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5.8 Ion channels and membrane potentials VIII
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來自 Peking University 的課程
Advanced Neurobiology I
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從本節課中
Signaling within neurons & Ion channels and membrane potentials
Let's switch to the electrical properties of the neuron.
與講師見面
Yan ZhangProfessor
School of Life Sciences, Peking University
Yulong LiResearch Fellow
School of Life Sciences, Peking University
That is good but their electrophysiology can tell
you you have one ion channel there.
It doesn't mean there's no other ion channel.
It could simply mean other ion channels are not conducting, are not opening.
Because of either they are well or while you apply,
you apply a lot of drugs to block them because you are only specifically
looking at the ones that you care about.
And therefore, even with this Nobel Prize winning work, you can grab a channel.
You can still not able to distinguish it structurally,
even if you have the ways to grab the and record it and
then immediately put it in a crien to look at it.
You still cannot find out which one is which one.
Or maybe there's some dust, some dirty things,
that when you're moving these around that go with you.
How do you know which one is which one?
So here's the problem.
I agree with her that she has this very
great insight to say looking at a gene.
But you need to identify that gene, isn't it?
And even with the EN studies, it will be
important to identify the protein first.
For example, it's present a lot and then purified.
Make sure you only enrich the ones that you care about and
then to look at its structure, right?
Then how do you go about it?
So through our discussion since that, It's important to identify it,
the molecular basis for this channels.
That is to getting to know which one includes which gene and
what is the protein sequence, for those channels.
And then, once we can identify the protein sequence,
then we can sort of express it and then trying to understand structure.
So, how do we go about identification of those genes?
What do you think?
Finding the new genes.
We want to find out the genes that encodes what educated channels,
how we know their property from Huxley's work.
We already know that property.
They all have a unique property,
but how do we go from function to the gene sequence?
What do you think?
So we can propose that we can use the model organisms through a genetic screen.
Is that what you are talking about?
You said fruit flies, genetic trackable system.
And then if you, we can identify the mutant, and
then through the counting of their mutant gene,
then we know which ion channel it is.
But the problem is,
how do you screen which phenotype?
Here's we can all do screening, at least in our brain.
We can go to screening, but with fruit flies,
which is a very powerful genetic model organism,
you can do a lot of sort of EMS render screening or insertion screening,
and then you can relatively easily to [INAUDIBLE] mutant gene.
But what kind of phenotype you are screening for?
How do you know which one is mutant?
What kind of mutant phenotype you are looking for?
Again, in 1984.
So [INAUDIBLE]
from the electrical fish, okay.
And this is, as Clay Armstrong called it, it's one of the Holy Grail.
Knowing in 1950s, 1952 to be exact,
the sodium potassium interpolate to lead to action potential.
And after 32 years, the first
sodium channels sequence was identified.
And so Sacu Newma again is really
breeding away his lab single handedly
Calcium channel.
So literally, in the 1980s, his lab is one after another,
in high profile journals that are publishing the important
results identified the molecular basis of those channels.
Well some faculty at least at on PKU are just
grad students in Stanford University and
those faculties one or four two like right.
We chat and we would always get shocked by how many papers,
Newma publishing in nature per year.
So, how did he identify this?
This is one of the works Affinity Purification.
It turns out, and we also know that sodium channels
are very sensitive and high affinity to TTX.
So one principal is one can use a high affinity probe to put out or
to enrich enough materials for sodium channels and
they actually it's not Newma’s work alone.
Other people have already, especially with biochemists,
they use the affinity purification [INAUDIBLE] or column and
use the electrical organ of some special marine animals.
It turns out that the way that generally the huge wattage of current,
you also need to conduct those currents through ion channels so
purification of marine species that enrich in protein of those ion channels.
And you see affinity purification to purify the homogenize,
relative of homogenized, homogenous protein.
And from those proteins, then you can do some [INAUDIBLE] in terminal sequencing.
And from a protein, you can get to know some of the peptide sequence and
from a peptide sequence, and there is one [INAUDIBLE] and
Newma’s lab is really good at, at least around that time.
Almost his last single handedly [INAUDIBLE] probably 20 laps in the U.S.,
just cloning all those ion channels.
From those peptides, then you can generate those
degenerative prions that can be used to hybridize either the genomic sequence or
cDNA sequence of those gene encoding for
this protein and from the hybridization.
And then you can further along to come either part of the cDNA, or the full cDNA.
If you think about it, after [INAUDIBLE],
it turns out that this [INAUDIBLE] sodium channel,
it has 24 trans-membrane regions.
And this 24 transmember regions.
So this structure, you can see that they
can be classified into four parts, and
each four has about six trans membrane unit.
And independent [INAUDIBLE] by fenobenzoyl and
fenobenzoyl's [INAUDIBLE] and [INAUDIBLE],
actually more than one [INAUDIBLE] almost simultaneously using fruit fly genetics.
People identified in close to 1987 the potassium
channel that encode a mutant called Shaker.
So it turns out that this Shaker potassium channel was a screening
from a behavioral assay, and that fly tends to like to be paralyzed.
There is shaking in certain behavior aspects, so they call it a Shaker.
And once they code it from a fly mutant, Shaker be what in this case?
And then they do the sequence analysis.
It found that this Shaker potassium channel
is as if just has six transmembrane region and
this six transmembrane region are very homogenous
to one of the six transmembrane region of one of
the four units of the sodium channel.
So now people generally believe, and
come to agreement that is that,
[INAUDIBLE] sodium channel are the ancestors for
sodium and calcium channels.
And is the [INAUDIBLE] sodium channels during evolution,
they sort of have the gene fusion together to have
the full copy fused together that they generate
a wattage sodium and calcium channel.
And now here's the question.
After coming off the sodium channel or potassium channel, and
later calcium channel, because once you identify one,
it's just easier to identify the rest of the family.
Now will be the next question you want to answer.
We already proposed, somebody already proposed he or
she is going to look at the cryeen to look at its structure okay?
But it's the simple on paper,
it's difficult in reality.
I don't think, until now, people have already using the for
sodium or calcium channel to get a structure yet.
So, what would be the next question?
How do you know?
This is fascinating properties of wattage channels,
that is namely wattage dependency.
The iron channels selectivity at this.
And then there's also this interesting channel inaccuration,
that is, up to wattage jump.
It opens up a little bit of time, and then it will close.
What is the molecule basis of it?
Will be important questions that signed his to find out.
And then we are going to discuss them in our next section, okay?
All right?
And a task for today's work.
Well, the TA have already sent three questions to the WeChat group.
So please, for one week please turn that in to
the TA's and then they will grade.
The second, last time we asked people to derive
the GHK Goodman Hawking Kass equation.
For those of you who have not derived to answer the question set for
the WeChat group, you need to know that, okay?
So please try to figure out the GHK equation, okay?
And, our next lecture is going to start with
what will be the important function of the ion channel.
What will be the molecular basis and
how do you design experiments to find that out?
Think about it.
What will be the wattage dependence it comes from?
What will be the ion channel selectivity comes from.
What will be the inactuation property comes from.
Then we are going to ask students to join me, to propose an experiment.
How do you design the right experiment to test the molecular basis?
