 Welcome back to the NPTEL lecture series on bioelectricity. So, today we are in the seventh class. So, as of now we have talked about the structure of the neuron and we have talked little bit about the ion channels and we have talked about the Nernst equation and in the last class I concluded the class showing that potassium ion is kind of slightly more leaky as compared to the sodium. So, today we will talk about the most fundamental unit of electrical activity which is the action potential. The most I should say the first electrical event which leads to the whole plethora of events of neural code is the action potential what really is action potential and how it was discovered. So, the story of action potential is much, much older than the story of understanding of ion channels, proteins and all these things. It was during 1940s and 50s that or much before that actually essentially 1930s and 40s. Some of the pioneering work by Hodgkin and Huxley, the two British scientist one of them was R. F. Piled before that to discover this phenomena. And they were working on aplicia one of the sea animals because it has a fairly long axon. So, that you can insert the electrodes into it and what they essentially discovered that at a certain stimulation they saw a tree something like this. So, let us start with action potential. So, I have already mentioned a cell in its normal resting state resting membrane potential of a cell is minus 70 to minus 90, minus 70 to minus 90 millivolt. So, essentially if I have if I draw the axis like this and x axis is still giving you the time and y axis is giving you the voltage in millivolt seconds. Then the cell is sitting somewhere. So, this is 0, this is minus 10, minus 20, minus 30, minus 40, minus 50, minus 60 and likewise. So, cell is sitting somewhere out here minus 90. This is the resting membrane potential of a cell and let us denote it by R M P resting membrane potential. So, they found a very interesting even if from the resting membrane potential a cell is excited. So, say for example, just to visualize what the kind of experiment they are trying to do something like this. Say for example, this is the axon out here and the part of the axon cell body is out there and you have an electrode out here and you have another electrode which is sitting outside. So, with respect to outside the inside is more negative. We have already discussed this something like this, this is the axon and this is the voltmeter. So, if this cell is excited it is been observed that if I go back to the trace there is a cell become more positive positive positive and then it overshoots the 0 and it comes back after sometime and something like this. This was the first initial traces which were observed and the way it was observed is something during those days there were no computers, there were no such programs or anything. So, these traces were being seen in oscilloscope and it was imaged. So, if you see those images it will be on black background you will see the dotted lines like forming like this. So, this was the first seminal discovery made by Hodgkin and Huxley and based on this trace at that time there was no idea of ion channels nothing was really clear protein first protein was not crystallized by that time. So, they would they did they got the stress and they did a curve fitting using different differential equations and based on that the kind of concept which evolved in last soon after that was that initially what is happening inside the cell. So, initially what happened because of excitation there. So, initially if this is the situation and let me show it using different kind of ions which are present. So, your sodium is fairly higher outside. So, the first event which takes place is this one there is a influx of sodium inside like this. So, as the sodium ions are increasing inside what essentially is happening is inside the cell it becomes more positive now go back to this picture. So, this is the zone where sodium is entering all the positive ions are getting inside the cell, but then there is a threshold zone that threshold zone gives rise to the gives rise to the concept of all or none. It means if you could reach a certain millivolt or a certain stage where this many number of positive ions have gone and the membrane has become this much degree positive. Then after that zone there is no stopping then if you reach to that zone. So, that zone lies if you see the picture out here that zone lies somewhere out here this is the zone this is the threshold zone. If the membrane become positive almost up to minus 40 from minus 90 from here starts this is the threshold for all or none. So, what does that essentially means if the membrane reaches this zone by because of the entry of the positive charges at this stage there is no stopping then this will promote more and more entry of sodium into the system essentially if you look at this diagram. So, this is followed by more and more sodium entry here and so much. So, it overshoots the 0. So, here is your 0 zone. So, at this stage in the beginning while it was sitting this is called the cell was in a polarized state. Of course, it is negatively polarized as compared to outside and here it becomes depolarized because there is no more polarity of the cell depolarized state after it overshoots the 0 out here this zone it is starts to come back to its original base line level like this. So, what essentially is happening here? So, as of now we were saying that there is a entry of sodium going on. So, as a lot of sodium ions get inside the cell. So, what happened essentially is because of too many positive charges inside the cell there is a mutual repulsion between the positive charges because mind it if you look at this picture out here there are already other positive charges of potassium which are present which is fairly high inside the cell. So, there is a positive charge positive charge repulsion starts and this positive positive charge repulsion leads to the next even where the cell starts to allow the potassium ions to get out of the cell. So, the next even what you see essentially is this even 2 now potassium is going out in order to balance the excess sodium which has entered and this is the part what you see in this picture out here where sorry this is where from the cell potassium is flowing out till it brings it back to its base line value. But during this process what essentially is happening as you could see the cell has excess amount of sodium and less amount of potassium. But cell has to bring back its homeostasis by maintaining if you remember in the previous class we are talking about the inner concentration of sodium should be around 5 millimolar with respect to outside which is around 150 millimolar. So, how it does so and there is a third event which comes into play that is the event there are some very interesting pumps which are sitting on this membrane shown by YOLO and these pump function in a totally different way these for these functions like. So, if an individual pump has to show it is something like this it is sitting on the membrane like this and if this is the membrane this pink one is showing the membrane line. So, what is essentially does it is so this is for example, if this is outside and this is inside the cell. So, it binds to the sodium from inside the cell and it binds to the potassium from outside the cell and it binds to the sodium and then this pump flips like this and this flipping action eventually what happens all the sodium which are present out inside these ones these ones these ones are being essentially thrown out of the cell then all the potassium which are present are restored back inside the cell. So, this is where the sodium potassium ATP is pump comes into play and this pump is ATP dependent phenomena. So, it needs a lot of energy to run this pump. So, now if we summarize the event. So, essentially if we go back while I showing the trace out here is essentially finally is happening is that cell is back into its base line. But this is where again inside the cell you have 5 millimolar of sodium and outside is 150 millimolar of sodium and the homeostasis is being maintained. So, this is one of the key point which has to be understood that there are 4 events if I had to summarize this whole thing. The first cell is sitting at minus 90 millivolt or minus 70 millivolt resting membrane potential and by some x y z impulse we will talk about the individual impulse. It could be a photon it could be a ligand it could be sound wave for mechanotransduction it could be a smell molecule it could be a odor molecule which comes and binds or it could be some kind of surface touch which leads to the opening of a bunch of sodium channels or channels which promote the movement of the sodium inside the cell. So, to tell you here membrane is asymmetric in nature what we meant by asymmetry is something like this. So, if I draw the membrane like this if so say for example, this is the membrane. So, this is outside and this is inside what we are trying to tell is that the membrane is asymmetric that essentially means the flow of ions is not reversible by the same route. So, say for example, sodium channel through the sodium enters I had to put them in picture. So, they are like this they are sitting out here in on the membrane like this and it only allows sodium to move in, but it does not allow it to go out through this route. So, in other word it opens from outside to inside vice versa if you look at the potassium channel which is sitting something like this potassium channel only allows potassium to move out from the cell to outside. So, potassium channel does not allow potassium to flow in through that channel then there is a third component which I was trying to describe here which is another asymmetric component of the membrane that is this component which is essentially a wonderful motor which functions to ensure that the sodium are bind here on the inner sport and potassium is binds on the outer surface and this has a property of you know flipping like this. So, even this one is asymmetric because it allows only sodiums to bind inside the cell and potassium on the outside and then it flips back and you have to get an analogy for these kind of pumps if you visit some hotels or some other places where you have these revolving doors where you enter on side and come out on the other side likewise they move like this the glass doors with partitions like this it is exactly similar to that. So, the molecules bind on one side it flips back on the other side and for this flipping movement for this movement circular motion of it it needs energy and this was one of the discoveries by Janis Kau and which for which he got a Nobel Prize. So, if you look at all these three components out which are responsible for executing action potential you will see all these are asymmetric in nature and that is what gives the membrane the asymmetry that this channel what I have mentioned here these are voltage gated sodium channel these are voltage gated potassium channel and these are sodium potassium ATPase pump these are the key players other than few of the leaky potassium channels which are present which allows to maintain the membrane potential at minus time minus 90 leaky potassium channels helps in maintaining RMP at minus 90 millivolt. So, these three are the major component which helps us in our understanding of bioelectrical phenomena at the level of ion channels. So, if we talk about this voltage gated sodium channel so what we are talking about is this if you look at this molecule now since I have introduced the membrane. So, if you look at this molecule across the cell like if this is the part of the membrane and the molecule is sitting like this something like you know this is the pore through which the sodium channels moves. So, what they essentially have is they have a segment which acts as a voltage sensor. So, they have a voltage sensor which could sense the voltage across the membrane. So, in other word they can they have a potential by which they can see the change in voltage like this voltage change out here influences this voltage sensor this voltage sensor is connected to another component which is the gate component like this. So, this gate component is something like this if it senses the voltage it modulates the gate and the gate moves like this and the gate attains a new position which is this position once again. So, the next position of the gate is like this and during that event gate is actually moving from here to here. So, whenever the voltage sensor since the necessary voltage it opens up the gate and followed by that is the flux of or the stream of sodium starts moving in at that point through this and these pores are. So, this is we are talking about the sodium. So, these pores what we are discussing out here are fairly specific it is just like a filter a filter which is regulated by a voltage sensor and a gate and these individual protein molecules are the smallest unit which generates excitability in the excitable tissues of the body which includes our nervous tissue all the neurons the muscle smooth muscle cardiac muscle and skeletal muscle and the neuro endocrine tissues which regulates the secretion of different kind of hormones. So, these complex structures are under intense investigation till date we have an idea about the sequence we have a fair idea about the structure, but at a very low angstrom resolution of 1 or 2 angstrom resolution we still we are waiting scientist are working very hard to figure out the structure because these are some of the most fundamental drug target for situations like pain neuropathy whole range of disease most of them have their roots in 9 channels. So, this is about the sodium same way if I had to explain you the potassium it is fairly similar it is just everything just gets reverse there if this is your membrane like this like this then you have the potassium channel sitting like this. So, these potassium channels which are just put potassium. So, they are essentially allowing the potassium to flow out. So, automatically they have certain sensor elements which are sitting out here somewhere and then they have these gate element as I discussed previously which opens up and allows the flux of potassium outside this. So, these are the basic structure before I explain some of the different variations of the potassium and the sodium similarly you have the calcium channels you have chloride channels likewise and so on and so forth. So, basically at this level the biology is all about these different ion channels and they could be voltage gated they could be ligand gated voltage gated or they could be ligand gated I have been that was the reason why I told you that I will first of all introduce you to the action potential and then I will come back to the ion channel structure. So, I told you in the beginning of the lecture that when ion when action potential was discovered there was hardly any idea about ion channels. It was a shared prediction of those two individual Hachkin and Huxley through their model that there are ports which allow specific ions to move in and it took mankind pretty much another 30 to 40 years almost 3 to 4 decades before the first ion channel recordings took place that was another breakthrough event. So, those are the stories what we are going to share while we will be talking about the different techniques will be dealing with because there are two three things which has to be highlighted here. So, one is what will be discussing in some of these classes is challenges to understand the structure of ion channel. This is definitely a big challenge and we will discuss this why it is so challenging second channel is second problem in the story is how to what are the different how to the story of different blockers what I meant by that say for example, if I talk about that there is a flux of sodium inside the cells while I was drawing it if you go back to my. So, if you look at it here I am showing you that here I am showing you in the slide that there is a flux of sodium which is taking place this flux of sodium how I prove that there is indeed a flux of sodium that I can only prove if I have some way to block the block these sodium channels. So, that is totally dependent on different kind of blockers and same way how could I prove that there are potassium channels. So, I will be needing the blockers to justify my claim that yes indeed there are potassium channels and top of that this field is also very much dependent on the advancement of electronics because the kind of current we are talking about are very minuscule current we are talking about 10 to power minus 12 likewise fairly very small currents which are because these are ionic current how to measure those how even to measure say for example, if you look at this picture how I could measure the current across a single ion channel. This is the story of bioelectricity where people have been successful in measuring current from single ion channel and that is where you will see the development of amplifier circuits. So, that is the reason why I was trying to highlight and especially the development of amplifiers they go hand in hand because the better the electrical tool electronic tools or electrical tools you have measuring tools you have better are the chances that you know you can do a quality recording otherwise to measure these kind of electrical phenomena could might as well be ruled out as nothing but you know electrical noise just measuring noise. So, it has taken mankind if you go back historical perspective if you look at it the birth of bioelectricity is much before the organized subject of electrical engineering during the time of Luigi Galvani and Volta long back when they discovered ionic electricity is or you know biological electricity. Since then from 1700 probably you know 16 to 1700 we have traveled all the way ion towards the 21st century and in that whole course of events there were this whole field has evolved hand in hand with electronics industry with the discovery of semiconductor devices in 1940s and 50s the discovery about in Britain and Shockley and the whole onslaught of development of very high profile electronic devices during 1970s amplified technology was really picking up during that time there were three two individual Irwin Nihar and Berth Sackman these are the two individual who were instrumental in you know recording from ion channels and they used a technique called patch clamp and we will be talking about these different patch clamp current clamp and different techniques in the technique section but in between I will kind of will slide it in and another interesting thing what we have to discuss about if we talk about this voltage gating voltage which is leading to the opening and closing of the channels. So, what will happen say for example, if this is a cell sitting out here and this is the inside milieu and this is outside if I have a electric voltmeter sitting here and say for example, this voltmeter could change also the not only it could measure it could measure voltage, but it could should have the ability to you know manipulate voltage across membrane it is a imagine it is a dual purpose tool if I change. So, basically it sits at minus 90 millivolt so if systematically or you know in a way I change this to say you know plus 0 or you know plus 10 or you know minus 30 how the ion channels are going to behave even without using blockers how the voltage gating is going to get influence. So, we will be talking more about this in subsequent classes. So, at this point I wish to introduce the action potential because that is the key. So, if you look back so these are the things. So, this is the basic action potential and this is the basic action potential of neuron. So, what we are talking about only the basic action potential of neuron out here and this action potential trace varies from cell type to cell type and we will come to the other series of action potential. Then we talked about what are the different stages of the action potentials taking place with stage one movement of sodium followed by stage two movement of potassium followed by the pump phenomena. So, these are the three events which are taking place then we talked about the three component which are involved in the game. We talked about voltage gated sodium channel voltage gated potassium channel and sodium potassium ATP is pump then we talked about the basic structure the voltage sensors and the gates in sodium and potassium ions and then we talked about the challenges we have to talk about sorry we have to talk about the challenges in a understanding the structure of ion channels and the advancement of the electronic and subsequent class we will talk about how to manipulate the voltage across the membrane which could lead to insight into the understanding of voltage gating. So, I will close in here for this class and in the next class we will resume further with the propagation of action potential and the variation of action potentials in other cell types. Thank you.