Transcribed by David Landsman Neuroscience 16 Action Potentials 1 by Dr.

Schiff

1/31/2014

No slides [Dr. Schiff] [] is the number of open channels to allow a particular ion to cross the membrane. The more open potassium channels, lets say, there are in a patch of membrane, the more easily potassium will be able to cross that membrane. Now what happens is in the membrane there are a large number of potassium channels -just to stick to potassium for the moment -- and what happens is these channels can exist in either one of two states, they can either be open or closed. Theres no such thing as the channel opening wider to increase permeability. When the permeability to something increases its because there are more channels open. So what you have are two states, closed and open, for this particular ion, lets say potassium. And theres a rapid equilibrium between the closed state and the open state. So closed channels are constantly opening, open channels are constantly closing. And theres a rate constant for a closed channel opening (K opening) and theres a rate constant for open channels closing (K closing). And what happens is the only way you can change the fraction of these channels that are open and thereby the number of open channels is by altering these rate constants. Now, so far, everything here, the Goldman equation, refers to any cell whatsoever. But what were doing in neuro, and youll later find it applies to muscle and some other cells as well, is were talking about what well refer to as excitable cells. And in excitable cells theres a sort of two way street here. In any cell, the permeabilities determine what the membrane potential is. But in an excitable cell, the membrane potential can alter the permeabilities. And the way that works is, for example, these rate constants of opening and closing depend on the electrical potential across the membrane. How does that happen? Well the way it tends to happen is these channels are proteins. Remember Dr. Kinnally described the typical transmembrane protein, which usually has like 7 transmembrane elements that together wrap around and form a channel down the middle. With some added stuff on the inside and on the outside of the cell. And all of these amino acids that make up these proteins have charges on them. So if you have some positive charges on one part of this channel molecule, and if you have some negative charges on some other part of the channel molecule, and if you change the electrical potential across the membrane that might tend to pull this part of the molecule down and this part, push that part up, depending on which way the charges are shifting and the electrical potential is shifting across the membrane. And that might change the chemical characteristics of this channel so that the rate constants for opening and closing will alter in response to the membrane potential. And so what will happen is, remember that the equilibrium constant, assuming that the, for a period of time these are both, these little Ks, are reasonably steady, then theres a rate of opening and a rate of closing and that determines the equilibrium constant, the ratio of open channels (number open over number of closed) will be so that the number of channels going in each direction is the same. This is KC times number O, thisll be K O times number C, they will be equal. So the ratio of open to closed channels will reach a steady level which is an equilibrium constant between which is the ratio of these rate constants. This is chemistry. Youve had this sort of thing when you studied enzymes in building blocks, right? 1

Transcribed by David Landsman

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The key here is that for excitable cells, these little Ks, these rate constants, are affected by the membrane potential. Lets take a look at these potassium channels in particular. Lets say you have a membrane potential of a cell and this is the resting state minus 70, or thereabouts, its different for different cells, but lest say on average its something like minus 70 millivolts. And lets also take a look at the permeability of this cell membrane to potassium. Lets do an experiment. Einstein used to refer to thought experiments, lets do a thought experiment. Lets say you depolarize this to -60 and then after some amount of time you repolarize the cell back to -70.Whats gonna happen to potassium permeability channels. Well you cant do this by thought experiment what you have to do is actually do the experiment and this experiment has been done countless times and what was always found is that in typical excitable cells you start off with certain potassium permeability and then when the cell is depolarized what you find is that the potassium permeability rises to a new level. Now here, were not letting the permeability affect the membrane potential through the Goldman equation, what were doing is holding clamping electrical, have all kinds of electronics and stuff, holding this membrane potential at -60. And you find that the membrane pot- the permeability of potassium stays at the elevated level and then when you repolarize the cell it gradually goes back to its baseline level. So now you know certain characteristics of the potassium channel molecules. And remember what were talking about is rate constants from the transition of closed to open and open to closed. You dont know whether its the opening the K open or K closed thats changing but the ratio between them is obviously changing so that when the cell is depolarized you get more channels in the open state. There are several things you can take from this one observation, several conclusions, one is the direction of the change in response to depolarization potassium permeability increases. But you can get a little more description out of it. One is that the change is relatively slow, might take a few milliseconds. And then when you repolarize it goes back to its other level, slowly, it might take a millisecond or two. The second thing that happens is that the permeability to potassium tends to depend on the membrane potential in other words once its reached its new level, it stays there. Because this is staying steady, now this stays steady. [And as a side comment, to those who might prefer to sit at home and play the itunes, if you were just listen to the audio of this, you wont know what I was pointing to when I said this and this! So its best practice to come!] OK, so we know something about the potassium channels. There is a tendency for a larger number of the potassium channels to be open while the cell is depolarized and so on and so forth. [microphone cuts out] OK, hello? OK. Now, what about the effect of this permeability change, because here I was talking about the type of experiment I refer to as voltage clamp, we are holding the membrane potential at a particular level. But suppose for some reason for a moment or so we depolarize the cell a little bit, what would be the effect on the potassium channels? They would tend to open. What is the effect of opening more potassium channels? You increase the potassium permeability, the membrane potential will, per the Goldman equation, tend to move closer to the potassium equilibrium potential or Nernst potential. This means that if we depolarize the cell a little bit the potassium 2

Transcribed by David Landsman

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increases. When the potassium permeability increases the membrane potential becomes more negative, moves towards the -80 Nernst potential for potassium and that will undo the depolarization. So basically this potassium permeability change is an example of negative feedback, you change the membrane potential (depolarize it) and this will tend to repolarize it back towards its resting level. So with the potassium channels you have negative feedback. OK, that sounds good. What about sodium channels? And if you remember from several of the powerpoints that Dr. Kinnally showed, they are different. But lets do this experiment for sodium channels. Normally the sodium permeability in a cell is lower than the potassium permeability so Ill draw it down here. And lets give a little depolarization to the cell, say to -60 mV, what happens to the sodium permeability channels? Well the sodium permeability increases [] its very fast compared to the potassium channel changes which are slow. You depolarize the cell that increases the sodium permeability. But now we hold it at this -60 and what you see is something very different from the potassium. The potassium channels, once the permeability increased with a depolarization it stayed up there. For sodium channels its declining. And finally when you turn off the depolarization it turns off back to its resting level and resumes. So the sodium channels are obviously very different in behavior from the potassium channels. Well whats going on with these sodium channels? For the sodium channels you have a closed state, in rapid equilibrium with an open state, but if you remember the diagram that Dr. Kinnally showed you, you have a sodium channel that looks sort of like this, this is the closed state because the channel is too small, closed off and a sodium ion cant pass through. And then its in equilibrium with an open state, which looks like that and a sodium ion obviously can pass through. But there was another feature that Dr. Kinnally showed that is this extra little flap or cap or something on the inner surface you get this little flap. This is a closed channel because nothing can pass through here. This is an open channel and sodium ions can pass right through. But there is a third state, or maybe a third and fourth state, in which once the channel is open, this flap can close. The door swings shut. And now sodium cant pass through. When I said a third or a fourth state, this part of the molecule can actually close its channel but as long as this cap is closed nothing can get through anyway so that functionally [there] is no difference between these two states. Sodium cant get through period. This is the closed state, this is the open state, and this one is referred to as either refractory or inactivated. [19:50] And this extra little flapper this cap on the inside surface of the cell is referred to as the inactivation gate because the gate closes the channel is inactivated, it cant pass sodium ions. And when a channel has the inactivation gat closed its referred to as refractory. So now your closed open equilibrium can also have a third state added. Does this explain the difference between the sodium and the potassium permeability curves? Actually, yes! Because what happens at every stage is there is a rapid equilibrium -- and by rapid I mean Im talking like microseconds the rapid equilibrium between open and closed states. Closed states are opening, open states are closing. Theres a certain ratio of their rate constants that determines what fraction of the channels that are oscillating back and forth here are open. And that 3

Transcribed by David Landsman

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determines the permeability. But, the total number of channels, once you start opening channels, the total number of channels that are open here, that are openable, that are participating in this equilibrium, is decreasing. Because some of these are becoming refractory. So the number of channels that are engaged in this rapid equilibrium that are determining the sodium permeability is decreasing because you are taking channels out of this pool and putting them someplace else. So the permeability declines. Once you turn off the depolarization, whatever remaining open channels there are will shift towards closed as you would expect, just as happens with the potassium except with the potassium its slower, this is faster. And youre back to the baseline permeability. But lets do one more thing with these sodium channels. Remember the potassium channels provided negative feedback, you depolarize the cell, you get increased potassium permeability which draws the cell towards -80 which tends to repolarize the membrane potential. So its negative feedback. Depolarization leads to repolarization. What happens with sodium? You depolarize the cell a little bit, the membrane permeability to sodium goes up. And whats that going to do to the membrane potential. Its going to tend to move it towards +60, which is the sodium Nernst potential. Once you start moving towards +60, that makes, if youre not clamping the voltage, that makes the cell even more positive, which opens more sodium channels. Which makes the cell more positive, which opens more sodium channels and so on and so forth. You get positive feedback. And depolarizing leads to further depolarization. So what happens then with the membrane potential? Lets stop clamping the membrane potential, lets just bring it up to -60. And you get this going up. So now you have an increase in sodium permeability, which leads to further depolarization, which leads to further increase in sodium permeability. And so on and so forth. As this depolarizes, it opens more sodium channels and brings this up to here. So you would expect [the membrane potential] to go to upwards towards the sodium Nernst potential. It never quite reaches that because the potassium permeability is never zero. So what happens? Well, the membrane potential is going to keep going up. As soon as you have enough of a depolarization this positive feedback takes over and all of the events that follow sort of go automatically. Its like you have a toilet with a tank, and you press the lever just a little bit you get a little water running, eventually it stops. But if you push it beyond a certain point you get a complete flush and the tank empties. What happens here is that once you get beyond a certain threshold, and the threshold in a resting cell is about -55 mV, the feedback is enough so that even though some sodium channels are turning refractory and getting out of the game the additional opening of the channels exceeds the ones that are dropping out of the game so you get positive feedback, further depolarization, further opening, further depolarization and so on. And you get this enormous shift in the membrane potential, which actually goes up to, well the Goldman equation would predict +40, its going towards +60 but not going to get there. So lets say it goes to +40, this never reaches +40, this membrane potential, why? It actually gets to about +10 or 15 or so, why? Well, think of the membrane, heres a membrane and lets say the membrane potential is -55 or -60 or -70, compared to the outside. See I have a sort of negative concentration of ions on one side, and positive ions close to the membrane on the other side, setting up this electrical potential. And now you want 4

Transcribed by David Landsman

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to get the membrane potential up to towards +40, which means you want to change that situation to something that looks like this. What happens? How do you get from this to this? Well, you have to move some ions, you have to take this positive ion and move it out here or in here. You to take this negative ion, perhaps its a chloride or something, and more it there. So how long it takes for this change in membrane potential to occur depends on two things, how much charge do you have to move and how rapidly can you move those ions across the membrane. How much charge is stored across the membrane? Thats a purely electrical function. The charge which for some reason is always abbreviated Q, makes as much sense as membrane potential being abbreviated E, depends on the potential across the membrane and the equation is written this way: Q = CE. C is the capacitance of the membrane. The membrane acts as an electrical capacitor. What does that mean? Well, depending on how much charge differential you have across the membrane, thats the Q, theres going to be a certain amount of electrical potential. Or depending on the electrical potential, theres going to be a certain amount of charge. Either way. If you want to change the electrical potential, make that a delta E because you are changing the E, you have to change a certain amount of Q. The capacitance is a essentially fixed constant property of a cell membrane, because capacitance of a membrane is essentially the area of a membrane divided by the thickness of a membrane times some number that relates to the composition, the chemical composition of the membrane itself. And generally that is referred to as epsilon. In the course of normal functioning of the cell, the capacitance depends on the area divided by the thickness of the membrane and neither of those are ever going to change, really. The thickness of the membrane is your 70 angstrom units. Thats what lipid bilayers are. The area is the surface area of the cell, thats not going to change, there are weird situations in which you might add or subtract lipids in the membrane and change the surface area, but rare, unusual, and not this fast. And youre certainly not going to significantly change the chemical composition of the membrane. Electrostatic properties. The epsilon. So the capacitance of the membrane remains constant for all intents and purposes. So therefore, for given change in the membrane potential, this delta E, that capacitance remains the same so the amount of charge you have to transfer across the membrane is going to be a sort of fixed quantity for that membrane potential change. How fast can you move that charge across the cell membrane? Well it depends on the permeability of the membrane, if the ions that youre going to move are potassium ions then its going to depend on the permeability of the membrane to potassium and the time. High permeability is low resistance, low permeability is high resistance. If youre talking about moving sodium ions same thing except for sodium permeability. Lets sort of lump all these things together without worrying about which ions we are talking about and refer to membrane resistance. So youve got the capacitance, youve got the membrane resistance (lets call it R), the larger the resistance the harder it is to move these ions across the membrane so the whole change in membrane potential will be slower. If you have a low resistance, a very permeable membrane a very leaky membrane, then the ions will just zip right across. Any change in the membrane potential will be relatively fast. So what we do, taking into effect the amount of charge that has to be removed depends on C. We typically multiply together the membrane resistance 5

Transcribed by David Landsman

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times the capacitance and we call that the time constant. As usually written as a lower case greek tau. And the tau is a measure of how fast the membrane potential can change. A smaller tau means a smaller resistance which means the membrane potential can change relatively rapidly. Lets put this all together. You depolarize the cell, heres the resting membrane potential, and you depolarize the cell to -55. Now the point is, whats going to happen now requires a depolarization to -55, to the threshold. If you dont depolarize to here, this is not going to happen, because youve opened some sodium channels, the open ones are becoming refractory at a greater rate than additional channels are opening so that you dont get the positive feedback. When you get above -55, the rate of channels opening exceeds the rate of channels becoming refractory so youre having a net increase in sodium permeability and that leads to more depolarization and more increase in sodium permeability and so on. So once you reach the threshold, the membrane potential wants to go up to lets say +40, the Goldman equation predicts its going to head towards +40 but it cant go that fast towards +40 because of the time constant. In order to get it to go to +40 you have to move some charges across the cell membrane. You want to make the cell more positive youre going to have to take something positive outside the cell and bring it in, thats sodium. So what youve done is youve increased the sodium permeability so sodium can move into the cell and make the cell more positive, but at a rate limited by the time constant. Now to be sure, during this phase of whats happening youve opened up an enormous number, essentially all of the sodium channels of the membrane. So the membrane has a very high permeability to sodium, and therefore as far as sodium movement is concerned the very low resistance and therefore the RC time constant is very low and the depolarization that results occurs fairly fast. So it wants to head up to +40. But what happens? The sodium channels that are open move into the refractory state typically at a rate of something like 1 millisecond, half millisecond to 1 millisecond, so the sodium channels that are all open are starting to close already and so the Goldman equation which has been predicting +40 now is predicting something lower. So now the membrane potential only gets up to about +15, by now the sodium channels have more or less closed. But what else is going on in response to this depolarization. The potassium channels are opening slowly, so the potassium channels are opening, the potassium permeability is increasing, and why is that important? Well whats happened is the membrane potential is up here at +15, the Goldman equation is now predicting that the equilibrium would be back around the resting potential cause the sodium channels that youve opened have closed. So now the sodium and potassium permeabilities are very close to what they are in the resting state. Except that youve got a higher potassium permeability which would tend to want to go to -80. So what happens is the membrane potential shifts back down and from positive its becoming more negative. Now, lets look at the shifts of ions across the membrane. Youve got to move positive charges in or positive charges out. Well youve got all this potassium inside the cell and youve just opened a whole bunch of potassium channels, so potassium can move outside of the cell fairly rapidly. So the potassium permeability gets big, but it never gets as big but it never gets as big as the sodium permeability was during the depolarization so 6

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the time constant here although its fairly small is not nearly as small, and therefore as rapid, as during the depolarization so the repolarization will be a little slower and this is caused by K moving out whereas this depolarization was mediated by Na moving inward. It goes down. Now if you watch this, this whole sequence of events is referred to as an action potential. It actually sinks a little bit more negative than 70 and then it comes back to -70. Why? Two things. By the time the voltage gets back to -70 the sodium channels are all back at their resting state, more or less, some of them that wouldve been closed are refractory but you have about the same sodium permeability as you do at rest. The potassium permeability was elevated by depolarization that had been going on and how does potassium permeability decline? Slowly. After you turn off the depolarization it takes a while for the potassium permeability to get down to its normal resting level. So during this time interval the potassium permeability is still somewhat elevated compared to the state at rest. Remember the state at rest is what produced the -70, the resting potential. But with the additional potassium permeability this brings the membrane potential closer to -80 which is the potassium Nernst potential. So this goes a little bit negative. Theres a second factor involved in getting the cell a bit more negative, and that is this: what happened over the course of this action potential? You moved sodium ions into the cell, you moved potassium ions out of the cell. What has to happen to restore the status that it had before hand? Think of running up/down an escalator. You have to do work to pump those sodium ions out and those potassium ions in. The sodium-potassium ATPase has to do work. Well its always working, but what happens in this situation? You have dumped a lot of sodium ions in, they dont distribute or significantly change the concentration of the cytoplasm in bulk. But a lot of these sodium ions that just came in are hovering in a thin layer sometimes referred to as the unstirred layer just inside the cell membrane, theres a layer of cytoplasm that has a slightly elevated sodium ion concentration compared to the cytoplasm in bulk. Consider the law of mass action. Rate constants, rate at which a reaction go, depend on the binding of the substrate. Sodium ions, the sodiumpotassium ATPase binds 3 of them, so if you just oversimplify a bit, by the law of mass action, you would expect the rate at which it pumps to be proportional to the sodium concentration cubed. Assuming the binding of the three sodiums is about equal and they dont influence each other. The fact that you have a layer of excess sodium inside the membrane will stimulate the pump to pump faster. Remember also that the Na-K ATPase pump is an electrogenic pump, it pumps 3 sodiums out for 2 potassiums in. So that tends to make the cell a little bit more negative, so that also contributes to that hyperpolarization here. So basically what youve got here is this sequence of events, increase sodium permeability is the fastest thing to happen, theres a depolarization related to sodium influx movement into the cell, you then have an increase in potassium permeability and a potassium efflux out of the cell which repolarizes the cell. And once youve reached this -55 threshold, the positive feedback takes over and it doesnt matter how quickly or slowly you reach this threshold, once youve gone beyond it, the feedback is positive and once the feedback is positive you end up getting an action potential. So the action potential is considered an all or none event, you either do or dont have an action potential. All

Transcribed by David Landsman

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of which leads up to, now that weve got an action potential, so what? As I said at the beginning the so what we will pick up on Monday.