Calcium control of neurotransmitter release

Question 1

Explain how calcium control influences neurotransmitter release

Answer 1

In the active zone are voltage-activated calcium channels present. In case of an action potential, these channels open increasing local calcium concentration. The cofactor complexin is the required, which is a small protein that binds to SNARE proteins and activates and clamps the SNARE complex so it will not induce membrane fusion unless synaptotagmin is going to displace complexin form binding to the SNARE complex. This displacement only happens if calcium and Syt bind. Calcium then binds to Syt on the synaptic vesicle via 2 C2 domains. Syt will then interact with phospholipids and SNARE proteins, resulting in the activation of the membrane fusion machinery (SNARE-complex.

Question 2

Explain the ‘kiss and run’ endocytosis

Answer 2

The synaptic vesicle (SV) partially fuses with the plasma membrane, so that the fusion pore is briefly opened and quickly closed. The SC is then pinched off and can be filled with neurotransmitters

Question 3

Explain what bulk endocytosis is.

Answer 3

Endocytosis of the largest parts of the presynaptic membrane leads to the formation of an endocidic intermediate from which potentially SVs could be regenerated.

Question 4

Explain what housekeeping membrane recycling endocytosis is

Answer 4

This involves clathrin-mediated endocytosis and involves canonical early endosomes (EE). EE can then be converted into late endosome (LE) and multivesicular bodies (MVB) that can travel to the cell body.

Question 5

Name 3 methods that can monitor endocytosis

Answer 5

• FMI-43
• synaptopHluorin (in combination with Bafilomycin)
• electrophysiology

Question 6

Explain the FM1-43 method

Answer 6

It is a method to monitor endocytosis by using a fluorescent styryl dye. FM1-43 has a hydrophilic and lipophilic side. Making it possible for one side of the dye to sin in the membrane and the other side to prevent it from completely entering. So the dye remains on the membrane, but because of endocytosis and fusion of the membrane. SCs formed by endocytosis also contained the dye on their membrane. The dye can then be washed away from the synapse membrane, while the dye stays on the SVs membrane inside the synapse.

Question 7

Explain the synaptopHluorin method in combination with Bafilomycin

Answer 7

SynaptopHluorin is a fusion protein of fluorescent protein Phluorin to the luminal portion of SV proteins. pHLyorin will fluoresce at a neutral pH, but because SVs have an acidic pH due to the protein pump, it will not fluoresce until exocytosis. During exocytosis, the luminal side of SCs will be exposed to the neutral extracellular matrix. After endocytosis, the fluorescence will stay for a very short period and will then be quenched. Bafilomycin prevents the pH from going down, trapping it in neutral pH after endocytosis. It does this by blocking the V-type ATPase that is required for the reacidification. This allows synaptopHluorin to remain fluorescent.