Vesicle fusion and transmitter release


Experiments to deduce presynaptic events

  • 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.
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The vesicular hypothesis

  • 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
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Neurotransmitter release

  • 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
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SNARE proteins

  • 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
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Reserve vesicles

  • 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. 
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Vesicle recycling

  • 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. 
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Kiss and run vesicle fusion

  • 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. 
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Studying vesicle turnover - FM dyes

  • 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
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Studying vesicle turnover - synaptopHluorin

  • 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. 
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