- Created by: 11APhillips
- Created on: 03-01-20 14:19
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.
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
- 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.
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.
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
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.