Molecular Mechanisms Of Exocytosis And Endocytosis Flashcards
What protein binds a reserve vesicle to the microfilaments?
Synapsin
From proximal to distal on a neuron what are the different vesicle pools?
Reserve
Proximal
Docked
What is the distance between the reserve pool and the active zone?
200nm
What is the proximal pool embedded in?
Cytomatrix assembled at active zone (CAZ)
What channels are the docked pools closely associated with?
Voltage gated calcium channels
What regulates the interaction of reserve poor with synapsin?
CaMKII
What happens no gene KO of synapsin?
Decrease in amount of reserve vesicles
But no effect on proximal/ docked pool
What is the active zone?
Essentially the pre synaptic equivalent of the PSD friend from - structural proteins (piccolo etc) - SNARE proteins - complexes involved in endocytosis
Name some important proteins within the active zone
CASK
Munc13-1
Piccolo and bassoon
RIM1(alpha)
What is CASK?
Member of the membrane associated guanylate kinase superfamily involved in membrane associated signal complex formation
What proteins does CASK interact with?
VGCC (may service to cluster calcium channels in active zone)
Cell adhesion molecules
Beta neurexin
Mint-1 via CamK domain
What does mint-1 interact with?
Munc-18 which is important for fusion of docked vesicles
What domains does munc13-1 have?
C1 domain (binds DAG ) 3xc2 binding domain (binds calcium)
Why is munc13-1 important?
Disruption decreases spontaneous and evoked NT release
What protein translocates munc13-1 to membrane?
Doc2(alpha)
What does munc13-1 do to munc-18?
Displaces it from syntaxin
Allowing core complex formation
What does RIM1 stand for ?
Rab3 Interacting molecule
What is RIM1 function?
Binds to rab3 in an GTP dependant manner
Translocates vesicle to proximal pool
Is a rab3a effector protein
Molecular weight of RIM1
180kDa
Molecular weight of piccalo?
530kDa
Molecular weight of Basson?
420Kda
Where are piccalo and basson found?
Cytomatrix assembled at active zone (CAZ)
What is the function of piccalo and basson?
Synaptogenesis
Tethering of proteins
How many domains do they have in common?
10 - the piccalo basson homology domains (PBH)
What is the difference between piccalo and bassoon?
Piccalo has a pdz and 2xc2 domains at c terminus (missing in bassoon)
Why are the piccalo zinc fingers important?
Bind to prenylated rab acceptor (PRA1) on vesicles
Since PRA1 can interact with V-SNARES it may be involed in endocytosis/exocytosis
What do the proline rich sequences in piccalo and bassoon allow for?
Tether proteins to SH3 domains of protein involved in endocytosis
What I the function of the vesicular ATPases?
Load NT along with H+ into 20nm vesicles
How large is a v-ATPase?
1000kDa with 13 subunits
One per vesicle
What are the stages of the vesicle cycle?
NT storage into vesicle Transition of active zone Docking Priming Fusion Endocytosis Endosome fusion Budding (Repeat)
Vesicular glutamate transporter transport glutamate into vesicles. How many subtypes are there?
3
Vesicular glutamate transporter transport glutamate into vesicles. How many TMDS?
8-10
Vesicular glutamate transporter transport glutamate into vesicles. How do they function?
ATP used to pump H+ into vesicle
Which creates an electrochemical gradient which drives glutamate entry (as negativity charged)
Vesicular glutamate transporter transport glutamate into vesicles. What is the effect of chloride ions?
High chloride inhibits channel function
Low chloride stimulates channel function
What occurs in the docking stage of exocytosis?
Vesicle in close proximity to membrane
What happens in the priming stage?
T SNARE and V SNARE interaction (core Complex formation)
What happens during fusion?
Membranes of vesicle and plasma membrane fuse to to opening of VGCC
What is the core complex?
Tight 4 helix bundle which pulls vesicle towards the plasma membrane in stepwise zipping manner from distal SNARE end to proximal
What does SNARE mean?
Soluble NSF attachment protein receptors
What motifs mediate core complex formation?
70 aminoacid alpha helix motifs
Synaptobrevin = R motif
Syntaxin = Qa motif
SNAP-25 (synaptosome associated protein of 25kDa) = Qa and Qb
What can inhibit SNARE complex formation?
Proteins which have similar SNARE formation motifs and compete for snare interaction such as amisyn
Same the t and v SNARES
T snares = synaptotagmin and synaptobrevin
V snares = syntaxin and SNAP-25
Other proteins such as munc13-1 / 18 and Vap33 are important in exocytosis
What is kiss and run?
Vesicles rapidly endocytose and recycle without clathrin mediated endocytosis
They do not remain in the active zone but undock and recycle locally and rapidly
Describe the role of syntaxin and munc protein interaction
Munc18 binds to and inhibits syntaxin function
Munc13-1 then displaces munc-18 allowing syntaxin to bind to other proteins
Core complex formation which may occur with or without munc 13 bound to the amino terminus of syntaxin
What domains of synaptotagmin bind calcium?
C2a (3 calcium) C2b (2 calcium)
What happens when calcium binds to synaptotagmin?
Allows association of synaptotagmin with snap-25 and syntaxin
2A and 2B domains then insert into phospholipid membranes
Is synaptotagmin a high affinity of low affinity calcium sensor?
Low affinity - causes synchronous release
From the presynaptic calcium current to the post synaptic action potential how long does it take?
Approx 1ms
What are the 4 mechanism of endocytosis?
Clarthin mediated endocytosis
Kiss and run
Kiss and stay
Bulk retrieval
Why might 2 mechanism of endocytosis exist?
A high frequency stimulus will deplete vesicles and from membrane cisternae. Vesicles reappear at expense of cisternae (CME)
Low frequency stimulation vesicles recycle fast enough there is no depletion of vesicles (KISS AND RUN)
What is clathrin mediated endocytosis?
Ap-2 binds to cargo patch in vesicle (?synaptotagmin) and recruit clathrin triskelions
Form clathrin coated pits
Dynamin needed to cleave vesicle from membrane
What are some of the characteristics of CME?
Very slow - cannot be sole mechanism of endocytosis
Occurs are peripheral synaptic membrane
Can occur from deep membrane infolds
In kiss and run does the vesicle collapse into the membrane?
No, forms transient connection in 5% of cases
Low probability of neurotransmitter release
Bulk membrane retrieval describes….
Large area of membrane are internalised following complete vesicle fusion with membrane
