Lecture 3 - Post synaptic events in neurotransmission Flashcards
What is the process of neurotransmitter release from the synapse?
1 - as an action potential goes through the synapse, membrane depolarisation opens the voltage-gated Ca2+ channels
2 - the in crease in Ca2+ tiggers synaptic vesicles to fuse with the plasma membrane
3 - The releases neurotransmitter diffuses across the synaptic cleft and interacts with the specific post synaptic receptors (either ligand gated ion channels or G protein coupled receptor)
4 - Neurotransmitter can also interact and activate presynaptic autoreceptors, and spillover into adjacent synapses
5 - Neurotransmitter is then eaither degraded or taken up by glia or presynaptic transporters
6 - The synaptic vesicle membrane is then retreived for recycling by endocytosis
7 - Neuropeptides are released from dense cored granules
What molecule is the trigger for neurotransmitter release?
Ca2+ increase
What is the size of the synaptic cleft and how is it held together?
~50nm
held together by many structural proteins
What are the two types of posy synaptic receptor and what are their properties?
Ligand gated ion channel -causes ion fluxes -depolarise the membrane G protein coupled receptor -slow -can undergo signalling -can also impact on ion flux
What must the synaptic vesicles be recycled?
-cell has a limited number of vesicles (~100)
therefore must be recycled via the synaptic vesicle cycle
How was it experimentally shown that neurotransmitter release is coupled to Ca2+ influx in the presynaptic terminal?
- used a voltage clamp technique on a squid giant axon that terminated on a muscle
- found out that potassium and sodium channels were not important by blocked with TEA (K) and TTX (Na)
- stimulated the pre with an influx of Ca2+ current
- this lead to depolarisation in the muscle
- concluded that depolarisation in the post synaptic membrane was due to presynaptic calcium levels
What is the structure of voltage gated Ca2+ channels?
-structure similar to Na+ and K+ channels
-like Na+ channels, Ca2+ are monomeric (evolutionarily younger than K channels)
-4 subunits
-
What are the features of Ca2+ channels involved in synaptic vesicle release?
- resting Ca2+ in the presynapse is ~0.1μm (compared to 1.3 mM in the synaptic cleft)
- level required to trigger synaptic vesicle release is 5-10μm
- release sites are clustered around Ca2+ channels
Why is calcium a good trigger for neurotransmitter release?
Because of the difference in concentration inside and outside of the cell
Extracellular concentration ~1.3 mM
Presynapse concentration ~0.1μm
When stimulated and get a calcium influx, pretty much a digital signal, as there is a massive change very quickly in the concentration (often increases from 0.1μm to 200μm when only 5-10μm is needed)
Either ‘on’ or ‘off’
What is MEPP?
Mini End Plate Potential
How was it shown that neurotransmitter release is quantal?
-recorded from squid giant axon at the outer muscles
-stimulated the axon, and recorded from the post synaptic cell (the membrane potential), measured the end plate potential (EPP)
-discovered that without stimulation they were measuring constant, spontaenous MEPP, which had a constant amplitude of 0.4mV
-when the axon was stimulated weakly (Ca2+ dependent), they discovered that they got EPP in multiples of 0.4mV
=corresponds to synaptic vesicle volume, showing that neurotransmitter release was digital (either released or not)
-also removed all the calcium from the synaptic cleft (from 1.3 mM to 0) and found that the EPPs stopped - however that the MEPPs did not stop, showing that these happen via a calcium independent process
= suggesting vesicles are constantly ready to be released and spontaenously fuse and are released from the plasma membrane
What is the Ca2+ sensor (discovered by Tom Sudhof) in the presynapse, and what are the fusion machinery?
Sensor
-Synaptotagmin
Fusion
-SNARE proteins
What is the process of the synaptic vesicle cycle?
1 - NT uptake
2 - Translocation of the neurotransmitter vesicle to the presynaptic membrane
3 - Docking of the neurotransmitter vesicle via SNARE proteins
4 - Priming prefusion (requires ATP)
5 - Ca2+ influx leads to Fusion Exocytosis and the release of the neurotransmitter into the synaptic cleft
6 - Endocytosis of presynaptic membrane, vesicle membrane coming back in again
7 - Translocation away from the membrane
8 - Endosome fusion with the early endosome
8 - budding off the early endosome
10 - NT uptake
How was the synaptic vesicle cycle studied in the past and more recently ?
Heuser and Reese (1973), Ceccarelli (1972)
-used electron microscopy to visualise the synapse and pre/post
Viewed:
-lots of mitochrondria (ATP dependent process)
-release sites much darker
-docked vesicles ready to release
-defined amount of neurotransmitter in the vesicle
More recently - fluorescent techniques combined with electrophysiology have allowed the synaptic vesicle to be analysed in real time
How was the structure of synaptic vesicles visualised by Tim Roffman?
Mass spectrometry analysis
How many proteins are attached to synaptic vesicles and why are they necessary?
~200 proteins Why? -trafficking e.g. endosome to plasma membrane -fusion of vesicles -detect calcium -ATPase -proton pump
What makes neurotransmitter release Ca2+ sensitive?
SNARE proteins for fusion are Ca2+ sensitive
1 - Vesicle docks, synaptobrevin on the vesicle interacts with Syntaxin and SNAP-25 on the plasma membrane
2 - SNARE complexes form to pull membranes together
3 - Entering Ca2+ binds to synaptogamin forming a v. stable complex
4 - Ca2+ bound synaptotagmin catalyses membrane fusion
Give three examples of neurotransmitters
Acteylcholine (ACh)
GABA
glutamate
What are the fast and slow receptors of ACh?
Fast - Nitotinic
Slow - Muscarinic
What are the fast and slow receptors of GABA?
Fast - GABAa
Slow - GABAb
What are the fast and slow receptors of glutamate?
Fast - NMDA, AMPA kainate
Slow - mGluR
What is the major inhibitory neurotransmitter (30-40% of synapses)?
GABA released by gabaergic neurons
What is the major excitatory transmitter (>50% of synapses)?
glutamate released by glutamanergic neurons
What type of channels are fast/slow channels?
Fast = ligand gated ion channel Slow = G-protein coupled 'metabotropic' receptor
Why is the synaptotagmin Ca2+ complex v stable?
The energy that is used to form the complex is use to pull and fuse the membranes to gether
How do inhibitory (GABAergic) and excitatory (glutamanergic) neurons act on a recieving neuron?
The SUM of the activity of all the neurons (inhibitory and excitatory) firing on a recieving cell must be over threshhold to initate an action potential
What are threee diseases caused by mutations in Presynaptic Ca2+ sites?
Spinocerebellar Ataxia
Familial Hemiplegic Migrane
Episodic Ataxia
How can presynaptic Ca2+ channels be sites for modulation??>
G protein By inhibition on the I-II subunit linker
Synprint site on II-III subunit linker (makes vesicles next to channel, instant release)
Calmodulin facilitation on the end C terminus (Ca2+ binding protein, channels open more easily)
What are three therapies for epilepsy?
Gabapentin
Levetiracetam
Dynasore
What therapies have been involved in treating epilespy in the past?
Gabapentin
- inhibits voltage gated channels and GABA transmission
- many targets, e.g. voltage gaated sodium channels, GABAa receptor, GAT-1 GABA transporter, GABA transaminase
What therapies are currently involved in treating epilespy?
Levetiracetam
- SV protein
- SV2A target
- regulates/inhibits neurotransmitter release when there is overactivity
What future therapies could be involved in treating epilespy?
Dynasore
- inhibitors of endocytosis
- dynamin target
- endocytosis is only needed when high activity not for normal everyday, therefore inhibition is good
What is epilespsy caused by?
Overactivity in the hippocampus
