 Welcome back to the lecture series in NPTEL on Bioelectricity. So, in the last lecture we talked about we initiated the patch clamp in the last half of the lecture I introduced the patch clamp. So, we talked about how you develop the electrodes and I told you that there are the name itself called clamp. So, essentially you are holding clamping a part of the membrane and at one part of the membrane by following the equivalent circuit model you can clamp two parameters either you can clamp the current that is why it is called current clamp or you can clamp the voltage it is called voltage clamp. So, we will resume our discussion from that point that what all parameters you can measure ok. So, once again we are into the lecture 11. So, resuming on patch clamp ok. So, this is where we were. So, in the last lecture while I was giving you the dimensions I told you that this technique helps you to approach a finite number of ion channels. So, just to have a recap what I was trying to tell you if we call this as a membrane this is the membrane structure you have and on that you have these embedded channel structures which are sitting along these are the embedded channels all along it just for the simplicity sake I am just drawing 7 or 8 channels out here and then your patch pipe it which has a dimension of around like you know 1 micron tip something like this. You can really hold a finite number of channels under the patch. So, what you do once you so this is just slightly advanced. So, let me come back what you do once you have the electrodes ready with you. So, now what you are having is essentially you have something like this you have pulled it and with a tip like this and something like this. Now, you have two options you can fill this electrode either with either with extracellular fluid or intracellular fluid extracellular fluid or depending on the configuration you want to follow intracellular fluid these are the two options. So, now once again let us draw the patch electrode here we have the patch electrode either intracellular or extracellular extra or intra then inside this you put a silver electrode. The silver electrode first is connected to a something called a head stage amplifier or the initial amplifier out here and from here it goes to the mean amplifier and whereas, you have another electrode which is your ground electrode which is let us show it like this which is essentially going here. So, here you have the mean amplifier and here you have the ground electrode and in between you have something called a head stage amplifier which is out here. So, the way it works you have to understand logic the way it works there are two amplifiers here one is a small head stage amplifier which is picking up all the signals then it transmits the signal to the mean amplifier and the mean amplifier all these analog signals are being processed. So, what is an amplifier amplifier is essentially there is a device inside it it contains a very powerful electro meter that electro meter is essentially measuring the charges and they are amplifying that current because since you are receiving current at a very small regime like you know nano nano ampere p q ampere. So, you have to really amplify that in order to distinguish it from the surrounding noise. So, those currents eventually these are all analog currents you are receiving these analog currents are now sent to a DAC card which is a digital to analog to or A D card analog to digital converter A D C analog to digital converter from analog to digital converter these and now this digitized signals. So, what is happening essentially is like this. So, what you are receiving from the pipette is analog signals these analog signals are converted into digital signal these digital signals are then sent to the computer for analysis you can really see it in the computer screen, but nowadays with the modern electronics what you essentially can do you can use the computer to give the command also. So, what you need for the signal which is coming from the pipette like this is coming from the measuring electrode if I just make it slightly more precise from measuring electrode to the amplifiers from amplifiers it goes to the analog to A D card and similarly the same computer now can give a command which will do the reverse route which it gives a digital command again a digital command these digital command is converted into analog command because that is what a cell will understand these analog command is eventually sent to the cell. So, it is pretty much a two way traffic you can use a computer both for recording as well as giving commands though initially when patch clamp was discovered computer was slowly like you know there is not very many computer PCs were nowhere there this was back in 1970s, but the modern electronics has equipped us with all these features that this whole process has become much more smooth you really can acquire a lot of data. So, coming back where I was stopping now you have this mean amplifier here. So, with this setup let me introduce let us assume to start off first with that we fill it with intracellular solution. Now this electrode is fit on a micromanipulator. So, this micromanipulator where it is fit it has the potential to make this electrode move in all the three axis. So, essentially if this is let us introduce the micromanipulator component here. So, it is connected to the micromanipulator where this knob here this knob and here this knob. So, that ensures that you can move it in x y and z axis in all the axis you should be able to move the micro move this electrode. So, essentially this electrode can move like this this electrode can go up and down and this electrode can travel a distance something like this also. So, the practical applications say for example, now I let us see a practical situation where we have the cell culture dish this is the cell culture dish out here and this is the matrix on which the cells will grow on this matrix. Now let us put the cells. So, let us represent the cells by these are the individual cells sitting. So, now we want to study this study the electrical properties of this individual cells. So, let us be more realistic to look through. So, this is the nucleus and the cells are sitting and here we have a patch electrode with us. Let us put the patch electrode in place and inside the patch electrode you have this silver and it is filled with the intracellular fluid to start off with. Now using the micromanipulator you are approaching the cell. So, basically going down on the cell eventually what will happen if I show you the stage 2 you will have the tip of the electrode touching on top of the cell and this is amplified. So, this is exactly the situation. So, if I represent all the ion channels on top of this top of this cell as like something like this if you consider these black dots are the ion channels on the surface of the cell. So, now you have finite number of ion channels in a very close proximity of the electrode this is very important because this is the diagram which I was trying to show in this slide. So, now what you have you have finite number of ion channels under that small pipette tip which is approximately 0.5 to say 1 micron at this stage there is a very simple technique which is being done along this I did not introduce that now I am going to introduce it. You have a small tubing which is connected to it for giving a gentle suction and we will see what happens when you give a gentle suction at this stage. So, let us move on to the next slide let us magnify this configuration. So, that it becomes it makes more sense here is our cell sitting here something like this individual cell and we want to patch this cell and what we have are a series of ion channels on its surface these black dots are the ion channels they were showing in the previous slide they are all over the place all scattered around on the surface of course and here you have the nucleus of the cell. So, now your patch electrode configuration let us look at it the patch electrode is sitting now much more magnified zone your patch electrode is sitting something like this almost touching on the surface. Now at this point you give a gentle suction that is what I was trying to show you in the previous slide you just give a slight suction what will happen this particular part of the cell will get inside the patch pipette essentially this is what is going to happen. So, now if I introduce the ion channels the ion channels are sitting on top of these now we have started when we started I told you that let us assume that this is filled with intracellular fluid and here we have the silver electrode which is moving to the amplifier. Now at this stage there are two options first option is that the first configuration is called the whole cell configuration when you have the whole cell within your control in the whole cell what you do you send a small impulse or a current pulse out here which is good enough to damage this membrane and what you are left with is this configuration in the whole cell this is it then this is the configuration you are left with that it is your electrode tip like this and here is the cell here is the is the ground electrode and this is the intracellular fluid filled in it mind it this is fairly thin I am just for your understanding I am showing it slightly bigger. So, this is since this is intracellular fluid inside the cell you also have intracellular fluid. So, these fluids are the same osmolarity. So, essentially now your electrode becomes part of the cell it becomes almost it is in continuous with the cell. So, what is our current which are either moving out or inside the cell out here through these ion channels now could be recorded ok. So, this is the first and first and most I should say most important configuration to understand the whole cell electrophysiology. Now, what all you can do let us see the power of this technique at this stage you can hold the membrane at different voltages you can hold the membrane at different voltage and you can measure the current or what you can do you can hold the sorry you can inject finite amount of current inside this cell and you can measure the change in voltage. So, first of all let us try to do that step 1. So, first of all do the current clamp and then we will do the voltage clamp. Current clamp essentially mean you are injecting finite amount of current inside the cell and changing its polarity. So, if you go back here let us see how the current clamp is going to work. So, once now we are in a current clamp motor. So, when we are doing current clamp what you are measuring out here you are measuring the change in voltage. So, your y axis is voltage in millivolt and your x axis is time and the cell we know is sitting at minus 90 millivolt. So, this is the resting membrane potential R n p resting membrane potential. Now, as you are injecting positive charges into inside the cell let us see here you are introducing say for example, imagine like this you are introducing positive charges inside the cell. So, what will happen is it will be a very similar situation as that of sodium getting inside the cell. So, this will immediately you will open all the voltage gated sodium channels and all the voltage gated sodium channel will lead to enormous flux of sodium ions inside the cell. So, as sodium ion is getting inside the cell this is what you are going to observe. The membrane voltage is start to go towards the positives and this is 0 it is going to go positive like this. And if it reaches something like say minus 40 or minus 30 we call this minus 40 here slightly over minus 30 then this should something called a phenomena called all or none. It is almost the membrane as if it looks like it collapses because then there is no way it cannot look back it will overshoot 0 like this that is called all or none, but it has to reach to that of minus 40 and between minus 30 and minus 40 millivolt. And if that happens all the surrounding voltage gated channels start opening up and they will shoot an action potential. And then of course, it again comes back because this is the time when all the potassium channels start opening and then rest is we have already studied. So, this is how you do a real current clamp measurements where you are seeing the action potential, but now the challenge is how we know at this part of the curve where there is influx of sodium there is enormous influx of sodium. How I can measure the influx of this sodium? So, in other word these sodium are nothing but sodium current how I could measure the sodium current and how I could measure the other current which is the out here the potassium current which is the going out of the cell. So, in order to do that now we will move on to the next technique which is called voltage clamp now we have to measure the current. So, in order to measure the current we have to fix the voltage at different level. So, our now the way the recording will work is something like this you are measuring the current in this axis in the y axis and the x axis you have the time current in pico ampere or nano ampere and on this you have the time in milliseconds. Assume the cell is setting out here the baseline. So, this is we are assuming at the zero current like in there is no interaction of current at this point. Now, let us again go back to the configuration of the cell. So, this is where we are injecting current now expose this membrane to different voltages. So, it is sitting at minus 90 I make it from minus 90 I started holding holding it across. So, let me draw this. So, if you have this cell out here and you have two electrodes like this. For example, one electrode like this and there is another electrode out here like this. Now, you are changing across it the voltage minus 90 millivolt. This is where the baseline value is at this point then I make it minus 80 millivolt. I am holding the membrane at minus 80 across it. Once it is minus 80 what I will see may be some of the. So, from the baseline I will see a small dip. So, then I move to minus. So, this is your let us give the corresponding number 1 trace 1 this is your trace 1 this is your trace 2. Then I put it to minus 70 millivolt and I saw trace very similar 3. Then 1, 2, 3 then I move to minus 60. I saw another trace coming like this 4 minus 50 5 minus 40 these are all millivolt something like this minus 30. This is where I will see this is where all the potassium channels starts to open 30 millivolt and simultaneously what is happening out here they are like this. Now, as I am going further say minus 20 minus 10 0 10 millivolt 20 millivolt what will essentially happen these sodium current which gets activated in a narrow window of out here they would not any further they will get closed down. So, now what will be recording is this side of the circuit. So, by axiom it is being followed that this current you can show this current on both on the upper side of the axis, but it is generally people follow it to show it like this. So, this part of the circuit what you are seeing out here of the current basically your sodium current or any form of inward current which is in entering inside the cell and out here what you see are the outward current or one of the major outward current is the potassium current. Now, what you see eventually as you are going up with your voltage clamp traces starts to start to see potassium traces like this and of course, they have a rain and these currents are much more delayed. So, these the sodium current generally these kind of sodium currents are called fast activating in activating sodium current which are ensuring the flow of this current and these currents are called slow activating or delayed rectifier potassium current or channels. So, just a word of caution there are several celtides where you do not have this fast activating or inactivating sodium current will come to those at this stage we are not getting into those will come to those later at this point we are just talking about most of the neurons what they have. So, they have this fast activating and inactivating sodium current out there. Now, how to ensure that these are sodium current this is the challenge question. So, how you can ensure is this say for example, one second you have this cell out here with let us show the sodium channel in red and show the sodium channel in red and show the potassium channel in any other color. The way you can show this is say for example, you have a specific very specific toxin which binds to which binds to the sodium channel. So, for example, it comes and it binds to the sodium channel. So, what you will essentially see is say for example, this yellow is the toxin what I am putting now and it is coming and binding to the sodium channel. What I will see out here in this trace is this part of the current will be abolished this is gone this will not be there instead the trace will look something like this what I will be essentially seeing is nothing and only the potassium current. So, what you have done is that you have block blocking sodium channels thereby aborting sodium flux and this could be done using toxins like tetrotoxin which is obtained from puffer fish which is in short it is also called TTX tetradotoxin puffer fish. This TTX could block the sodium channel similarly if I have another blocker which blocks the potassium channel what I will see going back to this second let me go back to the higher then what I can do is that I can abort this part of the this part of the current this will be lost. So, the way the trace will come is something like this the way it will come is that you will have the sodium current like this and that is it will have the all the sodium current like this. So, there would not be any. So, this is where you are blocking potassium and this you do using compounds like 4AP and tetraethylaminium likewise there are series of compounds which can block the potassium current. So, you essentially see now we are able to access the ion channels which was absolutely not feasible with the sharp electrodes or even with extracellular electrodes or any other known things that was the reason why this discovery 1970s change the way we look and it was the same time when pretty much the same time when PCR was discovered by Carrie Mullis. So, that opened up a flood gate of cloning. So, what happens is that that was the time when molecular biology was got a huge boost and electrophysiology got a very huge boost in terms of the discovery by Irwin and Nehaar on the patch clamp. So, all these techniques started you know marrying. So, people were developing cells where they could really specifically express sodium channels they could express potassium channels and then they could really access the individual cells with profound surety that yes that is what is happening. All the mechanisms are started to come into play and simultaneously that was the time when people started using molecular biology technique too. So, let us see how all these things are being done. So, for example, now one more thing which I have forgotten to tell you people is out here. So, when this cell comes in contact out here this is where it forms something called a when you pull the cell and go to this configuration out here it forms something called a giga ohm seal. This giga ohm seal is very important because this seal ensures that there is no leakage along this all the leakage are being prevented. It has been really measured and this was the ingenuity of the discoverer of this technique that you know because between the glass and the cell there is a really very strong seal which is formed and with the modern software you really can see physically whether the giga ohm seal has formed or not something like if this is this fingers of mine is an electrode and if this is the cell. So, it form something like that a fantastic strong seal out there which would not allow any leakage to take place. So, this is one thing which I just missed out while I was showing you this coming back. So, what are the techniques which are being used? So, we talked about the different toxin. So, this whole seal depends enormously on different kind of toxin and some of these toxins are also have a strategic importance because they could be used by the terrorists. So, there are a lot of restrictions in using this toxins, but these kind of toxins have open up a whole plethora of approach to understand ion channels. How they are binding? Are they competitively binding? Non competitive binding? A permanent blocker? They are likewise like this is a whole feel in its own merit where all these this toxicology merges with ion channel physiology. So, coming back how most of these so whenever we talk about ion channels. So, I told you one configuration which is a whole cell configuration. There could be several other configuration. So, say for example, I have this membrane out in front of me with the channels like this complete the membrane. So, and all these channels are sitting here on its surface. Now, I take take my patch pipette and patch pipette comes here. My patch pipette is on top of a bunch of channels by silver electrode. Instead of filling this electrode with intracellular fluid now I fill it with extracellular fluid. So, now in the fluid here is extracellular this is extracellular fine. At this stage instead of completely blowing away the membrane part of that membrane patch of that membrane instead of going into the whole cell mode what I did now is that I chop off I just pull the electrode because now it is in under the control of the micro manipulator I just gently pull the membrane. So, what I will essentially get is this configuration you have the electrode inside the electrode you have this is the electrode what you have is a part of the cell like this part of the membrane like this and a finite number of ion channels in it inside this you have the extracellular fluid which is filling it and then what you do you put it in a chamber or in a small dish which has intracellular fluid in it. So, here you have the intracellular fluid now what is this configuration essentially tells you is this you have a complete control of manipulating these ion channels a finite number of ion channels. Now this is called you are basically this is called inside out patch inside of the membrane is now exposed. So, these are special configurations which are being done to you know. So, these are special different kind of configurations which are being followed to you know to understand the ion channel behavior. So, what I will do now I will talk little bit more about these ones these individual channels how they are they were studied. So, I will close in here what I expect you people really to go through these slide very carefully because there are lot of informations here go through the patch clamp understand the concept understand current clamp and the voltage clamp and how you really can access the different ion channels from here what we will do we will talk about how the molecular biology techniques helped us in understanding the different ion channel gates, pores, voltage sensor and all other things. So, I will close in here in the next class we will discuss about all those things.