Mod-01 Lec-12 Bio electricity

Welcome to NPTEL lecture series on Bioelectricity.
So, we are into the twelfth lecture. So, the last lecture, we talked about the patch clamp
technique. So, we talked about the current clamp, we talked about the voltage clamp,
and I showed you graphically how you could have an access to finite number of channels.
And we talked about how we record the action potential that is basically varying the voltage
by injecting current which is essentially in technical term it could be called as current
clamp. Or voltage clamp where you are clamping the voltage at different level and you are
measuring the flow of current across the membrane. And after that I told you that there are several
techniques by which you can really manipulate these channels and study their voltage and
current or the electrical, over all the electrical properties. So, today we will be discussing that and we
will discuss about one more modification into the existing patch clamp. So, which is also
called planer patch clamp array. So, essentially what does that mean. So, before I get into
the channel, let us talk about the planer patch clamp array. So, let us draw
comparison between the existing patch clamp and the planer patch clamp. So, this is fairly
new technique. So, in the existing patch clamp what is happening that you have this electrode
which is coming from the top, and you have this three axis manipulator by which either
you can move the electrode like this, like this or like this or up and down. So, three
axis by where you can move the electrode. And this all you are doing either seeing the
electrode through a microscope from the top. So, essentially this is how it works.
So, if this is your micro manipulator, so this is where electrode is connected. This
can move like this, this can move like this, move like this. All the possible movement
are possible and here you have the cell, this is your cell and this is your electrode – E,
and this electrode can move up and down and likewise. And you are observing all these
things using a microscope either from top or from bottom depending on where your sample
is. If your sample is in a transparent sheet, you can see it from the bottom or you can
see it from the top. And a logistically speaking, this is very cumbersome procedure and it becomes
even more cumbersome when you have to out you have to give suction pressure.
So, for any specialized lab in this area, you first of all need an a need a specialized
electro physiologist, and on a given day that the best of the best efficiencies. They were
very small finite number of patch clamp recording, which could be done by any human individual.
So, what are our alternatives? One of the alternative which has been in the mind of
neurophysiologist as well as by electronics people could we automatized the whole process
somewhere or other. So, how to automatized the process, now that is where comes the whole
concept of automated planer patch clamp arrays, just like microelectrode arrays. These are
automated patch clam arrays. So, let us visualize situation what is happening,
here is a cell, you are approaching the electrode and you are creating a small hole on the top
of it, when you are going in to the holes cell mode. If you do not go in holes, so are
the other ways, how you can recreate that situation without approaching from the top.
So, one option is say for example, I have a substrate something like imagine a substrate
like this, where I have say one micron holes like this, and I ensures that something like
this, I ensure my individual cells are sitting on top of it like this. So, the cell is sitting
in three dimension on top of this small hole. So, where is small hole and ensuring that
the medium is not really flowing out because there is continuous supply of medium, which
is replenishing it or recalculating it. So, say for example, any medium which is present
there is getting which is coming out from there is kind of getting re-circulated into
the system back. And of course, fresh medium could be put here now underneath those small
holes you have imagine something like exactly like a patch pipette, you have already existing
structure which could be put and replaced something like that for simplicity saying I will be showing only three or four, so that you understand. This is come from underneath
all are from underneath. So, say for example, if this is the sheet
and top of that, imagine this watch is the cell what I have, this is what I am trying
to draw from underneath. So, such multiple things are underneath and exactly you follow
the same configuration inside that you have this electrode like this and you have the
ground electrode. So, this is connected to the amplifier. Now if you look at this configuration, what I have drawn here, and if I translate it in terms of patch clamp, you just reverse
this on the top. Imagine, it is coming from the top, it will be the again same configuration, here is the cell and here is the electrode. I just reverse the configuration to this,
so either you are here, it is the same configuration, it is just the upside down. Now you are approaching
the cell from the bottom. You really do not have to approach the cell it is already the
set up is already made. Now as soon as the cell touches on top of
this electrode, what you essentially you do is you follow the same protocol, but it is
completely automatized. So, at one point of time, you can provide it of course you could
ensure that the all the individual cells are sitting on top of those small holes, and that
could be done using modern lithography – part portal lithography where you can ensure that
you can ensure that only the cells sitting there. Say for example, so I put Yolo out
here, ensuring that the cells are not going to grow in these places. If I could ensure
that something like this, the cells will only sit on top of those small one micron volt
and if one could ensure that that essentially what you are what is happening is that now
you have a high through put planer patch clamp array just like high through put planer microelectrode array. So, this is one approach which is currently
underway specially in Germany, and some of the university in Germany, and some of the
companies has taken to over and there are fairly successful for cell lines where the
size of the cells are uniform and they could be put much more easily. But in terms of real
primary neurons directly taken out from that animal stilt it is possible, but it is not
a stilt full stream line there is enormous amount of work which is going on to ensure even that is feasible. So, what we are essentially seeing while summarizing this and of course again in this situation, you could have all the three different modes
you could have a hole cell mode where you have the electrode like this which is the
easiest one would you could see or you could have. Of course, one more thing, here I will
add. You may not be able to so easily study this kind of thing for individual channel,
where I showed you that the inside up out and all those things, where you have only
the membrane out there and you can study the membrane that may not be so easy enough. But again at least you could do high through put screening at least you do not have to you
know spend so much time for drug screening. Of course, if some of the drug really work
then you may go over and verified further using patch clamp, the regular patch clamp
arrays, where you know pull out the channels and you know study the channel dynamics and
everything. So, this is one of the most recent advancement of last five to ten years, I would
say slightly more may be you know of translating the traditional patch clamp into a high through
put screening system for especially this kind of things find application in the drug discovery industry. There it is being really one of the favorite candidate drug discovery and
toxin detection and and in the diagnostics; this is where this innovative technology or
designing problem or designing accomplishment finds a lot of applications. So, as of now we have talked about that we
could approach the individual channels. So, next what we will be talking about is these
individual channels, how those channels structure could be manipulated. Say for example, so
just before going that so whenever we are measuring individual channel in terms of its
something like this, what you see essentially is the channel opening and closing, you will
see something like this. These are single channel opening, what you are seeing. So,
these are the situation where you have this cell, and there is a patch out here, and you
have the finite number of channels out here something like this, and you are measuring
the conductance of individual channel. You can pull this out inside out or outside out
whichever ways you can measure the conductance of the individual channel.
What you essentially do, if really know the total number of channels on a you know within
this much area, you have this many then you back calculate and tell that for how many
channels you are getting it, form that you can back calculate and say single channel
how much will be the conductance. So, with this back ground of approaching the channel,
so I told you I am not getting the structure of the channels as of now, first of all I
want to introduce you to the channel how to measure channel electricity. Since now I have
introduced you the channel electricity, I will introduce you to one more technique which
will help you to appreciate the research of last 30, 40 years since the time patch clamp
has been discovered that how molecular biology techniques have helped in understanding bioelectrical
phenomena at the cellular level. So, whenever we talk about channels now I
will pick up the again let us see. So, this is the cell and we are talking about an individual
channel. So, this channel when you look at the molecular level, it is something like
a structure like, you know which we have already discussed in depth in detail something like
and you have the membrane both side of course running through like this. Now you have three
features, here you have a something called this zone, which is the selectivity pore.
So, this selectivity pore decides whether it will be sodium or it will be potassium
or it will be calcium or it will be you know chloride or whatever, then you have voltage
sensing element somewhere in this structure, which could sense the voltage. Voltage sensor and then this voltage sensor is somewhere rather is connected to a gate, which ensuresthe opening and closing, there is a movement in this. So, this is basically your gate. So, this is the overall channel architecture.
Now in terms of the molecular structure of this whole thing, this is the gross molecules.
In terms of the integrity details of the molecular structure if you look at it, so this is nothing
but this is a simple protein, which is occurred a shape like this. It could be a monomeric
protein, it could be a dimeric protein, it could be trimeric protein, it could be a tetrameric
protein, it could be a pentameric protein, it could be a hexameric protein likewise.
So, whenever we talk about protein, so what essentially this structure if it bussed down,
so it is basically there are amino acids like this. This individual circles are the amino
acids AA; and this is the peptide bond, which is attaching individual amino acid.
So, these amino acids join together and form this three dimensional geometries of large
large huge proteins, which are ten thousand, fifteen thousand amino acids structures. These
are fairly huge. So, how will you understand which part of this structure, so whenever
I am writing gate, voltage sensor, selectivity port which part of it is really involved in
gating or which part involved in voltage sensing, which part is involved in selectivity for
the specific ions, so how it is being determined. So, let us break down this problem – complete
problem into a array of amino acids first. So, it will be something like this. So, you
can break it down this AA stands for amino acids, and this is the protein. So, that there
is a N terminus of that protein, and there is a C terminus of protein. Now the way it
is being done is most tedious way. So, whenever we talk about this amino acids, they are coded
by the specific cordons. So, this structures are regulated by specific nucleotide sequences
from the nucleus from the DNA. So, now, the way it is being done individually replace
each one of them at a time or a chunk of them at a time or you remove them, delete them,
mutate them. So, mutate them means you replace it with something else, something like this
or you delete them, you delete a sequence. So, likewise you using mutation technique,
using deletion, using different kind of point mutation, replacing the amino acids. Over
forty years of research, now today we know at least for some of you handful of channels,
so in the mean time there are couples of things happens cloning as I was telling you discovery
by Cary Moilis which changed the way molecular biology is being done, the modern current
molecular biology. Then came the whole sequencing the first time it was Sishumunuma and all
these people who could you know sequence the whole channel. Once you this sequence then
you go back using genetic tools that you know that exactly how to mutate specific amino
acids. So, that way what you do now you have a control
on the structure. You can you can ensure, if this is a sequence of amino acids like
this, you can really ensure the this part is replaced or likewise or say for example,
this is involved. Let us take a single example, you know this sequence say for example, this
four amino acids one to the four amino acids are involved in same voltage sensing, just
for the hypothesis sake. So, what I do essentially is I kept on replacing each one of them one
at a time, and I express those mutated ion channels on some cell line, expressing those
mutated ion channels on some cell lines. And use those cell line in the mean time, cell
line technology was fairly straight forward now with that development over last forty years and the cloning and everything is fairly straight forward. So, I expressing mutated or genetically altered ion channels on cell lines. So, now, you have a cell lines which has genetically
altered ion channels. When you take this and you perform the electrophysiology, so that
where you will be able to figure out that how a specific change in a sequence or a mutation
at a particular part could influence its voltage sensing, could influence the selectivity port,
could influence the gate likewise. And it is a very very tedious process; as a matter
of fact, I mean think of it with in sodium channel there are so many subtypes, fast activated
inactivating sodium types; within them, there are types then you have slow activating channels.
Then you have so many potassium channels, then you have calcium, then you have chloride,
and even you have water channels, aquaperins. So, really to do it like this, and there is
not much other tool. Only the other tool which is available is bioinformatics tools where.
So, this is another thing which happen in middle mean time. So, you have electrophysiology going moving on. So, electrophysiology techniques where getting kind of electrophysiology and
going hand in hand with molecular biology tools; simultaneously came fairly late slightly
late in the game is something called bioinformatics, where you can start predicting the structure
function relationship by theoretical modeling. So, essentially you can tell the molecular
biologist that which particular sequence may should be or you can share with this both
of them which particular path may be involved in voltage sensing or gating or selectivity
port likewise and so on and so forth. So, if you see that timeline the way it is
moving, and if this is electrophysiology was there long time back, then came the molecular biology specifically with PCR, cloning, expression systems. And in the mean time, it is going
on hand with is toxicology, because as I was telling in one of the previous classes, you need certain specific compound which can block this channels. So, you have to have those
kinds of toxins like tetra detoxins, four a p trithyl-amonium likewise. So, toxicology
was also is which fairly old science moving with electrophysiology then you have the molecular
biology then you have bioinformatics coming into play.
Simultaneously there is another technique which people are on the structural set, this
is all about the functional aspect of ion channel. So, this functional aspect of ion
channels could be correlated with the structural aspect; and the structural side, simultaneously,
there are you are moving with technique like you know x-ray. So, one of the very hot area
is membrane channel crystallography then you have cryo electron microscopy – cryo E-M.
So, all this are adding more and more information about the smallest filter system or smallest
filtering machine of the biological world. If we look back from very historical perspective the way things are moved electrophysiology or studying bioelectricity was there for a
long period of time; within the biological system, it was known since the time of Luge
Galwani and Alsandro Volta that this techniques are existing. The techniques started getting
finer and finer and finer, and one of the important breakthroughs came in nineteen seventies,
when with the discovery of the patch clamp, where you really can access the smallest unit which is involved in mobilizing the charge or mobilizing the ion channels which is the
ion channels. Then simultaneously with the discovery of
PCR sequencing, cloning, the whole molecular biology world open up a total new vista, then
came happen the first marriage between the molecular biologist and the electro physiologist
along with the toxicologist who were putting the helping hand, you know blocking channels
as this was moving simultaneously there was enormous understanding about crystallography.
So, people started attempting could we crystallize this structures, really could we see those
filters which are so precise that they could only allow a specific form of ions to move
through. As crystallography was proceeding the discovery of cryo E -M or very low temperature
electron microscopy, where basically what we do is something like freeze fracture to do. If this is the membrane then imagine this
is the ion channel sitting on the membrane or you just using a cryo ion, if you cut it
at a very very very low temperature. So, essentially you can dissect out, you can separate out
that cross section of that membrane and you can really study the whole topology or the whole topographic feature of the membrane channel. While this was all happening, simultaneously
the cyber world was really flourishing; PCS were pretty much ruling the market and everything, and that is the time when PCS were about to come nineteen eighties, the whole field of
informatics or bioinformatics were all the known sequences of different proteins where all getting database. Now peoples are started predicting you know
what like this; so already the data, which are feeding through from those who are doing
this mutation and then electrophysiology. So, people started predicting you know these
residues may be helpful. So, instead of think of it, you can do random mutation out here,
you can keep on doing mutation forever, but if a bioinformatics or ion for theoretical
biologist with bioinformatics specialization comes into play, there will tell you you know
what these are say for example, these are meaningful ones or try this ones. So, they
could actually reuse your time for discovery instead of you know having a random walk all
throughout like you know mutated, this mutated, this mutated, this mutated, this and there
is no end to that. And then you do the electrophysiology and
then you say yes, you mutate this, this is how the voltage sensing kind of got hampered
or the ion selectivity got hampered or conductance reduces or the gating becomes little; obviously,
conductance reduces or something when the gating is not working. So, if you look at
it, the way the modern world is moving to solve one problem you need basic understanding
of all the different tools which are available at your disposal. You need really big team
effort to understand these different bioelectrical phenomena at the molecular level and could
we translate them to make a device those are even bigger challenges. So, as of now what all I have talked to you people is all about ionic electricity; and
there is one technique which is left which I have not talked to you where solid state
electronic device, especially the semiconductor devices like field effective transistor are
being used to measure these kind of ionic event. So, those are some of the pioneering
discovery by Peterframhers from Max Banks institute, Biochemistry. So, we will talk
about that in the next lecture. So, what I expect from you people, just kind of you know
open up your windows of looking at a problem from multitude of an angle, and that will
really help you people to appreciate this subject, and try to you know have a very broader
vision to solve a very fundamental problem. Thanks a lot.

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