The Synapse 2

Question 1

What does the guanylate like kinase domain of PSD-95 interact with in the PSD?

Answer 1

Guanylate kinase associated protein (GKAP)

Question 2

What type of experiment discovered that GUK interacts with GKAP?

Answer 2

Yeast two hybrid

immunohistochemistry shows colocalisation

Question 3

What does GKAP bind to in the PSD?

Answer 3

Shank

Question 4

What proteins does shank bind to?

Answer 4

GKAP

Homer

Question 5

What domains does Shank have?

Answer 5

Ankarin repeats
SH3
PDZ
Proline rich sequences
SAM

Question 6

What does the ankarin repeats in shank allow for?

Answer 6

Association with cytoskeletal proteins

Question 7

SH3 domains interact with….?

Answer 7

Proline rich sequences

Question 8

What domain on SHANK does GKAP bind to?

Answer 8

The PDZ domain

Question 9

Which does the proline rich region on shank allow for?

Answer 9

Interaction with homer and therefore indirectly to mGluRs

Interaction with cortactin which links to cytoskeleton

Question 10

The SAM domain of shank is important for….?

Answer 10

Shank mulitmerisation

Question 11

Give the three examples of the important evidence of scaffolding proteins

Answer 11

Couple of NMDARs to nNOS(alpha)
Homer permits coupling of mGluR to the IP3 receptor complex
AMPARs and stargazin and role in LTP

Question 12

Why is nNOS located in the PSD?

Answer 12

It can bind to psd-95

Question 13

Localisation of nNOS to NMDAR’s permits signalling that is?

Answer 13

Specific
Rapid
Efficient

Question 14

Briefly explain the significance of NMDAR localisation to nNOS

Answer 14

nNOS requires the calcium calmodulin complex to make NO
NMDARs are permeable to Calcium
Much NO can be produced

Question 15

Why is NO incredibly hard to measure?

What can we measure instead to study NO signalling?

Answer 15

NO has an incredibly short life span

Measure cGMP which is a molecule made down stream to NO signalling

Question 16

Who did the experiment which shows nNOS interacts with psd-95?

Answer 16

Brenmann et al 1996

Question 17

What does impairment of psd-95 and nNOS coupling do to levels of cGMP?

Answer 17

Decreases levels of cGMP

Since NO is neurotoxicity these neurons also survive longer with increasing concs of NMDA given to cell

Question 18

What happens to the NMDAR current with mutant psd-95 which cannot function?

Answer 18

NMDAR current is uneffected as psd-95 is not needed for postsynaptic localisation

Question 19

What are the different types of homer subunits?

Answer 19

Homer 1(a,b,c)
Homer 2(a,b,c)

Question 20

What makes homer 1a unique

Answer 20

Only contains the EVH1 domain

All other isoforms contain EVH1 and the leucine zipper + coiled coil domain

Question 21

What sequence of amino acids does EVH1 bind to?

Answer 21

PPXXFR
The c terminus if mGluRs have a proline rich motif
IP3 receptor also has a conserved protein rich motif

Question 22

What does homer allows mGluR(1-5) to couple to?

Answer 22

The IP3 R on the SER

Allow release of calcium

Question 23

mGluR 1-5 are coupled to which G protein?

Answer 23

Gq

Question 24

When talking about homer who will we reference?

Answer 24

Tu et al 1998

Question 25

What happens to homer 1a expression on increasing synaptic activity?

Answer 25

It increases

Question 26

What does increasing homer 1a expression mean?

Answer 26

Reduced mGluR to IP3r coupling as homer1a cannot multimerise

This will decrease calcium mobilisation on subsequent synaptic stimulation

Question 27

In what cells is this change in homer expression important?

Answer 27

Purkinje cells in the cerebellum

Important for LTD

Question 28

What did Chen et al discover?

Answer 28

Stargazin immunoprecipitates with psd95

Did this in 2000

Question 29

Prior to a high frequency stimulus, what is the difference between the EPSP between mutant psd-95 mice and WT?

Answer 29

Mutant mice show slightly diminished EPSPs

Question 30

What construct of mutant psd-95 were used by migaud et al in there experiment?

Answer 30

Psd-95 with only pdz 1 and 2

Since stargazin binds to pdz 3 they do not couple together?

Question 31

What was the purpose of the experiment by migaud et al 1998?

Answer 31

To observe is mutant psd-95 effects synaptic plasticity in a murine model?

Question 32

What did migaud et al 1998 discover after HFS for mutant and WT mice?

Answer 32

Mutant psd-95 show greater potentiation (increased sensitivity to a potentiation stimulus)

Question 33

What does an increased sensitivity for potentiation in the psd-95 mutants mean?

Answer 33

That there were a low number of AMPARs at the post synaptic membrane prior to the HFS.
I.E. There is greater potential for LTP in psd mutants
Stargazins interaction with psd-95 is essential for AMPAR localisation it membrane

Question 34

On an stimulus (1Hz) designed to cause LTD what happened to the EPSPs of the mutant mice?

Answer 34

They showed LTP

Question 35

In the Morris water maze what did mutant mice show? What does this mean?

Answer 35

They showed impaired spatial memory

Therefore, LTD is important for memory too and it is likely memory depends on bidirectional plasticity of neurons

Question 36

Why is psd-95 sufficient by not necessary for AMPAR localisation to synapse?

Answer 36

1. Even though psd-95 was non functional, mutants were still able to potentate the synapse (meaning they must have got to the post synaptic membrane in a psd-95 independent way)
2. Overexpression of psd-95 increases the amount of AMPARs made from GluR1 at the synapses. This means that there is less potential for LTP as it is saturable. Seen experiemtnally

Question 37

(FURTHER READING)

Name another protein which interacts with pSd-95 and state it’s importance

Answer 37

SynGAP which is activated by CaMKII
activates intrinsic GTPases of ras proteins making them inactive

Localisation to NMDARs important for efficient synGAP function (mediating LTP)