Vesicle fusion and transmitter release

• Bernard Katz did experiments on the squid giant axon.
• He found that above a threshold level, the post synaptic response depended upon the size of the presynaptic depolarisation.
• Blocking Na+ channels using TTX and K+ channels using TEA, he found that neither of these channels is responsible for neurotransmitter release. 

• He found that depolarisation opened voltage gated calcium channels.
• Internal Ca2+ must be kept low to maintain the concentration gradient:

                             Active Ca removal from the neuron

                             Internalisation of Ca2+ into organelles like the mitochondria or ER

• Experiments on the frong neuromuscular junction he found that spontaneous responses can be observed postsynaptically, always multipoles of a unit response spontaneous quantal release
• Using Mg2+ to compete with the Ca2+, evoked quantal release can be produced.

• Vesicles are the organelles of transmitter storage and release 

                             The two pieces of evidence are the quantal release discovery and the                                               observation of vesicles underneath the electron microscope.

• 1 quanta of acetylcholine is about 5000 molecules  - one vesicle creats 1mV of depolarisation

• Synaptic vesicles are clustered in the docked and reserved pools.
• They are clustered around the active zone,which has the protein machinery required for the vesicle to work.

Ca2+ influx causes....

1. The activation of vesicle fusion with the presynaptic membrane

2. The mobilisation of reserve vesicles towards the active zone.

• Vesicle fusion has never been directly observed, but has been seen through capacitance measurements and instant freezing...
  • This is where a preparation is put into "the slammer" and an electromagnet stimulates the tissue
  • It is then dropped into a copper block with cold liquid helium
  • This is the freeze fraction method, as the tissue snaps at the synapses

• The three types are:

                On the vesicle membrane - synaptobrevin

                On the presynaptic membrane - SNAP-25 // Syntaxin

• The fusion is driven by a calcium sensing protein on the vesicle called synaptotagmin.

1. A syntaxin and SNAP-25 complex attaches to synaptobrevin, forming a loose interaction (the C2B domain of synaptotagmin is also bound)

2. Ca2+ causes the SNARes to twist together and the C2 domains penetrate the membrane, catalysed by synaptotagmin. A core complex is created.

3. The vesicle is drawn very close to the membrane, forming a hemifusion intermediate.

4. The plasma pore that has been formed expands father, and the vesicle becomes continuous with the membrane.

• Tetanus and botulinum toxin are proteases that target SNARE protein

• Reserve viesicles are fused to cytoskeletal elements by synapsin.
• Ca2+ causes the phosphorylation of synapsin via calmodulin dependant kinase.
• The resultant conformational change uncouples synapsin from the vesicle membrane. 

• To stop the plasma membrane from getting too big, vesicles are recycled, which allows for fast sustained transmission
• Vesicle recycling happens via clathrin mediated endocytosis, which is where adaptor molecules absorb membrane elements and take them to the endosome for sorting. 

• This is thought to occur in central mammalian synapses.
• It is where a fusion pore is formed, which quickly shuts, allowing the release of less than one quantum of neurotransmitter. 

• These are sticky and so can be used to label vescicles, like for example FM4-64.
• Vesicle turnover causes the dye to be taken up, and after washing, the vesicles with dye can be seen. 
• It can be used as a functional assay

• This uses a genetically encoded protein, pHlourin
• The inside of the vesicle is pH5.5, allowing no fluorescence. 
• Vesicle fusion causes the pH to shift to about 7.3, causing fluorescnece. 
• There is also a newer variant called sypHy, which uses synaptophysin. It is better as it is more specific to the vesicle membrane. 

• Fluorescent dye techniques can be used to measure the rate of exocytosis, which comes out at 3 vesicles per second.
  
• Above 10 Hz, the vesicle availability becomes the rate limiting step.