CJH332H1 Lecture Notes - Lecture 13: Protein Phosphatase 1, Ampa Receptor, Protein Phosphatase 2
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)
find more resources at oneclass.com
find more resources at oneclass.com
• 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
find more resources at oneclass.com
find more resources at oneclass.com
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.