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7.11 Molecular mechanism involved in neurotransmetter release VII

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Advanced Neurobiology I

Universidad de Pekín

3.9 (271 calificaciones) | 29K estudiantes inscritos

Hello everyone! Welcome to advanced neurobiology! Neuroscience is a wonderful branch of science on how our brain perceives the external world, how our brain thinks, how our brain responds to the outside of the world, and how during disease or aging the neuronal connections deteriorate. We’re trying to understand the molecular, cellular nature and the circuitry arrangement of how nervous system works. Through this course, you'll have a comprehensive understanding of basic neuroanatomy, electral signal transduction, movement and several diseases in the nervous system. This advanced neurobiology course is composed of 2 parts (Advanced neurobiology I and Advanced neurobiology II, and the latter will be online later). They are related to each other on the content but separate on scoring and certification, so you can choose either or both. It’s recommended that you take them sequentially and it’s great if you’ve already acquired a basic understanding of biology. Thank you for joining us!

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3.9 (271 calificaciones)

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SH

Dec 19, 2018

An informative site which provides bunch of information.

SB

Jul 27, 2018

This was a true challenge and magically eye opening!

De la lección

Neurotransmitter release

Let's learn more about the neurotransmitter release.

7.1 How does Ca2+ control neurotransmitter release I8:23

7.2 How does Ca2+ control neurotransmitter release II11:46

7.3 How does Ca2+ control neurotransmitter release III7:27

7.4 How does Ca2+ control neurotransmitter release IV10:18

7.5 Molecular mechanism involved in neurotransmetter release I7:56

7.6 Molecular mechanism involved in neurotransmetter release II7:11

7.7 Molecular mechanism involved in neurotransmetter release III5:27

7.8 Molecular mechanism involved in neurotransmetter release IV11:00

7.9 Molecular mechanism involved in neurotransmetter release V14:23

7.10 Molecular mechanism involved in neurotransmetter release VI12:35

7.11 Molecular mechanism involved in neurotransmetter release VII17:53

7.12 Molecular mechanism involved in neurotransmetter release VIII12:40

7.13 Molecular mechanism involved in neurotransmetter release IX7:59

7.14 Molecular mechanism involved in neurotransmetter release X13:25

Impartido por:

Yan Zhang
Professor
Yulong Li
Research Fellow

Transcripción

We are going to switch gears to talk about another independent line of

evidence mainly lead by biochemist Jim Rothman, James Rothman.

Originally at that time was in Sloan-Kettering,

right now in Yale University.

And he independently identified the key components for transmitter release.

And again, I'm going to provide some background about his research and

then it's your turn to design experiments to fuller understand our process, okay?

So the title of his work is SNAP receptors implicated in

vesicle targeting and fusion, okay?

And look at the time,

this is March 1993, okay?

And the previous paper that we identify,

this is 1992, okay?

So the James Rothman's paper, again this is both of them are in nature.

James Rothman's paper is just half a year later than this one, okay, in nature.

Okay, let me just provide you a little bit background James Rothman, okay?

Who recently got a Nobel Prize by his work, okay?

And you can look at his sort of an educational background,

essentially collecting all the good training in famous universities, right?

Bachelor degrees in Yale, PhD in Harvard,

post-art in MIT and

then first become a faculty, assistant professor in Stanford.

And then become a chairman at Stanford and

then move to New York in the Sloan-Kettering.

And then be in Colombia as a faculty and then before getting Nobel Prize,

he moved back to Yale, okay.

So he's basically in good places okay.

And then this is before his Nobel Prize, he has been awarded many prizes.

Okay, so next time you can be predicting how a person can win

Nobel Prize by counting how many other awards they get.

Okay, and the most important one would be the Lasker Award, right?

Once a person get a Lasker Award, then more than about 50% of the time,

he or she might get Nobel Prize, right?

And this happened with some Chinese.

Okay, so let's just discuss a little bit the approach he did and then you

guys need to design for these parameters and how to interpret the results.

So James Rothman, he's very interested about the again,

the molecular mechanisms controls the vesicular fusion.

Initially, no transmitter release, but

a general principle controlling the membrane packet in a fusion.

Okay, in all the cells,

there is [INAUDIBLE] of the membrane protein from the circulatory pathway.

From the [INAUDIBLE] to the plasma membrane.

Okay he's very interested how those for example.

Can only specifically deliver to the plasma membrane

membrane to deliver some sort of membrane protein on surface rather

than the will be infused with other like mitochondria,

like [INAUDIBLE], like other organelles, you know?

There must be some specific controls for those trafficking,

if not then the cell will be in a bad condition.

All of that component will be randomized.

So he's trying to understand that.

And so his previous work, and

that is how the past that I'm going to provide you the background.

So previously he's about chemist.

Let's just take five minutes break, okay?

Now, let's get back to the James Rothman's work, okay?

James Rothman, he is a very smart and established biochemist.

He's interesting used biochemical approaches to identify

the key molecular components control the.

In his previous studies, he already

identified important factor called NSF, okay.

That is very critical for membrane to fuse with past membrane, okay?

How he do that?

He's a biochemist, so he used

ingredients to isolate the gorgi vesicle,

and isolated component passed

the membrane extract, okay.

And here is the experiment, he designed an essay.

He put enzymes inside the Golgi vacuoles.

And pour substrate inside the plasma membrane components, okay?

And only when these two fills,

the enzyme will meet in the substrate and then he can see the reaction, okay?

So in the essay he found that there is

ATVs, NSF is record.

How did he do that?

Well, he found out using the Golgi and plasma membrane and

if you simply put them together they will not fuse, but

rather on top of this you add a cytoplasm

extract, cytosol extract.

Okay, basically,

some components inside the cytoplasm.

And with ATP and GTP,

then these two will fuse, okay.

As long as when this extract works.

And then if this extract contains a protein,

he can isolate the extract into different components.

You can separate the extract by molecular weight.

Okay, by the by the by the chemical sensitivity,

okay so basically we can separate this extract into individual components.

Eventually he found a very critical component, NSF, ATPS,

but it was very critical for the [INAUDIBLE].

That's all his, more than 15 years work in five minutes, okay.

He found the NSF, okay.

But this NSF is an ATPase, okay.

But it's a cytosolic protein, meaning that it doesn't not bind to membrane, okay.

How do they work?

How does it work?

The problem is if you want to get membrane to fuse

you only have a cytoplasmic protein.

It needs to somehow attach tot he membrane,

somehow handle the membrane to cause membrane to fuse, okay?

So, he hypothesized there will be

an important receptor for NSF, okay?

That there will attach to a membrane, okay?

So, this NSF is a cytocolic protein,

you need to work on some membrane components to cause membrane fusion.

That will make sense, okay?

That's all he know and that's how he design experiment.

Okay let's see how he do that and

then you guys can help me to answer why he do that in this way.

Okay so he identified NSF okay.

So in his experiment he wants to identify what is the receptor

in the plasma membrane?

So how did they do it?

Well first for the identification what they did was

they first enrich the NSF-Myc, okay?

So what is Myc?

Myc is the is after that you confuse with NSF.

And then you can recombinantly express this protein for example in coli.

You get a lot of proteins, okay.

And there you can get this commercial available link with the antibody, okay?

So, once we use this piece, you can enrich NSF by to this piece, okay?

And previously they already identified snap, this two proteins.

Actually, they are the accessory protein or

the binding protein for the cofactor for NSL that they already know, okay?

For NSL to work, it requires the snap, okay?

So then one idea is they are trying to use this.

Please to put out NSF, this SNAP, okay?

And then they want to add some print,

some cell extract to put on the seeds, okay.

Similar as some of the students say, I just need to okay?

And then they want to identify which things they put up, okay?

So, now here's the question.

When they use this antibody,

they add the and they add the.

Why do they want to ATP gamma s and EDTA why do they want to do that?

Okay, that's great so it's exactly like that.

So NSF is ATPA we already talked about this,

we already talked about we talk about kinase.

Whenever you have an enzyme and

how it works with suction.

Usually its a reversible process, the request turnover right?

So for example the proteins will cut the substrate and

the substrate will get released, the LSF is ATPAs.

Why it does just like any ATPAs, you will catalyze the hydrolysis of ATP and

then undergo some confirmational changes to a fair substrate.

Usually we'll break the substrates or

make the substrate undergo some confirmational changes, okay?

So if you are trying to identify the substrate for

NSF, in this case the receptor for NSF.

Then, In the presence of ATP we will always turn over okay?

Has ATP, you will change the cell substrate, and

a new cell substrate coming, and then you will get changed.

So you will have a low affinity fighting to the substrates, okay?

So what it did is using the ATP analog.

ATP-gamma-S, okay?

ATP has three phosphate, okay?

The gamma is the part that one

of the oxygens has been replaced with sulfate, S, okay?

So it is the perfect analog for ATP, except

this ATP comments because the S has been replaced, you know, with all the oxygen.

It cannot follow be hydrolyzed, okay?

It can feed it in to the ATP by the pocket to allow, to the,

[INAUDIBLE] to adopt a confirmation to buy to the [INAUDIBLE].

It will not follow get hydrolysis to restop actual original state,

so in essence, the ATP is a lock into an active

confirmation just to grapple the substrate, okay?

In a irreversible manner, okay?

And the way they added the EDTA it's, I think,

it's as somebody pointed out, let, this ADPA will [INAUDIBLE] magnesium.

Okay, because when you synthesize ATP [INAUDIBLE].

Maybe there's some contaminant of ADP.

Or maybe the cell, since you are going to get something extra from the cell.

The cell might contain some ATP, okay?

So, they added the EDPA to all the magnesium, and magnesium, ATP,

they work together to work as a cofactor for the NSF, okay?

So, in this case they can lock the LSF in this active confirmation and

then they want to put out this component, okay?

And how did it get this extract, okay?

Get this extract,

what are you going to do, where are you going to get this extract?

Okay, great.

First you need to get a lot of material for

biochemical analysis you need to have a lot of material.

The reason being, the proteins, they are not regenerative,

they are not like PCI you can amplify the DNA.

Well now we lose some proteins, they get lost, okay.

And, secondly, you neet to retent the membrane fraction.

How do you do it?

Because that we, want to study the receptor,

they caught, in the membrane that, been recognized by NSF.

Okay so when they extract the component,

they got to attend to the membrane faction.

How do they do it?

Well you can use some special detergent that

can solubilize the membrane component to retain their confirmation okay?

Detergent.

That is hydrophobic and hydrophilic in a way that solublize the membrane and

including protein components to keep their native structures, okay?

This protein?

Yes indeed you can get a cultured cell.

But what it did interestingly that they get from

a brain not why do they wash magnesium ATP gamma s and

saying this is a non-specific elulate okay?

Why do they want to do that?

Okay so the reason that we are using ATP gamma S,

because as we mentioned ATP gamma S is the analog of ATP.

That it cannot be hydrolyzed in by ATPAs.

Okay, so even if you add the magnesium ATP gamma S.

We were trying to wishing with it, the sense that the key is loaded,

would be the non specific sense of vice to it,

because the ATV [INAUDIBLE] exist, you will still lock the NSF

[INAUDIBLE] confirmation to buy to the specific packet, okay?

Only the non specific packet which cannot be hydrolyzed.

That it will get eluded.

And then they will add excess amount of magnesium ATP.

Because ATP and ATP compounds are analog are similar right?

So ATP will compete out, its going to replace.

And once the ATP replace the the ATP can be hydrolyzed by the ATPAs and

then the NSF is going to undergo some conformational changes to release,

to go down from an active state, to release the binding component,

and this would be the specific.

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