CJH332H1 Lecture Notes - Lecture 13: Protein Phosphatase 1, Ampa Receptor, Protein Phosphatase 2

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25 May 2018
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Lecture 13: Synapsefulness Meditation
Moving receptors does that actually happen while animal is
learning? (not checked after the mouse is dead)
NMDARs as Coincident Detectors
Vast majority of our receptors are glutamate receptors (80
glutamate, 20 GABA)
NMDA and AMPA are coincident detectors
The basis for this
A) Early development silent synapses (before you are born)
- Only NMDA present cannot respond since at rest, it is
blocked by Mg
- Although glutamate binds, the channel cannot open
B) After birth functional synapse
- The movement of AMPA is directed towards the silent
synapse
- At the presence of glutamate, NMDA is capable of
responding (opening up channel)
C) Synaptic strengthening same amount of glutamate released but
bigger response on the postsynaptic cell
Are these synapses in other animals and is it possible to convert
NDMA to functional? (after tetanus, etc)
Molecular correlates of LTP NMDAR
Activation of NMDARs allows Ca2+ entry into the dendritic spine
- NMDARs are the only glutamate receptors that allows the influx of Ca2+
Ca2+ activates CaM (calcium-calmodulin)
CaM in turn activates CaM-dependent protein kinase II which is auto-
phosphorylated enabling it to stay active after Ca2+ have returned to rest
- Signal remains after the inputs have stopped
Phosphorylation causes recruitment of AMPA to traffic to cell surface more glutamate responsiveness
AMPA receptors already on surface are phosphorylated to increase responsiveness
CaMKII LTP NMDAR dependent
CaMKII has 2 main effects on the synapse
- It phosphorylates AMPARs increasing their conductance
- It mobilizes reserve AMPARs to the membrane
Still somewhat controversial and contentious
The process of movement
LTP = insertion of AMPARs
LTD = removal of AMPARs (dephosphorylation)
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Both are NMDA and Ca2+ dependent but the processes differ
In LTD the Ca2+ may activate phosphatases such as PP1 (protein phosphatase 1) AND PP2A and calcineurin (all
phosphatases)
All Ca-dependent phosphatases block insertion and enhance endocytosis of AMPAR on cell surface
AMPA receptors must move into certain position after input into the surface
Tracking receptors using antibodies
Key points in visualizing proteins through staining
Subcellular localization of proteins?
Epitopes
GFP itself is difficult to see whether receptors are inside or on the surface
Triton x-100 (non-ionic detergents) are used to punch holes in the cells to allow antibodies to move across the
membrane (usually cannot move across membranes)
Creating tags
1990s and early 2000s saw the investment in a series of different artificial epitope tags
Allowed for easier visualization of the receptors
- Where do you tag them and why cant you use other traditional markers?
GFP tagging of proteins to see expression
GFP is limiting even newer variants such as photoactivatable forms (PA-GFP)
First series of tags PCR based
One of the subunits of AMPA receptors (heterotetramers) have HA-GluR2 (newly GluA2)
GluR2 is the old name for GluA2
Used tags to track the receptors subunits
- C-Myc (EQKLISEEDL)
- Hemagglutinin (HA) (YPYDVPDYA)
- 6 His tail
- FLAG (DYKDDDDK)
HEK cells antibody labeling/staining
Human Embryonic Kidney cells used looks like neurons (similar family)
- Have proteins needed for AMPA insertions, endo/exocytosis of receptors
Two different antibodies to know movement of receptors permeable and non-permeable stain
- Antibody tags GluR1 and R2 on the surface (no holes in the membrane)
- Antibodies also inside (using triton x-100)
Shows that there is two different subcellular localization but not movement of receptors
To see the movement:
- Label the receptors on surface with antibody (freeze at 4 degrees for no movement)
- Endocytosed due to antibody binding favors endocytosis of these receptors
- Stain cell surface in non-permeable state
- Stain cell using triton to punch holes in membrane with different color (internalized receptors stained)
- This shows LTD receptors recruited INSIDE
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Document Summary

Lecture 13: synapsefulness meditation: moving receptors does that actually happen while animal is learning? (not checked after the mouse is dead) Nmdars as coincident detectors: vast majority of our receptors are glutamate receptors (80 glutamate, 20 gaba, nmda and ampa are coincident detectors. The basis for this: early development silent synapses (before you are born) Only nmda present cannot respond since at rest, it is blocked by mg. Although glutamate binds, the channel cannot open: after birth functional synapse. The movement of ampa is directed towards the silent synapse. Molecular correlates of ltp nmdar: activation of nmdars allows ca2+ entry into the dendritic spine. Signal remains after the inputs have stopped: phosphorylation causes recruitment of ampa to traffic to cell surface more glutamate responsiveness, ampa receptors already on surface are phosphorylated to increase responsiveness. Camkii ltp nmdar dependent: camkii has 2 main effects on the synapse. It mobilizes reserve ampars to the membrane: still somewhat controversial and contentious.

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