Ca2+ REGULATION - SNARE-mediated fusion Flashcards
What is important regarding APs and NT release ?
Important that the induction of an AP and the release of Neurotransmitter (therefore vesicle fusion) occurs at the same time / is synchronised
Munc13 - function and relationship with syntaxin-SNAP25?
Syntaxin/SNAP25 - function and mechanism in vesicle fusion?
Munc13 is involved in vesicle priming by regulating syntaxin-SNAP25 acceptor sites
Synataxin/SNAP25 - first dimer complex to form before vesicle fusion zippering can occur. Once this dimer forms, VAMP2/synaptobrevin can be recruited which forms the SNAREPIN.
Proteins that make up the composition of the vesicle and their functions?
GLUTAMATE – always seen in vesicles
RAB - not an integral membrane protein but distinguishes vesicles from other endosomes
Synaptotagmin - Ca2+ sensor which aids in regulated secretion
Synaptophysin - glycoprotein that is an integral part of the neuroendocrine secretory granule membrane - often used in imaging as it is paired to an antibody
What must synaptic vesicles do for efficient transmission?
What is the importance of proteins being recovered?
Vesicles must dock at the right place in the active zone so that they are ready for diffusion upon Ca2+ entering
All fusion proteins must be recovered so that the process can repeat
Features of synaptotagmin – number of isoforms – C2 domain (function and mechanism of action) – purpose of the fusion clamp
- 16 isoforms of synaptotagmin with different Ca2+ affinities and different localisations to the membrane
- Synaptotagmin triggers fusion upon binding to Ca2+ at its C2 domain – inserts its C2B domain into the phospholipid bilayer which SOMEHOW facilitates the opening of the fusion pore
- Synaptotagmin behaves as a fusion clamp for regulated exocytosis –» Ca2+ binding to the SNARE complex is important for docking and Synp works with COMPLEXIN as a fusion clamp
What effect can altering the C2B domain have and why?
Altered Ca2+ sensitivity to fusion as the amount of Ca2+ in the active zone is subject to change
Synaptotagmin – what is it?
Major Ca2+ sensor for neurotransmitter release at the central synapse
Synaptotagmin K.O mice - what was shown?
- Vesicles still load with NT and fuse with the membrane
- NO SYNCHRONY with the action potential is seen
- No apparent docking defect but asynchronous vesicle fusion
K.IN of mutant Synaptotagmin1 mice - what was shown?
- No depletion in the supply of readily available vesicles but a DECREASE in the number of PRIMED vesicles (a decrease in the release probability of vesicles)
- Ca2+ cannot bind to stimulate NT release (and fusion)
How many synaptotagmin isoforms are there and what is meant by different Ca2+ affinities (low and high)?
Many isoforms of synaptotagmin»_space; these have different Ca2+ affinities
1,2 and 9 have low Ca2+ affinities meaning a high concentration of Ca2+ is required for these forms of Synt to trigger fusion
Other isoforms have high affinities which means they trigger fusion at a lower concentrations of Ca2+
COMPLEXIN - where is it found? and where does it interact?
Cytosolic protein that has a clamping function.
Interacts with SNAREs via the C2B domain of Synaptotagmin
Outline the mechanism of action of Complexin?
Binds C2B domain, ‘flips’ an arm open > SNARE complex tightens and this has a knock on effect
It bridges SNAREpins in a zig-zag and prevents the zippering reaction from occuring
Relief of this clamp (upon Ca2+ binding) allows the zippering reaction to occur
Syntaxin1 K.O – what does this allow us to do? What was the result?
Allows us to investigate the function of the protein in regulated exocytosis
The K.O interfered with Ca2+ binding to the synaptotagmin/SNARE complexes
Synaptotagmin (-/-) - what effect was seen?
What did this allow us to conclude?
Mutant cannot bind Ca2+ but there was NO DOCKING DEFECT - only a FUSION DEFECT
Allowed us to conclude that synaptotagmin has docking and fusion roles
THE BIGGER PICTURE - what needs to happen for a synapse to work well?
Why does this link in with Synaptotag’s key feature of low Ca2+ affinity?
Vesicle needs to be docked in the right position next to a Ca2+ channel so that when Ca2+ is sensed and a channel opens, recieves sufficient Ca2+ – this needs to happen because the sensor has low affinity so this will only happen when the [Ca2+] is HIGH!
