 In this section 2 where we started with membrane physiology of nerve and muscle, we designated to have 3 lectures. So, we are done with the first lecture where we talked about the lipids which are present in the membrane and the classification of the lipids and few other details. Today, what we will do in this part second lecture of this section will be talking a little bit about the structure of the membrane and the techniques which are being used. So, before I move on to this lecture there is a small carry over which I missed out in the previous lecture. We talked about within the membrane you have the presence of the glycolipids and the phospholipids. There is one more component which is present in the membrane and which has a very profound role to play that is called cholesterol. So, now I will just add that and then I will start this today's section lecture. What exactly is the cholesterol among the lipids? We talked about the phospholipids and the glycolipids. Now, this is the third moiety in that structure. This is a very profoundly important molecule in biology and evolution. This is how the structure of cholesterol looks like. It is a fairly big structure with the X. I will mention what X is and you have one CH3 attached here. You have another CH3 attached here and you have OH hydroxyl group attached here and this X equals to CH3H and this is where the X is actually connected CH2, CH2, CH2, CH3 and CH3. So, this is the structure of the cholesterol and in this lecture we will come to see where this cholesterol is playing a very important role in the fluid nature of the membrane and what are its other physiological function. As I will work through the course, we will talk about it where the cholesterol plays a major role. With this small backlog which I missed upon, I will now enumerate the topics which I will be taking up in this lecture. There are 12 small topics which I will be taking up in this lecture under the heading of membrane structure and dynamics. So, some of we have already covered around 10 topics in this structure and dynamics. The topics which I am going to cover today in this section, this includes lipid bilayer, first topic lipid bilayer and techniques to study lipid bilayer. This is the first topic I am going to techniques to study lipid bilayer, just putting it as BL as a short cut. Then we will be talking about the permeability. This is the second topic I will be touching in this lecture permeability of lipid bilayer. Then I will be talking about the flow of ions through lipid bilayer and how this could be detected. The fourth topic what I will be covering will be the membrane protein. The next topic I will be covering is the experiments to prove. If you remember in the last lecture I told you, I will be talking about the experiments to prove the mobility of proteins and lipids in the membrane. Next topic I will be talking about the fluid mosaic model of the membrane. Then we will be talking about the asymmetric nature slightly more detail, asymmetric nature of the membrane followed by that we will be talking about factors controlling membrane fluidity and this is where we will be talking about the role of cholesterol membrane fluidity. Then we will be talking about presence of carbohydrate in the membrane. Last we will be talking about the use of detergents in studying membrane proteins. So, these are the topics which I am going to pick up in this section. So, having said this let us talk about the lipid bilayer. So, in the last class I told you that lipid bilayer, the bilayer formation is directly related to the presence of phospholipids and the glycolipids and now I have added the cholesterol. So, now what are the different techniques which could be used to in order to understand this. So, under this heading we will be talking about let us draw the lipid bilayer how it looks like, drew it like this. So, this is one side of the lipids, the other side of the lipids and here you have the hydrophobic chain. So, is there a way how we can recreate this lipid bilayer in the lab. So, this is there are two different ways how you can do it. So, we will be talking about one of the techniques first which will help you. So, that is basically fall under the preparation of lipid vesicles in order to study the lipid bilayer. So, how it is being done preparation of lipid vesicles. So, what is generally done in the labs is that you take a beaker like this. In the beaker you have a layer you make a layer of a phospholipid let us put the phospholipid you can see it in green this is the phospholipids. On top of that you add water likewise and you can use any kind of this case I am adding these are small amino acid called glycine this is water. What you do with this you sonicate this sonicate means you agitate it at a very very high sound frequency sonication. So, once you sonicate it. So, let us follow it up following sonication what you see it forms something like this what is the color code I used fine. So, post sonication what you see these kind of structures which are formed and the water all over the place outside and within that you see the trap molecules of glycine and there are some glycine which are present outside. So, from here what you do you do a simple technique called gel filtration and following gel filtration what you obtain are pure lipid vesicles. You get rid of all the glycine from the suspension and there are glycine which are trapped inside the lipid vesicles this is how you create the lipid bilayer this is one such techniques of creating pure lipid vesicles. You can use the kind of which server phospholipids you can use instead of phospholipids some mixture of glycolipids you can use colostrol you can vary the concentration of colostrol you can do several things in this situation which will help you to create different kind of vesicles. There is another technique which is slightly more advanced technique that is called second that is called black lipid membrane technique of studying lipid bilayer black lipid membrane. In short sometime it is also referred to as BLM technique black lipid membrane apparatus. So, what really black lipid membrane apparatus is? So, just imagine you have two beakers like this you have a jar where there is a small partition fine and within that partition you have a small hole like this sorry let me redraw it like this. Now what you do? So, you see this a small hole out here mark this hole for your understanding. So, this is where the hole is and what you do? You take a paint brush all of you have seen a paint brush you take a paint brush like this and dip it in this is the paint brush and the dimension of this hole is approximately 1 millimeter you take the paint brush and you have the lipid solution here it could be a phospholipid PL it could be a glycolipid it could be cholesterol whatsoever and then you take the paint brush out here just the way you do it like this you rub the paint brush out along that hole. So, once you do so what will happen is this if I redraw this and here you have the cap it forms a lipid bilayer here. So, something like this what you will you see if I observed it under the microscope it will look like this either side. So, in other word you form a membrane across that hole something like this this is what is going to form here redraw it on top of it it will be like this just for a simplicity sake I am following a color code, but it could be all mixed up something like this now what you do is you fill this side with water and you fill the other side with water and you add different kind of solutes into this say example you add sodium potassium calcium you do the same thing at different concentration calcium plus plus sodium plus potassium plus at different concentration you can have sodium potassium higher on one side potassium lower on other side you have to have sodium higher on other side potassium other side and then what you can test is actually you can connect it with two electrodes you have electrode like this and you have another electrode which is like this and these electrodes are connected to some kind of volt meter which will measure the voltage or you could kind it to some other gadget which could measure the voltage. So, any kind of mobility of the next slide so that it is not crowded for you to understand. So, here you have the bilayer already formed and here is the electrode on one side here is the electrode on other side and this is all connected to some kind of measuring device. So, this is the electrode this is the electrode and this is that hole where the lipid bilayer is formed and here you have the fluid and with all the different so any kind of movement of ions across this could be recorded by this voltage sensor or any kind of current devices which are present there. So, this is how the black lipid membrane is so very useful in understanding the movement of any kind of ions or any kind of molecules across the membrane. So, this is one of the techniques which are being used from here we will move on to the permeability of lipid bilayer this is very important this is the second topic of this section permeability of lipid bilayer. So, one of the fundamental thing about lipid bilayer is that these are highly impermeable to ions and most polar molecules. The molecules which are having some form of charge out there or some form of ion like sodium potassium they are exceptionally impermeable they cannot really directly pass through the membrane. In other word what I mean is this say for example you have a lipid bilayer like this thing like this and you have this it is very tough for these kind of ions or some kind of polar molecules you know some kind of say polar amino acids which we have already talked it is most unlikely that it can pass through it this will block the movement of these polar ions from either side which over every you tried it is going to block the entry of the polar molecules. So, polar molecules or the ions polar molecules and ions are very impermeable to the lipid bilayer and if I kind of draw a scale of about the permeability of different component through the lipid bilayer in terms of the increase permeability. If I have something a scale like this increasing increase permeability unit will be permeability centimeter per second that is it then if you look at it 10 to the power minus 14 just putting the values 10 to the power minus 12 10 to the power minus 10 likewise if I move to 10 to the power minus 2 10 to the power minus 4 10 to the power minus 6 10 to the power minus 8 10 to the power I cannot just draw this scale ok 10 to the power minus 8 if this is the permeability values I am. So, you will see most permeable will be water then you have something around 10 to the power 4 you have something a molecule called indole which is very similar to another molecule which will come very soon then around 10 to the power 6 you have urea which is more permeable urea or glycerol then around 10 to the power 8 you have tryptophan tryptophan tryptophan is one of the amino amino acids and around 10 to the power 8 is glucose then comes around 10 to the power 10 is chloride ions 10 to the power 12 is potassium ions and near about is the sodium ions which is very least permeable into the game. If you look at this figure just it raises a very simple question how come this tryptophan which has a very similar structure as compared to indole very very similar structure how come in indole is more permeable as compared to tryptophan. So, in other word the question is why indole is more permeable through lipid bilayer as compared to tryptophan this is one question I will leave for people to think over it what is the difference between the two structure which makes tryptophan less permeable to lipid bilayer as compared to indole though their structure is very very similar and your answer lies in some of the polar nature of it as I have mentioned in this small section that polar molecules or the charge ions or the ions are very less permeable across the lipid bilayer. So, now the question arises a very serious question now arises what makes an ion or polar molecules to pass through there must be some way because as we have already seen the previous scale that most of them most of these are not that very permeable and this is where comes the role of proteins or the membrane protein this is where comes the role of ion channels ion channel proteins which is a very very significant area of research there is a huge amount of budget which has been allocated by national institute of health in United States and several other countries to understand the structure and function of ion channels and we will talk more as we will moving through the course here comes the role of pumps. So, these are those proteins which are embedded within the lipid bilayer which allows the flow of polar molecules ions and many other molecules which directly cannot pass through lipid bilayer or the permeability is very low and with that permeability no physiological function can take place. So, now we will in order to understand this we will talk before we get into any kind of membrane protein which are very very complex structure and very little structural information about it is available currently very few are really known in at a very low resolution crystallographic structure we will talk about some simple molecules which have the ability to form channels or pores along the membrane. So, this section falls under transport antibiotics as carriers or channels. So, now this is what we are going to deal with transport this is what we are going to deal with transport antibiotics carriers or channels talking about this let us move on to the next slide let us redraw the membrane again these are the polar head groups in red and another set of polar head groups which are facing inside the cell like this and here is the hydrophobic tail and keep on repeating it. So, that it kind of engraves into your thinking and understanding some kind of continuously repeating all these things for you guys. So, a membrane protein or anything which has to form a passage has to stand like this has to form something like this structure like this which has may be an inner core like this through which any of the molecules can pass through something like this this is the molecule which is passing through. So, this is outside this is inside. So, what are those nature of these kind of molecules this is where we are going to talk about some of the smaller simplest one before we get into the one which are forming ion channels and everything. So, one such example is carrier transport antibiotics are something like this is one such carrier transport antibiotics which has a hydro hydrocarbon periphery and within the hydrocarbon periphery it forms it has something like this. So, this is called carrier transport antibiotics I am just putting a short form antibiotics as A B. So, that you will understand these are the oxygen molecules and in the center you have a potassium and potassium ion form coordination complex with 6 oxygen there and this is the zone which is this is hydrocarbon. Or hydro phobic part of it and this part what you see out here is the hydrophilic core. So, these kind of molecules when they get inserted inside the membrane. So, it is something like this again I have to draw the structure. So, that that makes sense of the membrane. So, this is where these kind of molecules get inserted into the membrane and here they have the potassium core through which any kind of water or any other any other ions can pass through or any other molecules can pass through this is one such example which is carrier transport. So, there are other examples which will be talking about it one of them is one of them is your valinomycin this is this is another cyclic molecule it is something like this all the residues and then I will tell you A B C D and this A B C D stands for different residues I am going to come to that A B C D D. So, some total there are 1 2 3 4 5 6 7 8 9 10 11 12 there are 12 such residues where A stands for L it is an L isomer L lactate B stands for L valine C stands for D hydroxy iso valerate D hydroxy iso valerate and D stands for D valine and A stands for D if you if you go through the structure of it it is something like this. So, C H O minus C H 3 C double bonded with O. So, this is A. So, now come to the B B is N H C H and you have C H you have C H 3 you have C H 3 here and you have C double bonded with O and this is your B. Then you have the C molecule which is oxygen C C H C H 3 and you have C H 3 C H 3 C double bonded with O H this is C and this another one here is N H C H C double bonded with O C H C H 3 C H 3. So, this is D and this is C. So, this is how it is being calculated in the valinomycin and when this valinomycin molecule I showed you that in an A B C D likewise let us see A B C D A B C D and then again A B C D and within the center of it what you observe is something very interesting again there is an potassium which is present in the center and which is kind of coordination complex and it is this potassium which is there which allows ions to move through it. In the same line there is another molecule these are kind of the classic molecule which is which have been used all these years is called gramacidine A. Gramacidine A is another such channel forming molecule. So, gramacidine is even much more complex structure of put the structure for you guys so that you kind of get a feel of these kind of structure these are the small structure these are easy to understand before we understand. So, this is an alternate L and D isomers of amino acids. So, this is the L isomer glycine glycine then you have the alanine which is the L isomer here then you have I kindly request you people to kindly go through the structure of the amino acids from the nodes from alanine is L is up to 5 then you have the valine which is in D conformation and you have another valine which is in L conformation and you have another valine which is in D conformation you have tryptophan which is in L conformation you have a leucine which is in D conformation. So, you see there is an alternate L and D and then you have tryptophan which is in L conformation you have leucine which is in D conformation another one which is in L conformation you have leucine in D conformation you have in L conformation and then it is attached to a carboxylic group NH, CH2, CH2OH. So, this is how the gramicity in molecule looks like and all these molecules have alternating L and D isomer and they are the one which forms the channels. And these are some of the probes much before the membrane structure could be derived and it was very challenging task. These are some of the molecular probes by which ion channel structures have been studied for several years and now also they are being studied. These are small molecules they act as antibiotics, they act as toxins there are series of them I just picked up two or three examples like valentomysine, gramicitydein and carrier antibiotics which could help you to appreciate how the membrane must have developed. So, from here we will move on to the fourth topic of this section which is the flow of ion through. So, I told you I showed you that diagram in the beginning of the black lipid membrane. So, say for example, let me redraw it. So, you could recreate the membrane like this and here you have this lipid bilayer formation and here you have the electrodes and you have a very high gain amplifier to record the change in voltage across it and there are different concentration of say sodium, say may be high on this side sodium ions may be low chloride may be high here or chloride may be high here or say for example, potassium may be low here potassium may be high here whatsoever the flow of this could be the flow of ion through single channel in a membrane can be detected. So, basically single channel can be detected how it is being done this is very straight way. So, I showed you that using a brush let me go to the next slide. So, you use a brush to create that membrane structure. So, along with that brush you make some of these channel forming peptides or channel forming molecules. So, what will happen is this now out here you have some of the channel forming structures which will form and they will allow the flow of ions across either one of the sides and that will help you to record the flow of ions through those channel. So, this is how most of these channels are being studied and the recording kind of comes like this if this is the base line and you will see something like. So, this is when it is going up is basically the channel is getting open you can measure the density of channels you can do a whole bunch of complex experiment and you can understand how the channel looks like where x axis is showing you time and y axis is showing you conductance. And we will talk more about it about the single channel recording as we will move through go to the ion channel where we will be talking about some of the fundamental discoveries ion channel recordings which were done by Irvain Nihar, Bert Sackman, Alan Marti and all these other people which won them a Nobel prize and many other things. But just for your interest I can tell you that across these gramicity in channels and everything almost 10 to the power 7 cations could travel per second this is almost the rate of migration which is the huge number. So, with this we will move on to see. So, we kind of give you an introduction about how the how probably have been bigger structure. So, we talk about very very small structures very very small structures which are forming channels. Now, think of it a huge protein like structure how it will look like there are several conformation and geometries how it looks like. So, let us draw the structure of the membrane to tell you how the proteins look like. So, now we are entering into the area of membrane proteins which are embedded within the lipid bilayer let me again redraw the membrane. So, the proteins I will draw it in black to give an understanding some of the proteins span like this from the one side to another side they are like this. Yet there are proteins which sits on top of it like this like this it could be on the outer side it could be on the inner side in both sides. Yet some of the proteins just have something like this one part and may be attached to another part like this. So, this is how all these different kind of proteins are sitting all along the membrane structure. And these proteins these proteins could be could be on the peripheral side on the periphery it could be integral protein it could run all across the membrane run all across the membrane. How these things were discovered that is very important one for you people to appreciate. So, there are certain techniques by which these have been done there is a technique called freeze fracture etching technique. So, what do you do say for example, let me take some say for example, this is the bilipid membrane just think of my first is the bilipid membrane let me see how I orient it. So, imagine underneath is inside the cell and up it is outside the cell what I do I take this stuff and I what I do I freeze it down freeze it down at a temperature of say liquid nitrogen exceptionally cold temperature. I freeze it down as soon as I freeze it down this is what will happen the membrane. So, this is your membrane structure what I was trying to show you and here is your protein of choice. So, now I bring down the temperature to liquid nitrogen temperature ok. So, temperature is of liquid nitrogen and then what I do is at this stage I bring a sharp knife which is called a microtome microtome knife and I cut the membrane split up the membrane. And this is called basically the whole technique is called freeze etching etch H freeze etching electron microscopy. This is how it was discovered that they are integral proteins which run from one side of the membrane to the other side of the membrane electron microscopy. And what you get out of this is that doing this deep etching is something which I will show you through the purse using my wallet. So, say for example, this is the outside and this is the inside and this is the lipid bilayer. So, when you put this at liquid nitrogen temperature and you cut it. So, you take the knife say for example, let me see. So, say for example, this is the knife and I take the knife and I cut it through like this or I cut it through like this. What you see is that it opens up like this and then this is exposed in front of the microscope in electron microscope. And there you could see if there are some proteins which are suppose these my fingers are the proteins which are there. So, I can see the structure of the proteins. So, this is how it is being done. So, this technique is called freeze etching electron microscopy and it follows the deep etching process. This is how most of these integral proteins have been discovered that they run from one side one end to the other end of it. So, this is one set technique which I wish to expose you people. So, from here we will move on to another technique. You remember in the last class we talked about that we talked about that the lipids can move across the membrane laterally and very rarely it do a flip flop movement. It is really tough for it to do. So, how it was been proved and I told you that I will give you some of the techniques by which membranes have been studied. So, one such technique is like this say for example. So, let us put the title so this is in this section we will be talking about lipid and many membrane proteins, brain proteins diffuse rapidly in the plane of membrane. So, how to prove this? So, we need experimental proof to do so. So, this is how it is being done say for example, I have one cell A. So, for example, cell A like this and I have another cell and let me put a different color cell B like this. These are the two cells and on these cells I have some kind of a tagging of the lipids and the proteins. So, for example, these are the tag you see these are the blue colors are the tags and other one I have this black tag. So, if the blue tag you have the black tag the next what I do is let me pick up another color to explain it easily. I fuse these two cells if I fuse these two cells so the structure will form like this I am fusing these two cells once I fuse these two cells. So, what will happen? This black will remain here and all the label things will remain here and after some time what you will observe is this after you just give it some time and then you observe the new structure which is almost this big and if I give the real color something like this like you know just let me check through and red fine green. So, if this part is the green one and this part is the red one what I will see is all those black dots and all those blue dots have mixed together likewise. So, that is one of the indirect proves or kind of a crude prove to say these proteins and the lipids are moving all across the membrane there is another way to prove it which is much more quantitative. So, this diffusion so this diffusion takes place. So, there is a diffusion of lipids and proteins there is another way to do so that technique is much more quantitative. So, that is called fluorescence photo bleaching recovery technique photo bleaching recovery technique what this technique is about. This is a very interesting technique. So, this technique is like say for example, I have a cell like this I have a cell like this fine and in the cells. So, this is the nucleus and all those things and this is the membrane on this cell what I do I tagged it with some kind of a fluorescent probe. So, these are the fluorescent probe all these are fluorescent fine. So, that what I do I pick up one spot say for example, I pick up this spot and I put my microscope at this spot. So, here is my microscope you could see here is my microscope I am seeing this spot. So, what I do at this point I record say for example, you know I have x axis y axis. So, I am recording fluorescence intensity on y axis fluorescence intensity on y axis and x axis is time. So, initially what I see at this spot there is a fluorescent intensity like this and put it in red that will make more sense. So, then what I do next thing I bring a very strong laser beam and here is a laser beam coming some kind of a strong beam which kind of you know bleach the whole surface or in other word bleaching the whole surface means it get rid of all the fluorophores which are present out here. So, all the fluorophores which are present out here. So, it is free from all the red. So, what you will see in the graph is that immediately the fluorescent immediately the fluorescence intensity will fall down like this sharp fall because of this is where you are bringing the this is where bringing the bleaching coming into play after sometime what you see is that there is a recovery it started pulling back what exactly is happening white recovered it recovered because all the surrounding these molecules what you see these molecules is started moving to the bleached side like this. In other word there is a mobility on the movement of the different proteins and the lipids which are present. So, these are two standard techniques by which it has been proved that there is a mobility of the lipids and the proteins across the membrane and very rarely you will see a flip flop movement which I explain in the last class. So, membrane proteins do not rotate across the bilayer membrane bilayer bilayer, but sometime the lipids do so, but very very slowly based on all these we moved on to the so there are over the last century several models of the membrane has been proposed as we are advancing as we started understanding many of the older models got discarded and the most current model which is fairly acceptable is called fluid mosaic model. This was proposed by in 1972 by Jonathan Singer and Garth Nicholson. So, fluid mosaic you understand mosaic mosaic is just like in your house you have the mosaic floor. So, it is kind of mosaic like this is the mosaic of proteins in the lipid something like that how you can in 1972 by Jonathan Singer Garth Nicholson. These are the two people who proposed that Nicholson what they proposed is that was their basic definition on it is the membrane are two dimensional solution of oriented lipids globular proteins. Lipids globular proteins this is their central theme about the model and which consists of the lipids like phospholipids glycolipids which acts as a solvent to embed the proteins in them and there is a lateral diffusion which we have already discussed. From here we will move on to the next topic which is the asymmetric nature of the membrane. We have already discussed this we will not discuss much on it except the fact that as I have mentioned the proteins never flip flop and that is what helps them to maintain the asymmetric nature of the membrane. So, it is something like that there are certain pumps called we have been talked one of the major pump is called sodium potassium ATP is pump. So, this pump has the ability to throw sodium outside the cell and takes potassium inside the cell and this only does when ATP is present inside the cell always remember ATP has to be present inside the cell and this is the membrane which I draw this is the membrane and this is the pump and this offers an asymmetry this offers an asymmetry in terms of the ion concentration inside and outside the cell. So, there are certain toxins which binds from outside which open is one of them which binds outside and block this block this pumps and these pumps and these channels they offer a lot of asymmetric nature to it. So, now what controls membrane fluidity? Membrane fluidity is controlled by the fatty acids composition and cholesterol. How it does? So, say for example, if the fatty acids are arranged like this if they have a all straight chain like this the hydrophobic tail is very straight and that is only possible when the hydrophobic tail is in trans configuration. So, that situation it will be a very compact structure whereas, think of it if the hydrophobic tail is something like this where there is a lot of cis configuration coming into play. So, this will have this will be not very compact this is much more disturbed or much more random structure. These structures have low melting point T m which are present the melting point they will have a low melting point. So, in other words and whereas, as compared to a very rigid structure. So, there are three parameters which decides the length of the fatty acids the kind of bonding they have saturated or unsaturated saturated or unsaturated. In other word the presence of single or double bond and on top of that the presence of cholesterol where we started this class the cholesterol decides a lot about how the membrane fluidity is going to change. The last one minute I am going to devote now about the carbohydrate. So, if this is the membrane most of the carbohydrate points on the outer side. If this is O is out and I is in the carbohydrate moieties are outside either attach on top of proteins like this if this red thing is the protein or they are directly attached to the lipids they could be a glycolipids or they could be a glycoproteins. And apart from it there is another technique last technique which I just want for your information these membrane proteins could be isolated using several detergents like Triton DDAO they have several full forms that is dodecail, dodecyl, dimethylamine oxide, sodium collate and octyl beta glucoside. These are the different detergents which could be used to isolate the membrane proteins. So, I believe this is what is essential for you people to understand that membrane is a very dynamic structure it is asymmetric in nature it is extremely fluidic. And all these parameters changes whenever there are some changes in the body and with this we will move we will move on to the we will we are closing on this lecture. And in the next lecture we will talk about the membrane of the nerve and muscle. Thank you.