion
improved in line with affinity
why does complexin clamp SNARE pins in a zig zag array?
CPA can bridge 2 snares bind to create a zig zag, (fusion pore closed)
forms ring of vamp molecules coming out and SNARE underneath (fusion pore open)
What happens if you displace one arm of one complexin molecule?
It allows SNARE to tighten and zipper
What did they discover in 2011 about ring of vamp coming out of snare (open)
relief of clamp sufficient to allow snare zipper but allows synchronised multiple snares
What did the crystal structure of syt1-SNARE-CPX-syt1 complex show
- snare complex interacts with synaptotagmin in one of coils
- complexin inserts into complex and interfere with synaptobrevin making complex
Rationally designed mutation and expression in Syt1 ko animal show what?
Insight into protein function in regulated exocytosis
- na open so depolarises
- ca open= ca in, increase conc in active, will exist at mouth micro domain
What is synaptotagmin function in docking and fusion?
Docking - ca independent interaction with SNARES
fusion - calcium dependent functionn
What does low affinity of synatotagmin 1 and 2 ensure?
Only primed vesicles docked by VGCC released by AP
How is tight coupling of Ca2= channel and vesicle proteins accomplished?
interaction of syntaxin, SNAP25 and synaptotagmin with the SYNPRINT site on the calcium channels pore forming alpha subunit
Syntaxin- Snap25 acceptor complex by synaptotagmin and synaptobrevins forms?
Lose snarepins to dock vesicles
- m18 = regulates availability of syntaxin and formation of acceptor site
- m13 = regulates opening of syntaxin
What primes vesicles for rapid synchronised fusion?
snare pins and clamping by complexin
What does ca2+ binding to c2B domain of set trigger?
fusion- pore may expand to induce full fusion
Tethering by Rab3 ensures?
vesicles docked to pm at correct location
