chapter 4 - calcium control Flashcards
4.1 [calcium control] Explain mechanisms by which ca2+ controls nt release
draw the timescale of neurotransmission and check the image
only local increase of ca2+ levels at pe-synapse trigger synchronized release
- we focus on how ca2+ triggers exocytosis
Ca2+ binding to synaptotagmin triggers synaptic vesicle exocytosis
How it goes in pre-synapse:
> action potential opening of Ca2+ channels transient increase in local Ca2+ concentration
> Ca2+ binding to synaptotagmin via two C2-domains
> interaction of synaptotagmin C2 domains with phospholipids and SNARE proteins
activation of the membrane fusion machinery
> In triggering exocytosis, synaptotagmins require an obligatory cofactor called complexin, a small protein that binds to SNARE proteins and simultaneously activates and clamps the SNARE complex for subsequent synaptotagmin action
> This mechanism operates in most, if not all Ca2+ regulated forms of exocytosis throughout the body, including degranulation of mast cells, acrosome exocytosis in sperm cells, hormone secretion from endocrine cells, and neuropeptide release
4.2. [calcium control] describe the structure of the three groups of synaptotagmins and draw it
check drawing.
16 genes encoding canonical Syts are expressed in mammals
* Synaptotagmins (Syts) contain a short N-terminal sequence, transmembrane region, a central linker sequence, and two C-terminal C2-domains
* C2-domains: Ca?+ binding domains found in a large number of signal transduction and C membrane trafficking proteins
* Syt1, Syt2, Syt9 and Syt12 are expressed on synaptic vesicles
* Not all C2-domains bind Ca2+
* Syts are classified in two groups:
Ca2+-dependent: further subclassified based on the presence/absence of disulfide-bonded cystein residues in N-terminus
> Both C2-domains of Syt1 bind to phospholipids in a Ca2+-dependent manner, and to SNARE proteins:
* Syt1 binding to phospholipids requires Ca2+ and negatively charged phospholipids, with phosphatidyl-inositol phosphates being most effective
* the Syt1 C2-domains seem to directly interact with syntaxin-1; this interaction is greatly enhanced by Ca2+
* Syt1 also binds to assembled SNARE complexes in a Ca2+-dependent manner
4.3. [calcium control] which synaptotagmins function as synaptic ca2+ sensors for NT release (a) and how this has been discovered and demonstrated (b)
syt1,syt2,syt9 function as synaptic Ca2+ sensors for nt release
A. Deletion of Syt1 in cortical neurons blocks fast synchronous NT release; note that not all Ca2+ stimulated release was abolished, a delayed asynchronous form of release is retained; release induced by hypertonic sucrose (thought to cause Ca2+-independent exocytosis of all vesicles in the readily releasable pool) is unchanged
-> Syt1 KO did not interfere with vesicle fusion as such, only with the Ca2+ triggering of fusion.
B. A systematic screen of all Ca2+-binding Syts for rescue of the Syt1 KO phenotype revealed that only Syt1, Syt2 and Syt9 were able to rescue
C. Syt1, Syt2, and Syt9 mediate Ca2+ triggering of release with distinct kinetics: Syt2 exhibits the fastest rise and decay kinetics, whereas Syt9-mediated IPSCs are two-fold slower. This fits well with Syt2 primarily expressed in synapses requiring very fast transmission (e.g. auditory system or NMJ) and Syt9 primarily expressed in the limbic system
D. Quantification of increase and decay of the IPSCs
syt1 specifically
Syt1 Ca2+-binding point mutations in the C2A-domain:
* D232N: increases the amount of Ca2+-stimulated SNARE complex binding, without altering phospholipid binding
* R233Q: greatly decreases the apparent Ca2+ affinity of Syt1 during phospholipid binding, without altering SNARE complex binding
* D238N: modestly decreases the apparent Ca2+ affinity of Syt1
> Conclusions:
* Svt1 is a true Ca2+ sensor
* Both SNARE- and phospholipid-binding by Syt1 are involved in release
4.4. [calcium control] explain and draw how complexin and synaptotagmin function together to mediate ca2+-induced synaptic vesicle exocytosis.
what are Complexins?
> small soluble proteins (=120 amino acids), evolutionary conserved in mammals, which bind to the partially assembled SNARE complex
> Four complexin isoforms in mammals, with complexin-1 and -2 widely distributed in the body and abundant in the brain
> Deletion of complexin-1 and -2 in mice induces a milder phenocopy of the Syt1 KO phenotype (e.g. partial loss of synchronous NT release).
mechanism of action of synaptotagmin and complexion in Ca2+ triggered exocytosis
Current model: complexin binding to primed SVs containing partially assembled SNARE complexes “superprimes” the SV into an activated state, and subsequently clamps them
Ca2+ binding to Syt then triggers Syt binding to the SNARE complex and the phospholipid bilayer, dislodging the complexin clamp and pulling on the SNARE complex, thereby opening the fusion pore
Priming: partial SNARE/SM protein complex assembly
Superpriming: binding of complexin to partially assembled SNARE complexes
Fusion pore opening: triggered by Ca?+ binding to Syt
summary
* Complexins act both as activators and as clamps of NT release
* SNARE complex binding by complexin is essential for its function, and the complexin N-terminus is crucial for its activating role
* How does complexin act to promote Ca2+ triggering of SV fusion?
- the central a-helix of complexin and Syt1 bind to SNARE complexes at overlapping sites
- Ca2+ binding to Syt1 triggers displacement of complexin from the SNARE complex
