iGluR 1 Flashcards
What is the structure of iGluRs?
ATD (No effect if deleted)
LBD
TMD
CTD
What iGluRs is glutamate an agonist for?
AMPAR
KAR
GluN1
What iGluRs is glycine/D-serine an agonist for?
GluN1
GluN3
How can we determine the structure of iGluRs using X-ray crystallography?
- Sample crystalized
- Make an electron density map
- Make an atomic crystal structure model
What are the pros and cons of X-ray crystallography?
:) High resolution
:( Uses crystals
How can we determine the structure of iGluRs using cryo-electron microscopy?
- Sample is frozen in an electron microscope
2. Make 2D pics > 3D map > 3D model
What are the pros and cons of cryo-electron microscopy?
:) Can use single particles
:( Difficult to get high resolution
How did we work out that iGluRs are tetramers?
- Electrophoresis and sedimentation used to separate proteins from neurons
- Western blot analysis used to show AMPAR formed tetramers
What are the subunits in AMPAR, KAR and NMDAR tetramers?
AMPAR: GluA2 assembles with 1, 3 and 4
KAR: GluK 4 and 5 assembles with 1, 2 and 3
NAMDAR: 2 GluN1 with 2 GluN2 or with GluN2,3
What did they discover in 1950 and 1970 with regards to glutamate?
1950: injected into brain = convulsion
1970: injected into brain = depolarisations (thought they were ion channel)
What did they discover in 1970 and 1980 with regards to glycine?
1970: NMDAR + conditioned medium = depolarisation
1980: realised it was glycine
How did they discover that NMDA and glycine are co-agonists and when?
1988:
1. Extracted RNA from rat brains
2. Injected into frog oocytes
3. express for few days
4. measure electrophysiology of cells with NMDA and Gycine = large depol
What did they find in 1979 and how?
- Rat brain slices isolated and synaptic membranes washed
- Add kainate and glutamate serum = little radioactivity because they compete for same site
- Add kainate alone = lots of activity
- Add kainate and NMDA = lots of activity because they have different sites
What blocks NMDAR and when did they find this?
- Magnesium
- 1980
Describe ion permeability in iGluRs
- AMPAR: Na, K, Ca (least)
- KAR: Na, K, Ca
- NMDAR: Na, K, Ca (most)
Describe an experiment to test antagonists of non-NMDAR
- Stimulate synapses = depolarisation
- CNQX + YDDG antagonists given = no depolarisation.
- AP5 = depolarisation
Conclusions: CNQX and YDDG are non-NMDAR antagonists whilst AP5 isn’t
Describe an experiment to test antagonists of NMDAR
- NMDAR given tetanus = high depol (LTP)
BUT - When AP5 is given followed by tetanus = normal levels of depolarisation
Therefore, AP5 must be a selective inhibitor of NMDAR not non NMDARs.
How can we stop seizures (give an example)?
- Block glutamate receptors
e. g. block AMPAR with CNQX
Where are different AMARs found in the brain?
GluA1 = not in entorhinal cortex GluA3 = in hypothalamus and amygdala GluA4 = olfactory bulb/cerebellum/auditory pathway
What type of neurons are permeable/impermeable to Ca?
Interneurons - permeable
Principle neurons - impermeable
Where in the brain are different NMDARs found?
- GluNB (embryonic) and GluN2A (adulthood) in the hippocampus
- GluN2C (adult) and GluN2B (embryonic) in cerebellum
What is the iGluR channel conductance equation?
G = i/V
G = conductance (ability of channel to pass current) I = current V = voltage
NMDAR has higher conductance current vs AMPAR
Describe microscopic currents
- Caused by rapid transitioning of opening/closing channels
- Detected using patch clamp technque
- Difficult to record tho cos its so small
Describe macroscopic currents
- Caused by activation/deaction (when agonist binds/comes off = conform change)
- Desensitisation = when agonist binds to closed channel but it is still stable
- Resensitisation = channel stays shut but becomes unstable so agonist comes off
DIDN’T INCLUDE:
- CLONING
- MEASURING GENE EXPRESSION IN NEURONS
