 Welcome to NPTEL lecture series on Bioelectricity. So, we are into the 12 lecture. So, in 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 axis 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 overall 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 planar patch clamp array. So, essentially what does that mean? So, before I get into the channel let us talk about the planar patch clamp array. So, planar patch clamp array. So, let us draw a comparison between the existing patch clamp and the planar 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 these three axis manipulator by which either you can move the electrode like this like this or like this or up and down. So, there are 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 micromanipulator. So, this is where the 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 this electrode can move up and down and likewise fine. 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 on the top. And logistically speaking this is a very cumbersome procedure and it becomes even more cumbersome when you have to out here you have to give a suction pressure. So, for any specialized lab in this area you first of all need and need a specialized electrophysiologist and on a given day with the best of the best efficiencies there are 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 bioelectronics people could we automatize the whole process somewhere or other. So, how to automatize the process now that is where comes the whole concept of automated planar patch clamp arrays just like microelectrode arrays these are automated patch clamp arrays. So, let us visualize the 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 into the hole cell mode if you do not go in a holes. So, what 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 ensure that something like this. So, I ensure my individual cells are sitting on top of it like this. So, the cell is sitting in a three dimension a cell is sitting on top of this small hole. So, very small hole and ensuring that the medium is not really flowing out because there is a continuous supply of medium which is replenishing it or recirculating it. So, say for example, any medium which is present there is getting which is coming out from there is kind of getting recirculated into the system back and of course, a fresh medium could be put here. Now, underneath that those small holes you have imagine something like exactly like a patch pipette you have a already existing structure which could be put and replaced something like that. For simplicity say I will be showing with only 3 or 4. So, that you understand this is coming from underneath all of them are underneath. So, say for example, if this is the sheet and on 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 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 the 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 and you really do not have to approach the cell it is already the setup is already made. Now, as soon as the cell touches on top of this electrode what you essentially do you follow the same protocol, but it is completely automatized. So, at one point of time you can provide it of course, if you could ensure that all the individual cells are sitting on top of those small holes and that could be done using modern lithography and photolithography technique where you can ensure that you know you can ensure that only the cells will you know be sitting there. So, for example, I put the yellow out here ensuring that the cells are not going to grow in these places if I could ensure that something like this. So, the cells will only sit on top of those small one micron hole and if one could ensure that that essentially what you are what is happening is that now you have a high throughput planar microelectrode array sorry high throughput planar patch clamp array just like high throughput planar microelectrode array. So, this is one approach which is currently underway specially in Germany and some of the universities in Germany and some of the companies has taken it over and they 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 the animals is still it is possible, but it is not still so streamlined 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 you could have a whole cell mode where you have the electrode like this which is the easiest one what 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 things for individual channel where I was showing you where I showed you that you know the inside 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 a high throughput screening at least you do not have to you know spend so much time for drug screening. And 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 5 to 10 years I would say slightly more may be you know of translating the traditional patch clamp into a high throughput screening system for especially these kind of things find applications in the drug discovery industry there it is being really one of the favored candidate drug discovery toxin detection and in the diagnostics this is where this innovative technology or designing problem or designing accomplishment find 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 channel structure could be manipulated. So, say for example, so just before going to that. So, whenever we are measuring from individual channels in terms of 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 which other way you can measure the conductance of individual channel and what you essentially do if you really know the total number of channels on a or you have an approximate and you know within this much area you have this many and then you can back calculate and tell that for how many channels you are getting it and from that you can back calculate and say for single channel how much will be the conductance fine. So, with this background of approaching the channel. So, I told you that I am not getting the structure of the channel as of now because first of all I want to introduce you to the channel how to measure the channel electricity since now I have introduced 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 channel. So, now I will pick up that 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 like something like and you have the membrane on both sides of course, running through like this. Now, you have three features here you have something called this zone which is the selectivity pore. So, this selectivity pore 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 a voltage sensing element somewhere in this structure which could sense a voltage voltage sensor and then this voltage sensor is somewhere other is connected to a gate which ensures the 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 nitty gritty details of the molecular structure if you look at it. So, this is nothing but this is a simple protein which has occurred a shape like this it could be a monomeric protein it could be a dimeric protein it could be a trimeric protein it could be a tetrameric protein it could be a pentameric protein it could be an exomeric protein likewise. So, whenever we talk about protein so what essentially this structure if it boils down. So, it basically there are amino acids like this these individual circles are the amino acids A and this is the peptide bond which is attaching individual amino acid. So, these amino acids join together and form these three dimensional geometries of large large huge proteins which are 10,000 15,000 amino acid structures these are fairly huge. So, how really you have to understand which part of the structure. So, whenever I am writing gate or voltage sensor selectivity port which part of it is really involved in gating or which part is involved in voltage sensing which part is involved in selectivity for the specific ions. So, how this 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 A A stands for amino acids and this is the protein just not. So, this there is a N terminus of that protein and there is a C terminus of that protein fine. Now, the weight is being done is the most tedious way you. So, whenever we talk about these amino acids they are coded by the specific codons or you know. So, these structures are regulated by specific you know nucleotide sequences from the nucleus from the DNA. So, now the weight is being done you 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 40 years of research. Now, today we know at least for some of handful of channels. So, in the in the meantime there are a couple of things which happen cloning as I was telling you the discovery by Carrie Mullis which change the way molecular biology is being done the modern current molecular biology. Then came the whole sequencing the first time it was Shishimu Numa and all these people who could you know sequence the whole channel. Once you know the sequence then you go back using genetics tool that you know that exactly how to mutate specific amino acids. So, that way what you do now you have a control on this is structure you can you can ensure if this is a sequence of amino acid like this you can really you know ensure that this part is replaced or likewise or you know if say for example, in this is involved say for example, let us take a simple example I say you know this sequence say for example, this 4 amino 1 to the 4 amino acids are involved in same voltage sensing just for a 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 line and use those cell lines. So, in the meantime cell line technology was fairly straightforward now with the development over last 40 years and the cloning and everything is fairly straightforward. So, I express the expressing mutated or genetically altered ion channels on cell lines. So, now you have a cell line which has genetically altered ion channels when you take this and you perform the electrophysiology. So, that is 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 pore, could influence the gate likewise and it is a very very tedious process. As a matter of fact, I mean think of it within sodium channel there are so many sub types fast activating in activating sodium channels 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, then you only have water channels aqua parenes. So, really to do it like this and there is not much other tool only other tool which is available is a bioinformatics tool where. So, this is another thing which happen in the mean time. So, you have electrophysiology going moving on. So, electrophysiology techniques where getting kind of electrophysiology 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 functional relationship by theoretical modeling. So, essentially you can tell the molecular biologists that which particular sequence may should be you know or you can you can share this with both of them which particular part may be involved in voltage sensing or gating or a selectivity port likewise one and so forth. So, if you see the timeline the way it is moving and if this is. So, 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 is toxicology because as I was telling in one of the previous classes you need certain specific compounds which can block this channels. So, you have to have those kind of toxins like tetradotoxin, 4AP, triethyl ammonium likewise. So, toxicology was also which is a fairly old science moving with electrophysiology then you have the molecular biology then you have the 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, these functional aspect of ion channels could be correlated with the structural aspects on the structural side simultaneously there are you are moving with techniques like you know x-ray. So, one of the very hot area is membrane channel crystallography then you have cryo electron microscopy cryo EM. So, all these are adding more and more information about the smallest filter system or smallest filtering machine of the biological world once again and if you look back from a very historical perspective the way things have moved electrophysiology or bioelectricity studying bioelectricity was there for a long period of time with in the biological system it was known since the time of Luigi Albany and Alcindor Volta that these techniques are existing. And the techniques started getting finer and finer and finer and one of the important breakthrough came in 1970s when with the discovery of the patch clamp where you really can access the smallest unit which is involved in mobilizing the charges or mobilizing the ions which is the ion channels. And then simultaneously with the discovery of PCR sequencing cloning the whole molecular biology world opened up a totally new vista then came happened the first marriage between the molecular biologist and the electrophysiologist along with the toxicologist who were putting helping hand in a 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 em or very low temperature electron microscopy where basically what we do is something like a freeze fracture to do if this is the membrane if imagine this is the ion channel sitting on the membrane or you just using a cryo knife you cut it at a very very low temperature. So, essentially you can dissect out you can separate out the cross section of the membrane and you can really study the whole topology or the whole topographic feature of the membrane channel. And 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 1980s the whole field of informatics or bioinformatics where all the known sequence sequences of different proteins were all getting data based. And now people started predicting you know what like these. So, already there are data which are feeding through from those who are doing this mutation and electrophysiology. So, people started predicting you know these residues may be helpful. So, instead of think of it instead of as I was showing you can do a random mutation out here you can keep on doing mutation forever. But if a bioinformatics or ion for theoretical biologist with a bioinformatics specialization comes into play they will tell you know what these are say for example, these are meaningful ones or try this one. So, they could actually reduce your time for discovery instead of you know having a random walk all throughout like you know muted this, muted this, muted this, muted this and there is no end to that. And then you do the electrophysiology and then you say yes you muted 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, conductant reduces or something when the gating is not working fine. So, if you look at it the way the modern world is moving to solve one problem you need a basic understanding of all the different tools which are available at your disposal. And you need really big team effort to understand these different bio, 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 are 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 devices specially the semiconductor devices like field effect transistors are being used to major these kind of ionic events. So, those are some of the pioneering discovery by Peter from Hertz from Max Planck institute of 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 angle and that will really help you people to appreciate these subjects and try to you know have a very broader vision to solve a very fundamental problem. Thanks a lot.