Lecture 4 - Molecular Postsynaptic Plasticity Flashcards
What does an ionotropic glutamate receptor look like?
- 3 transmembrane domains, 1 sits in the membrane (M2)
- extracellular ligand binding NH3 from M1
- Intracellular C terminus from M4
Features of NMDA receptor
- Can be blocked by Mg2+
- Gates sodium and calcium
- Inhibited by AP5 OR mk8o1
What can you use to inhibit AMPA receptors?
NBQX
What is the Mg block on the NDMA receptor dependent on and what is this for?
Resting membrane potential
So you need glutamate, glycine and depolarisation to open it
What is yeast 2 hybrid?
Tests protein protein interactions
- Have a bait protein attached to half a TF
- Make a library expressing different proteins (prey) attached to half a TF
- If the protein binds the TF forms and get a blue readout
What did Kim and Sheng (2004) discover and how?
Used Y2H to show there is lots of proteins with PDZ domains that bind to the C terminus of the NDMA receptor showing there is lots of intracellular signalling going on in post synapse
What did Seth Grant do?
Characterised all the proteins under the post-synaptic membrane by using an antibody to the AMPA receptor and pulled out 200-300 proteins that make up the post-synaptic density
What disability is associated with 6 mutated proteins in the PSD? Examples of proteins
Autism
SynGAP, Shank
Outline an LTP experiment
- Record from cell body of CA1 pyramidal cell
- Stimulate axon from CA3 to CA1
Why do you record from the cell body and not the synapse?
A synapse is 1 micron across so its too small to record from
What is postsynaptic LTP also known as?
Schaffer collateral-CA1 LTP
What is MK801
An inhibitor of the NMDA receptor at a particular voltage which wipes out learning
What was the 90s model of LTP?
- Depolarisation ejects Mg and opens NMDA receptor
- Ca entry activates second messenger pathways
- Cell becomes more sensitive to glutamate and retrograde signalling causes enhanced neurotransmitter release
What else was found about LTP in the 90s which was strange?
LTP causes fewer synaptic failures (when the synapse doesn’t fire) which suggested some synapses didn’t have receptors in them
How were synapses shown to develop?
NMDA receptors come first followed by AMPA
What experiment did Lledo et al (1998) do?
- Stained AMPA receptors green, NMDA red and this makes yellow when mixed
What were Lledo’s results and what did this suggest?
Never had AMPA receptors with out NMDA but had NMDA on its own.
Suggested LTP traffics AMPA receptors to the membrane
How did Lledo’s results explain silent synapses?
The NMDA on its own would have Mg blocking it.
What treatments did Lledo then use on the synapses to block exocytosis
- NEM inhibits NSF
- N19
- Botulin toxins cut SNAREs
What did blocking exocytosis show?
Prevented AMPA receptors going into the membrane and activating synapses
What is immunogold electron microscopy?
Way to image proteins
Have an antibody to protein, then a secondary antibody has gold particle.
Can use different sizes of gold to differentiate between proteins
How did Shi et al (1999) show AMPA receptor exocytosis?
GFP to AMPA receptors and looked at silent synapses before and 30 minutes after applying tetanus
What proteins are involved in endocytosis of AMPA receptors in LTD?
Calcineurin, p38, Rap, Arc
What does Richard Huganir do?
Makes phosphorylation mutant mice.
What 2 proteins did Richard Huganir show bind to the PDZ domain on the C terminus of AMPA receptor?
PICK1 (protein interacting with PKC)
GRIP1 (glutamate receptor interacting protein)
What do PICK1 AND GRIP1 do?
They fight for binding to the PDZ domain.
When the receptor is in the membrane GRIP1 stabilises it
When the receptor is internalised grip falls off and PICK1 pulls it in
What happens when you remove the last 7 amino acids from the end of the C terminus?
No LTP and no LTD
What is the mechanism for changing from GRIP to PICK?
Phospho-switch.
PKC phosphorylates GRIP causing it to let go, and phosphorylates PICK causing it to pick it up
