Huganir article

Question 1

Why did they use drugs? What drugs did they use?

Answer 1

They used isoproterenol, forbol ester (PMA) and forskalin (FR) to increase the phosphorylation of the GluA1 receptors.

Question 2

What is the pathway of Forskalin?

Answer 2

Forskalin activates adenylyl cyclase, which converts ATP to cAMP. cAMP activates PKA. PKA phosphorylates its targets.

Question 3

What is the pathway of Forbol Ester (PMA)?

Answer 3

Forbol ester acts like DAG and directly activates PKC without the use of calcium.

Question 4

What is the pathway of isoproterenol? What is the result of this pathway

Answer 4

Isoproterenol binds to beta-adrenergic receptors, which is its GPCR. Which activates the same responses as forskalin does. PKA binds beta-adrenergic receptor kinases, which activate the beta-adrenergic receptors. They phosphorylate the beta-adrenergic receptors.

Question 5

What is isoproterenol an analog of?

Answer 5

Epinephrine

Question 6

What is an advantage of isoproterenol?

Answer 6

It is a natural way of stimulating Adenylyl Cyclase because it activates a G protein.

Question 7

Why do the researchers use an Ab against VGLUT1?

Answer 7

VGLUT1 is a glutamate transporter and is everywhere in the spines, and it packages the glutamate inside the synaptic vesicles. You want its staining to overlap with the staining for the phosphoisoforms and the GluA1.

Question 8

Even if you see basal level of phosphorylation, why can’t you definitely conclude that you are seeing lots of phosphorylation?

Answer 8

You can have one strong Ab against one strong AMPAR that might be giving rise to all this fluorescence. Therefore proportion of phosphorylation vs amount of phosphorylation might not correlate

Question 9

How do the researchers know that the Ab 845 is specific?

Answer 9

Look at the figure in powerpoint.

Question 10

How do the researches know that the Ab 831 is specific?

Answer 10

Look at the figure in powerpoint.

Question 11

How do the researchers know that the CIP phosphatase works?

Answer 11

Look at slide 10

Question 12

Why is it okay that CIP only slightly decreased the FR staining?

Answer 12

You want to show that the staining is related to the phosphorylation and not anything else. So even though you want less staining, you decreased it with the CIP, which is what matters.

Question 13

How do you know the CIP doesn’t destroy proteins?

Answer 13

The other labeling procedures such as GluA2, VGlut1, were not altered by the CIP and they served as controls. So you know that the phosphatase isn’t destroying proteins.

Question 14

What is the purpose of the immunoprecipitation?

Answer 14

You can measure the proportion of the phosphorylated AMPARs to the unphosphorylated ones.

Question 15

Process of immunoprecipitation?

Answer 15

1. Have an input
2. centrifuge it
3. add antibodies to pull the phosphorylated AMPARs down. Add igG to serve as control where nothing gets pulled down.
4. Supernatant has unphosphorylated AMPARs. Pellet has phosphorylated AMPARs.
5. Measure proportions.

Question 16

What is the significance of the IgG control?

Answer 16

Gives you a baseline of how much total AMPARs you have.

Question 17

Why can you not compare the different antibodies to each other?

Answer 17

They each have different affinities for their proteins.

Question 18

What does it mean when the supernatant GluA1 decreases after FR and PMA treatment?

Answer 18

Figure E, slide, 12. It means that more GLUA1 subunits got phosphorylated and went into the pellet, since the supernatant only shows the unphosphorylated GluA1.

Question 19

Why is using CIP on fixed tissue hard?

Answer 19

Fixed tissue crosslinks proteins and makes them denature. Since phosphoatases can only do their jobs when they recognize a specific motif, phosphatases are not as efficient at recognizing the denatured tissues and they can’t reduce phosphorylation.

Question 20

What is the function of tubulin?

Answer 20

it is a loading control to ensure you have the same amount of protein in each condition. Therefore, any changes are related to the experiment and not other variables.

Question 21

Why did the researchers look at the pellet during the CIP treatment?

Answer 21

They wanted to see if there was a reduction in phosphorylated GluA1, which would show you how efficient the CIP is

Question 22

How does the CIP treated experiment raise the possibility that the AMPAR is phosphorylated on two sites?

Answer 22

When you pull down one phosphoisoform, the antibody against that phosphoisoform also recognizes the other one. Slide 15.

Question 23

What was different about the second experiment using CIP to test for dual phosphorylation? What can you conclude from this experiment?

Answer 23

Greater exposure times. You can conclude that there is double phosphorylation.

Question 24

Why did the antibody against p845 recognize the S831A in the pellet?

Answer 24

The mutation is only on the 831 site, not the 845. Therefore, the antibody against p845 knows that in that condition, everything besides 831 (including 845) is phosphorylated.

Question 25

Why should both inputs when mutating serines into alanines show the presense of GluA1?

Answer 25

Mutation of S->A does not prevent the expression of GluA1 receptor.

Question 26

Why did the researchers test double phosphorylation under denaturing conditions?

Answer 26

You can have 2 glua1 subunits that each have one phosphorylated site so that one glua1 never has two phosphorylated sites. Therefore, 1 glua1 is never doubly phosphorylated.

Question 27

What did the researchers conclude from the denaturing test for double phosphorylation?

Answer 27

They found that the graph C (slide 17) was similar to the previous one where they were testing just phosphorylation. This shows that individual subunits are doubly phosphorylated.

Question 28

How did the researchers induce LTD in the cultured neurons?

Answer 28

They added NMDA receptors without glycine, and kept the Mg2+ block in.

Question 29

What induces LTD?

Answer 29

Calcium influx through NMDA receptor.

Question 30

Why can calcium enter NMDA even if there is an Mg2+ block?

Answer 30

It is not glued into the receptor so calcium enters because there is a driving force on calcium.

Question 31

How did the researchers induce LTP in the cultured neurons?

Answer 31

They added glycine and removed the Mg2+ block.

Question 32

What did the researchers conclude after the LTP and LTD protocol?

Answer 32

LTD decreases phosphorylation and LTP increases phosphorylation.

Question 33

How you know if LTP increases phosphorylation?

Answer 33

Less is retained in the supernatant when they tested the iso version of LTP. Slide 19.

Question 34

How does EE affect serine phosphorylation in the PSD fraction?

Answer 34

It increases phosphorylation.

Question 35

How does EE affect serine phosphorylation in P2? Why?

Answer 35

Not much of a difference, but still an increase because the P2 is less pure since it is a membrane fraction.

Question 36

How should and how does EE affect other scaffolding proteins?

Answer 36

If you have LTP induced by EE, then you should increase scaffolding proteins. Which it does.

Question 37

Does EE increase serine 845 pplation through the immunoprecipitation method in the P2? How do you know?

Answer 37

It does, but the decrease is not significant. You have slightly less supernatant, but not enough to say that there is a significant change.

Question 38

What does the PSD fraction say about EE?

Answer 38

EE significantly decreases phosphorylated S845 in the supernatant, which means that EE generates more phosphoisoforms of GluA1, decreasing the amount of supernatant.

Question 39

Do the EE pellet fractions show evidence of double phosphorylation?

Answer 39

Yes. Go through slide 23

Question 40

How does Amphetamine increase LTP?

Answer 40

Amphetamine blocks the reuptake of norepinephrine, which activates beta-adrenergic receptors, which increases PKA, which increases LTP.

Question 41

Does amphetamine treatment increase p845 levels in mice forebrain membrane fractions? How do you know this?

Answer 41

Yes. Slide 24.
- Amphetamine treated GluA1 decrease in the supernatant, which means more are phosphorylated in the Amph condition than the Veh condition. This difference is significant.

Question 42

What can you conclude about the phos-tag SDS PAGE?

Answer 42

The Huganir group found phosphorylation in their Pho-tag SDS PAGE, like Hayashi group did. The only difference is that the present study found more phosphorylation in the control.