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BIOL 4315/6315 Neuroscience Tue Th - NEURO Lecure 07 Memebrane Circuits, IV Plots, Action Potential II
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00:00 Right away. This is lecture seven . It's Thursday September 15 and we're
00:11 continue talking about the action potential. we're also gonna talk about membrane properties
00:17 more. So we already talked about membrane properties, membrane potential addressed and
00:24 introduce the action potential. And I that there are several concepts that I
00:29 like for you to know about when talk about membrane potentials and these are
00:39 . Right? So number and equivalent . And that's the first topic that
00:43 going to discuss today. We call you have this diagram to follow up
00:49 the action potentials and you have all your notes in the lecture notes.
00:54 so uh this is from the following lecture six and seven marked in your
01:02 . This is from the following lecture . Last lecture. Now, what
01:08 seeing here is some of the things we've discussed in the past, but
01:14 didn't discuss the circuits behind or representation those things within the membrane equivalent
01:21 So, what we talked about is said, look if there is a
01:27 where you have electrical equipment and that equipment is an electorate, a micro
01:33 , that micro electorate can inject We talked about current injections. So
01:38 like a switch, you can turn the electrode activity and you can turn
01:43 off and you can do it very . Okay, so you have current
01:46 that can produce these currents into the . And we discuss that these currents
01:52 square wave like and that indicates that zero. You turned on the switch
01:58 you produce the square wave like Okay and that stimulation is coming from
02:05 electrode. This is the micro And let's say this micro electrode is
02:09 a positive charge into the neuron that attached to. But this is
02:16 So instruments. Okay instruments if you on the current will produce the square
02:23 like positive current on. Okay switch switch off. However what you see
02:33 is the cellular member in response to current. Whether it's positive D.
02:38 in this direction or it is hyper . So the response from the south
02:45 the square wave like current is The response from the cell has a
02:52 bit of delay before it reaches the and when the signal stops you can
02:59 it also takes time for this membrane . This is cell versus instrumentation.
03:08 response has this kind of a rounded before it reaches the maximal deep polarization
03:14 the maximal change in the membrane And also it takes time for it
03:20 re polarize. So after the stimulus . There is a delay before you
03:26 full re polarization and these are the of the cells that they have and
03:33 properties are resistance and capacitance. Okay you look at the plasma membrane,
03:40 top diagram represents plasma membrane with potassium . So each one of the potassium
03:50 . Oh maybe I shouldn't be using marker. It's a little bit too
03:56 . We can also should have the in zoom. It's okay, let
04:04 see if I can. A Yeah, I'm probably gonna hear about
04:14 the department chair something just kidding. is my one. Let me just
04:25 this uh little household issues going on a little clean up following it.
04:34 when you look at this diagram in and the slide on the top,
04:39 talked about the fact that you have membrane and that plasma membrane is not
04:44 for the ions to freely cross. it's basically it's resistant. Okay to
04:50 flux of current. And that current if you have channels in the plasma
04:56 and those channels are open. So you think about these channels, those
05:02 and the membrane equivalent circuits can be and things for example like this is
05:11 symbol for resistor. For resistance. . Uh if you recall, resistance
05:22 the inverse of conductance. So this also a symbol for a conductor.
05:29 G or r if you see an going through it, that means it's
05:34 variable resistor, variable conductor. And what you will learn today, sometimes
05:40 channels are open and sometimes they're starting close and during those moments when they're
05:45 open, the conductance is quite high maximal. But then if you're starting
05:51 close the channel, that conductance starts . So there's variability in the conductance
05:57 on the opening and kinetic properties of channels in the membrane. The other
06:03 is you can see this symbol is symbol for battery. Okay, because
06:16 talked about the electro motive force and talked about the fact that ions are
06:20 . So you can see that there a battery sodium, you have a
06:24 distribution of sodium on the outside versus the potassium which is dominating on the
06:30 . And so you have this battery here. So each one of these
06:34 , not just potassium channels, but one of these sodium potassium calcium chloride
06:39 can be represented by these member and circuits which are built by basically comm
06:46 resistors, batteries. And talking about of the other subject matters that we've
06:53 discussed. If you remember this electrochemical force, we called driving force,
07:00 is electrochemical driving force. We talked the sports being the difference between VM
07:07 the equilibrium potential for in this case mine that it can be equilibrium potential
07:11 sodium minor. The difference in the between the Librium potentially to measure with
07:17 equation and the number of potential which measure with Goldman equation will determine the
07:23 force and the size of this driving . So this is the same arms
07:27 . V equals ir except that it D. M minus E.
07:32 E equals Ir. The change of driving force and the voltage. We
07:36 talked about conductance being the inverse of . If we rewrite arms law I
07:44 B. Over R. And Is equal one over R. That
07:49 i. Is equal conductance is the force. And we talked about it
07:54 we started discussing the rising phase and following phase of the action potential and
07:59 you through and I said that the flocks is the current flocks is conducting
08:07 the driving force. So even if channel is open is conducting but there's
08:11 driving force the driving force is The current value is still going to
08:16 zero, current is not moving in one direction. Netting into in and
08:21 of the south. So you have of these channels and each of these
08:27 have their own conductance. So you rewrite this I. K.
08:32 Tansy conductance minus conductance times D. . And you can also imagine that
08:40 you know the individual channel current And know the conductance is for individual channels
08:48 you will say well I want to the overall conductance for the cell for
08:53 of the potassium channels. So you overall conductance for potassium is calculation where
09:01 have the number of potassium channels. depends 200 500 channels times the individual
09:08 through each channel. Pretty basic. you have to basically calculate the overall
09:15 versus the conductance through one channel, calculate overall conductance for all of these
09:21 . So let's look at how the response. We already talked about this
09:28 let's look about here how the input RN what it depends on. It
09:37 on the wrestling channel density. So many channels there are. And membrane
09:43 area. And the resistance to the is the resistance of the membrane over
09:53 pi A squared. Where A. the radius of the spherical neuron.
09:59 means that the smaller the neuron, smaller the A. The larger the
10:05 the systems. So if you increase A the larger cells the larger cells
10:12 have smaller input resistance. So resistance radius here. The relationship for capacitance
10:23 what are some of the features of capacitor? For capacitance? The symbol
10:29 capacitor is this symbol here, It's to the battery but two plates are
10:38 in size should be when capacitor is with charge storage as is shown
10:49 And what are the features of a capacitor? The features of a good
10:55 that it can store a lot of to store a lot of charge.
10:58 need surface area. You have a of surface area. The other good
11:04 of a good capacitor is that the plates of the capacitor that hold the
11:09 and negative charge are located very close each other. So the closer they
11:15 , the faster that discharge can happen exchange of charge. Okay. Otherwise
11:20 they're separated by several layers. And you have separation of the capacitor by
11:26 a possible lipid bi layer, The membrane. So cell membranes are really
11:32 capacitors and if you look at this year, the capacitance of the cell
11:38 on the capacities on the membrane times by a square. So in this
11:45 the larger than your on the larger input capacity. So really large cells
11:51 be able to capacity state to store lot of charge based on the surface
11:57 and smaller cells will have smaller Then we come to this last uh
12:06 here on the right and I'm going discuss that in a second but I
12:14 to finish talking about member an equivalent . So what you can do is
12:18 can represent the plasma membrane using these and equivalent circuits. The resistors of
12:24 and the batteries. This is for showing the active activated circuit. And
12:32 does the active circuit have? That's ions? Well, it probably has
12:36 coming in from outside to inside because chloride is abundant, cellular. So
12:42 the current direction of positive ions. course every battery here fluoride is not
12:49 much, let's say during action And you'll see another example why it's
12:54 doing much in a couple of potassium potentially is going in from inside
13:00 out from inside the south to the of the south. You have an
13:05 pump that always works as long as is a T. P. That
13:09 always work. And notice that the here is going in the opposite direction
13:13 going against the concentration gradient to sodium potassium and then finally have this capacity
13:20 . So this is a really good of an active circuit using these
13:25 An equivalent circuits. And there's significant to these. You can use them
13:32 engineering world, you can use it electronics world, you can use it
13:36 computational world. You can build models models and change something about the properties
13:44 these elements. The resistance availability, open there and so on. Change
13:49 things and see how it changes the member and potential. Uh And what
13:55 you see in the membrane potential when altering these terms. So let's go
14:01 to this other slide on top. for the exam, what I would
14:06 for you to know from uh this and equivalent circuits. As you should
14:11 these symbols. You should be able recognize this is a resistor, this
14:14 a capacitor, this is a And no no the circuit for example
14:20 it's not very complicated but it should total sense to you because of everything
14:25 we're talking about resting membrane potential. we're talking about the fluxus through these
14:30 and these channels that we're talking Remember already mentioned these channels are
14:38 The permeability through these channels is And if you recall the permeability during
14:46 member and potential which is uh calculated and potential using portion of the nurse
14:57 and then introducing the premier ability term for potassium including potassium sodium and chloride
15:06 showed potassium and sodium. And I chloride doesn't really have much influence on
15:11 change in the resting membrane potential. so this top line shows that during
15:17 resting or when the cell is not active and not firing an action
15:23 potassium is leaking out of the cells has these leaky channels. And that's
15:28 the way the nature is it has leaky channels and potassium is leaking
15:32 So it's dominating and permeability and it's because equilibrium potential for potassium is
15:40 And the membrane potential addresses about Which is closer much closer to potassium
15:46 than for example sodium the second major . The equilibrium potential for sodium Positive
15:53 positive 60. And this is an of permeability, how the conductors change
16:00 through those channels, how much they conduct based on probabilities it switches where
16:07 that's our sodium becomes 20 times more than potassium. And this is during
16:13 rising phase of the action potential. the third uh line item here is
16:22 from your building and as you can first of all, chloride across this
16:31 , 0.45. And during the rising or the action potential is 0.45.
16:38 doesn't change. So it tells you . Chloride really doesn't contribute the permeability
16:44 chloride doesn't change and doesn't contribute to action potential. And if it has
16:50 contribution to the resting membrane potential it's little. And you can calculate it
16:55 you want to the D. By adding in the chloride outside versus
17:00 of the selling And these permeability ratios you're interested uh the greater concentration of
17:07 particular island and the greater its the greater its role in determining the
17:12 number of potential. So what happens the rising phase of the action potential
17:19 and potential goes up And that's because permeability for sodium has increased and sodium
17:24 ruling the number of potential for just couple of milliseconds in time.
17:31 we finally come to this diagram here is some of the things that we
17:39 about, we'll all come together hopefully the next couple of hours. But
17:47 diagram here that you're seeing is what call an I. V.
17:54 And what you're seeing on the Axis is voltage in millennials membrane potential
18:03 . M. Which stands voltage of memory and on the Y axis you're
18:08 current I which is a nanogram So all of these channels that we're
18:16 about as it relates to rest except leaky channels. They are voltage
18:22 That means that the current flocks and conductance through these channels will depend on
18:28 change in the voltage. They're sensitive voltage. Give it another 30
18:35 So there are channels that have linear . V. Plot. So I'm
18:43 make this drawing. And this is . M. This is current in
18:51 amperes. Okay this is Miller this is -80, This is -40
19:07 plus 40 Plus 80 million holes. I don't know if I'm gonna draw
19:15 diagram for you. So you may to draw it with me or take
19:19 picture. Uh But as you see the racing stuff, so maybe I'll
19:23 to take a picture if you stop before I erase everything. So and
19:29 on zoom. Can you see the ? Okay. Yes, we can
19:34 it. Okay, good. Because the best I can do with the
19:39 . Um Okay so here we have one nana one plus 29. After
19:47 here we have minus one and we minus two. And we have a
19:56 of things that are written here. of all, this is hyper
19:59 right? So from resting membrane potential more negative values is hyper polarization.
20:06 resting membrane potential. So resting membrane would be similar around here. This
20:12 resting membrane potential value. This is little the chair. And by convention
20:21 nana emperors measurers and downward deflections. is an inward car. This is
20:31 . Word card and this is outward . This is awkward and what this
20:47 . It actually shows a linear plot this linear plot is referred to as
20:59 or linear ivy plant. I stands current, the stands for voltage.
21:09 it's I. V. Plot. ivy plots are sort of like representations
21:16 how voltage controls current flux through these . S. I. V.
21:23 is a representation how the current depends the changes in the voltage. Just
21:32 same thing. Paraphrase. So this a linear plot that you're seeing
21:40 throw it in here and it's linear we're the same in the change in
21:46 membrane potential and the value of the you always get the same change always
21:56 the same change. And I should drive it to one here unless you
22:05 so one here too, you get same change and in voltage And you
22:13 the same change here also. you get 40 million volts change and
22:18 have negative one and you have positive million volts change and you have positive
22:24 nana empires. Everybody follows this. this is the same on both
22:34 So this is linear I. Plot on the exam. I may
22:39 you a question like this. I'm show you a plot that looks like
22:50 right? Or something similar and I'm ask you to think about something.
23:00 the reversal potential or equilibrium potential for is -90 million volts. Mhm.
23:10 is the driving force? If the of potential is that -90 million balls
23:17 and the equilibrium potential, potassium is v. M. is -90.
23:29 is VM -E. K. equal zero. So this would be a
23:40 of a potassium channel that has linear plot because potassium will be going out
23:52 the cell right once you raise this . And we also call these reversal
24:00 . Because if you were to hyper the plasma member, remember, this
24:05 sort of a normal regime around resting potential and there might be enough of
24:12 negative input. There might be something on with this potential becomes even more
24:17 but it's not going to stay there physiologically the state the state of the
24:24 is to fluctuate around this resting membrane back and forth produce an action potential
24:30 come back to this resting membrane So it's not going to stay in
24:35 negative potentials for a very long time it's more likely to be more and
24:40 hyper polarized negative potentials and it's definitely going to stay in the positive potentials
24:45 a long time. Because if you the cell and have it be polarized
24:51 million balls for a long time, cell will die. So it's non
24:56 . But this is what happens The other plots that I may show
25:01 and recall that whenever I'm talking about values for the reversal or equilibrium potentials
25:12 other such things you should go back this and these are your values I'm
25:21 on the exam follow these values equilibrium for potassium minus 90. I started
25:27 it reversal potential. Also started calling reversal potential because if the cell actually
25:32 polarizes below minus 90 potassium instead of there's a lot of it on the
25:40 instead of cohen going outside is gonna coming back inside. Okay so the
25:46 of the current flow through these channels now. I'll show you another plot
25:54 on the exam question that looks something this and I'm already gonna give it
26:00 by putting this value here. But say it's gonna look like something like
26:07 And I'm gonna ask you do you this is likely to be the potassium
26:13 . V. plot or sodium ivy sodium. And you would answer that
26:18 by first of all seeing where is current? Where is the current
26:23 zero. The current value is zero positive 55 million balls. Which is
26:30 reversal potential for sodium or equilibrium potential sodium. If the cell is addressing
26:37 in potential year sodium channels open, is sodium doing? There's a lot
26:43 sodium on the outside of the sodium is coming inside its inward inward
26:48 And then at positive 55 if you to de polarize or have the cells
26:54 these really positive potentials sodium current would reverse and it would become outward,
27:01 inward, that's why we call equilibrium reversal potential. So it's a real
27:08 of the direction of the flux of ion outside to inside versus inside to
27:13 of the cell. Mhm. So I should take this picture because it's
27:19 to turn into an abstract piece of . Um Okay. How many of
27:32 ivy plots, how many of these gated channels? There's a plasma member
27:40 having one south? Yes. That's a guess for each for each
27:48 and that's a good guess for the ions. However there's and it's a
27:53 good guess but there's variability in these and there are subtypes of both educated
27:59 and one cell may have 34 subtypes both educated sodium channel. Four or
28:04 subtypes of both educated potassium channels and on. So a single cell and
28:10 guess is great because this is the ions that we're talking about but the
28:16 will have variations of this channel. remember we talked about roderick mackinnon and
28:21 said how important amino acid structure is we talked about splice variants and things
28:26 spliced differently sometimes with slight variations in structure. Well that structure of the
28:32 and three dimensional structure is also going determine how much of the ions are
28:37 through, how fast it's opening. it's a channel we actually have
28:41 How fast it's opening. How fast those gates closing the gap? So
28:46 cell may have up to 10, even sometimes 20 different subtypes of voltage
28:54 channels and not all of them are have the linear I. V.
28:59 right? Some of the channels are to conduct their little inwardly but then
29:07 will conduct a lot of it So you can see that this 40
29:12 volts negative change just produces this level current but positive produces a much higher
29:19 of current. So this is outwardly outwardly rectifying channel. We call it
29:25 rectifying. It prefers to conduct. there are some channels that do the
29:32 . They prefer to conduct a lot current inwardly. And they're not so
29:37 at conducting that current outwardly. So can see that there's a lot of
29:43 here in current. Okay. And very little change if you came up
29:50 , you wouldn't even see this Okay, so this current this current
29:55 to conduct inwardly or inwardly rectifying currents just by definition what they are.
30:04 you will see these kinds of variations I. B. Plots. And
30:09 what do you do with 10 or or 20 different ivy plots? I'm
30:16 gonna use my fantasy here. You have to drive. Yeah this is
30:34 you have. So he's a two cells present in a very abstract ways
31:00 will have multitudes of voltage community channels multitudes of the curves. Some of
31:06 are linear only curves. Some of are non linear. They're rectifying
31:11 But when we talked about the dialect we said that these different subtypes,
31:16 the inhibitory cells that produce such diverse of action potentials. That diversity comes
31:25 the fact that you have different voltage channels expressed by those cells. And
31:30 different voltage gated channels will have slightly properties. These I. D.
31:36 properties and these I. V. properties will play into the overall response
31:42 the cell as it produces. You the stuttering frequencies of action potential.
31:46 bursting or continues are fast or slow so on. So maybe I should
31:53 a picture of this. Okay, Alright, the ivy plots a lot
32:18 the things that we're talking about. they're gonna be coming back up in
32:23 next hour and a half that we still about to cover of the new
32:29 . But in general we want to about action potential. The rising phase
32:34 by sodium, the following phase dominated potassium. So we talked about the
32:40 of the action potential. Okay, I'm gonna before I go here,
32:47 actually gonna go back into your action diagram and I'm gonna one more time
32:54 about it. Everything that's here. should know that everything that's on the
33:00 . UK chloride. RMP wrestling number potential threshold of action potential generation.
33:07 that's happening here in the number of fluctuations. So the cell gets excited
33:11 inputs in the dendrite glutamate. The potentials to de polarize the cell and
33:17 the potential a little bit and then inhibitory inputs come in and they hyper
33:22 yourself. And if there's enough of , excitatory input, so single excitatory
33:27 in the C. N. Produces a deep polarization of about 0.5
33:34 balls. So one active synapse deep of 0.5 million balls. 10 active
33:41 , deep polarization of five million 20 active synopsis 10 militants, 40
33:50 synopsis 20 million volts. Which is be enough to drive the cell to
33:56 threshold for action potential generation. So are greater inhibitor excited during inputs inside
34:02 cell. And graded deep polarization and polarization until you reach the spawned and
34:08 the response becomes all or not All none means that you will always produce
34:14 action potential of -45. You can it. People are as the cell
34:18 always produce action potentials. If you the stimulus, it will produce
34:23 Eyes action potentials throughout the sustained stimulus the cell gets exhausted, which does
34:29 to during the continuous stimulus. So we talked about this resting membrane potential
34:36 the south membrane is doing a random . A little bit up, a
34:40 bit down a little bit up a bit down. If it's up and
34:43 the threshold will fire the action Now this stay here, potassium is
34:51 most permissible to plasma member, potassium dominating here at rest. Once we
34:58 the threshold for action potential, we bolt educated sodium channels. Why do
35:03 open them? Because we de polarize south through the synaptic inputs enough into
35:10 positive potentials from negative 65 to It's enough now to open the sodium
35:19 educated channels. So the change in voltage is what opens voltage gated channels
35:25 then the membrane is dominated by leaking conductance. Is once the sodium channels
35:33 sodium takes over sodium with deep polarization more sodium channels which causes more deep
35:40 which opens more sodium channels. So is a positive feedback loop. And
35:45 would say great sodium is dominated and conductance drives the overall D.
35:52 member and potential to its own equilibrium value. It's saying I'm gonna take
35:57 home to Vienna which is positive 55 it fails to do so and fails
36:04 do so for two reasons. There's about the channel kinetics sodium channels when
36:09 open actually very quickly closed and you about this today. Number one.
36:13 channel kinetics number two as the number potential D polarizes. What happens to
36:19 driving force. The driving force is difference between E. N.
36:24 And the number of potential the driving shrinks here for sodium and those are
36:29 two reasons why the peak of the potential does not reach the E.
36:34 . A. Or equilibrium potential for . Now at this point when the
36:39 member it is so deep polarized, is a huge driving force for potassium
36:45 this is their russell potential for potassium number one, number two sodium channels
36:52 started closing because of the kinetics. they open they have to close,
36:57 starts ruling the game and potassium selfishly to drive the DM number and potential
37:06 its own equilibrium potential values and it succeeds. So it almost succeeds.
37:12 you have this hyper polarization that's lower the firing of action to control the
37:18 membrane potential and you have this re that slowly happens. The sodium potassium
37:24 slow wear that are working with against gradient with A T. P.
37:31 A. T. P. During area here, under the curve of
37:37 action potential, which is pretty much to the threshold with the action
37:43 it's an absolute refractory period, which that you cannot devote another action potential
37:50 the absolute refractory period. But during re polarization, when it crosses back
37:56 about -50 or so close to this is relative refractory period. And
38:02 you stimulated the cell or the cell an incoming stimulus to continue stimulus,
38:08 can produce another action potential during relative period. But not during the absolute
38:14 you cannot have another action potential on of this action potential. So that
38:19 doesn't work. There's not the way brain cells work. And some of
38:25 South will have very short relative refractory which will allow them to produce action
38:32 . High frequencies and some of them take time to recover longer and the
38:37 potential frequencies are going to be Mhm. These are the reversal
38:44 This is the reversal potential for So in fact if you were at
38:49 membrane potential and you were just talking driving force, what ion has the
38:57 driving forces, resting membrane potential? the driving force. It's council.
39:04 Because calcium reversal potential is positive. and plenty. But it's a cell
39:10 permeable to calcium. No. Is a lot of calcium on the
39:15 10,000? More than that. It's permeability is key. Calcium channels are
39:20 open or they're not located with sodium potassium channels are. So where you
39:26 action potentials you will have a lot voltage gated sodium and voltage gated potassium
39:33 where you release neurotransmitters, external You will have a lot of voltage
39:37 calcium channels. So this is important cell has its own strategy of placing
39:46 channels, voltage gated channels and receptor channels in different locations along you know
39:52 own cell body and processes. Did have a question. So for the
39:59 relative doesn't harm the cell or having stimulus during that period. No,
40:09 the cell does need time to So you cannot have neuron sustained stimulus
40:16 a long time neuron without starting to some other physiological problems. And the
40:24 have the way to come back and this, you know, shifts to
40:29 potentials back to resting moment of If they lose the ability to maintain
40:34 separation of charge, that's probably something a dysfunction in the channel and now
40:41 can have a shift in resting membrane , it becomes more positive and those
40:46 will need less excited to start And this could be a part of
40:51 too. It's also part of as I mentioned themselves, will have
40:58 , that's -70 -65. But if took to, you know, equivalent
41:03 circuits same age, the same but one of them had a mutation
41:08 channels, you're likely going to see membrane properties change the resting membrane potential
41:13 a shift the action potentials. They be faster than maybe slower, depending
41:19 on the on the dynamics of these can be longer, you know,
41:24 say if sodium channel remains open longer action potential would be longer.
41:32 very good questions. Okay, this the undershoot that I'm talking about the
41:40 over drives by dominating the membrane. , another important concept to understand this
41:50 clan. And I mentioned this yesterday this diagram looks scary but it's actually
41:59 . It's pretty old and rudimentary. there isn't gonna be scary questions in
42:04 diagram either. But do we need understand and know what voltage plant
42:10 Absolutely. Because this will really explain us how the action potential that we're
42:18 member and potential that we're measuring. can also measure individual currents for sodium
42:22 potassium and to measure individual currents for and potassium and other ions. We
42:28 to be able to manipulate the member potential because these channels are vaulted
42:34 So if we could manipulate the membrane , if we could manipulate the voltage
42:39 the membrane experimentally, we could answer lot of questions about the kinetics of
42:44 currents and which currents are dominating during rising phase, the falling phase and
42:49 on. And so the the setup is illustrated here is sort of a
42:56 to the squid giant axon watch the on that. It has two
43:03 One of these green electorate is a electrode. So this electrode or measurement
43:10 measures the membrane potential inside the south . And it is also connected to
43:16 voltage clamp amplifier. So measures BM volt meter and it's connected to an
43:25 . This green plate here is a electrode or the ground basically. Which
43:30 it's zero from the outside as we . The difference between outside and inside
43:34 the cell address is about 1965 million or so. So yeah, this
43:39 elector. Now the number two. information from the cell gets fed into
43:44 voltage clamp amplifier which has a certain voltage. What is command voltage,
43:55 voltage is what you are commanding it be. So, this is the
44:00 to command that. I want this to be minus 90. I want
44:04 membrane voltage to be minus 60 I it to be zero. You're the
44:11 , you're commanding this voltage. in other words, also because its
44:16 clamp your clamping a voltage at the member and potential value minus 17 minus
44:23 minus 30 and so on. So information is going in here and you
44:28 commanded desired potential. So you're commanded certain desire potential. And in order
44:35 hold that certain desired potential, there's be fluxus of currents that this axon
44:41 in the dish. But if this was activated by the stimuli, there
44:45 be fluxus of currents across. Or you added some chemicals that would be
44:49 of these ionic currents across. So voltage clamp amplifier is injecting current.
44:57 it's always saying basically keep this at seven to keep this at minus
45:02 This guy, the green guy is this one is dictating or commanding clamping
45:08 planted at minus 70. This one measuring measures minus 70 minus 70.
45:14 , minus 70 minus 70 Good, have a deviation to minus 40 flux
45:20 happened, inputs came in channels So in response to that, when
45:26 becomes different from the command potential, when the member and potential now shifted
45:31 minus 40 I said at the state 70 then the clamp amplifier will inject
45:36 current into the axon through this second . This feedback arrangement causes member and
45:43 to become the same as the command . So you're holding it minus 70
45:49 goes to minus 40 you bring it to minus 70. So you can
45:53 see what's going on at minus 70 what current might be flexing at that
45:57 potential value. So the current flowing into the acts on invested across this
46:04 can be measured here. And any current deviations which might be because
46:09 ions flexing, not because of the , because of the synaptic inputs on
46:13 flexing all of these deviations can be . These deviations will be actual currents
46:20 these currents can be action potential currents you snap the currents and uh in
46:27 way this is a negative feedback like the air conditioner temperature goes up
46:32 have it set at 70 goes up 75 says, no go back to
46:37 A. C kicks in negative This is the same way minus 70
46:42 minus 40. No go back to 76 if it goes from minus 70
46:47 minus 80 it's a smart system. like a sea that can either cool
46:53 heat at the same time. because I guess it's called, what
46:57 it the nest? Uh It's a control that they see that that they
47:03 these days. And I think it's enough to recognize that the temperature went
47:07 and down and kicking either cool or heater, which in Houston, I
47:11 it happens. We have this, know, we go from summer into
47:15 sometimes, or from winter into summer there's this few days where you're using
47:21 C and then wait a second, getting cold and you're turning on the
47:26 . So, but this is a feedback system, it's just feeding,
47:29 know, to the deviations from this of potential from this clan.
47:34 the voltage clamp techniques for studying membrane in the squid, giant axon,
47:40 how it started. That's how it with two electorates. Modern electrophysiology,
47:45 did with one electrode. We can it in much smaller accents and much
47:50 cells. Remember, these are one in diameter. So you can see
47:54 clearly with the naked eye and even with tweezers. Yeah, So now
48:04 and Huxley, if you remember where 1939 were the first ones to publish
48:10 action potential. And in 1963, and Huxley received Nobel Prize in Physiology
48:17 Medicine for their work on the action . So they published the first one
48:22 1939. You can see that 24 later they did a lot of work
48:29 that period of time. And after recorded action potential, they had this
48:35 . Well, what are the important is because remember they squeezed the ions
48:40 of the squid and then later they the ions out of neurons. And
48:44 said, oh these are the other . And then we're able to poke
48:50 and finally recorded this action potential. we know this. So, you
48:55 this potassium chloride, no calcium, islands are important, which are the
49:00 that produce that blip on the silla . And to answer that question,
49:05 needed the voltage clamp because the voltage allows you, as I mentioned,
49:10 clamp these potentials different what we call values. So Hodgkin and Huxley contributed
49:19 our understanding to this day of the and dynamics of sodium and potassium during
49:26 action potential. They came up with model Hodgkin and Huxley model for action
49:33 . If you're in computational neuroscience, know about it or you learn about
49:37 and most likely even use it What they did with voltage clamp has
49:43 here is that they d polarize the 2 -26. Again, this is
49:49 that the cell wouldn't do physiological, cell wouldn't sit at positive 50 to
49:54 positive 65 for the sustained period of . But this is an experimental manipulation
50:01 allows us to basically discover how these and how these channels were potassium and
50:07 channels. And if you recall this deflection and in current is inward
50:15 As I mentioned on the plots its current. So once you clamp the
50:22 from resting to -26, you do it. You're seeing this little blip
50:28 is an inward current and that blip away. And then you have the
50:33 outward current and blue and then you polarize the south zero. Lock it
50:40 zero. You can see even larger current but you also see much larger
50:47 term. The features of this inward are interesting because it turns on and
50:52 turns off and then the dominating current the outward current. You do polarize
50:58 this case to positive potentials. Now have positive 26. You start seeing
51:03 decrease in the similar current. This current, the sodium coming inside the
51:11 . Why are you seeing a decrease positive 26 As opposed to zero.
51:19 is this current here smaller than it a zero value because you reduce the
51:26 force for sodium and it's positive Which I told you go by my
51:35 , which is positive 55 which is close to positive 52. But is
51:40 any inward car that positive 52? , but there's still stimulus here.
51:45 still be polarization and there's still massive con And then look at positive
51:53 You see this little blip here. this little blip here is this current
52:01 now has reversed that sodium that's And why has it reversed? Because
52:09 65 is on the other side of equilibrium potential which is positive 55.
52:16 you still those currents have very sustained prolonged potassium conductance. So what they've
52:25 is that there is an early and conductance in the late sustained conductance.
52:32 early inward transient conductance of sodium rushing the south. And this late outward
52:40 is potassium leaving the cell. And you look then in this diagram illustrates
52:48 lot of these teachers here that this the stimulus of the polarization and you
52:54 see that during the rising phase of action to control you have several
53:02 These are voltage gated sodium channels. simplicity reasons. You have three channels
53:07 are shown here in reality activates tens hundreds, sometimes thousands of channels depending
53:14 areas of the number and so But you can see that the sodium
53:18 almost immediately after the deep polarization they . The deep polarization is still happening
53:27 . In fact there's even more deep here than when this channel was
53:33 But this channel is closed already. this is the transient nature of the
53:39 gated sodium channels as they open for close you'll understand why in a little
53:46 . And if you average over these trace system, this is the average
53:50 conductance or the some of the channels it looks smoother this inward current coming
53:58 . And these dash lines that you're here also correspond hear to potassium
54:04 So this is during the deep What you're seeing is that potassium slowly
54:10 activating individual channels. The other teacher that in biology, not all channels
54:18 participate in national potential will open at same time and close at the same
54:21 , slight variation and duration, which gonna be open the time at which
54:27 going to open. Uh And if look at the potassium channels, once
54:34 open the potassium channels, they're long and sustained conductance is so very different
54:41 these transient opening and closing civilian channels potassium channels. As long as there's
54:47 polarization is prolonged sodium potassium current So, if you were to break
54:55 the action potential, that rising phase dominated by sodium influx and then the
55:03 phase is dominated by potassium b flux potassium beating yourself and this is inward
55:10 sodium coming in. This is potassium current the same as as you're seeing
55:17 . And that's what Hodgkin and Huxley . And to do that, you
55:21 a voltage plan. So what questions you expect from this diagram, what
55:27 voltage clamp do. It allows you clamp the potential allows you to isolate
55:33 current conductance is but you're not going be asked to draw this diagram.
55:39 is it a positive and negative feedback ? Well, it acts more like
55:42 negative feedback system. So and the is, is a negative positive feedback
55:48 for sodium influx during the rising phase the action potential is it's it's positive
55:54 system because more sodium radicalization, more more deep polarization, but it's ineffective
56:00 positive feedback system because these channel they shut down very quickly as soon
56:05 they're open. So in order for to understand really how voltage gates the
56:14 , we have to understand the sodium structure. And so we'll spend about
56:19 next 10 minutes or so talking about , you recall the obvious polytrack tied
56:25 you know, I have to change building blocks. We have sub units
56:30 gated sodium channels have 1234 subunits. one of these subunits has six trans
56:39 segments as 12345 and six as As you can see Sheldon Purple has
56:48 lot of positive charge. So it contains a lot of positively charged amino
56:53 residues. What does that mean? we talked about the positively charged amino
56:58 residues inside the inner channel lumen that interacting with sodium ion stripping in the
57:03 of hydration and propelling it through the . Is because as you make this
57:09 ordinary complex structure three dimensional structure of proteins, you'll have some amino acids
57:15 will be positively charged. It will negatively charged. And it just so
57:19 that S. Four and the voltage sodium channels at the accumulation of these
57:24 charged amino acid residues hanging in this . Four. And that becomes important
57:29 sensing the voltage and opening this channel between us. Five and a
57:35 As was discovered by roderick mackinnon and channels. He talked about the hairpin
57:41 . So this is the hairpin which is from one subunit is one
57:47 loop, another subunit, another pin . 3rd 4th. This is the
57:53 filter, the innermost lumen of this that will be controlling and selecting for
58:01 in in this case this voltage sensor here as four and it actually is
58:10 here because it is drawn by the currents that are accumulated. This is
58:26 inside of the south and addressing member potential. The inside of the south
58:35 is negatively charged and the outside is charged and opposites attract each other.
58:43 so this positively charged amino acid It's like a voltage sensor. It
58:48 attracted by a negative charge that's accumulated the membrane and in this position developing
58:57 and the gates for the sodium channel closed. So this is an illustration
59:03 the gates and will have more details that. So what happens to bad
59:11 the projector is too high up. what happens during the resting number and
59:20 . You have negative charge when you polarize the cell, you have accumulation
59:26 positive charge. Guess what happens when accumulate this positive charge? And they'll
59:31 , well, what are these deep is coming from? Remember we talked
59:34 membrane going up and down synaptic excitatory inputs, We polarize the
59:39 So if they do polarize the cell , if there is enough of the
59:43 charge accumulated here, it's going to this positively charged amino acid residues And
59:52 amino acid residues are going to this sensor in S. four, it's
59:59 to slide up through the coating as is being repelled by the build up
60:06 positive charge here. It now slides through the protein and it changes confirmation
60:13 that three dimensional structure. So you a confirmation all change in the protein
60:19 which through this conformational change allowed for channel to open the gates. So
60:25 need deep polarization, its voltage gated is getting this channel, what is
60:31 the gate open or closed voltage. there's also certain kinetics in this
60:38 And these kinetics are such that if have this sustained deep polarization here from
60:45 to -40 number, that voltage gated channels will be very well activated at
60:51 threshold of action potential which is So if you d polarize the south
60:57 if you climb the potential at you should activate sodium channels now voltage
61:03 sodium channels and what you're seeing is as we saw in the previous
61:08 these individual channel traces and these channels up and they don't always open at
61:14 same time. And once they open the very closely very quickly closed within
61:20 a millisecond or so. So you four positions here. When the sodium
61:28 is closed, sodium channel actually has types of gates. These arms that
61:34 closed, we call them activation gates this ball and chain here that's hanging
61:42 in the cytoplasm, we call it activation gates. So this is in
61:48 gate. So when there is enough polarization and you have the vaulted sense
61:54 sliding up sodium channels, you have opening of the activation gates and now
62:02 have the flux of sodium through the channel. And number two showdown is
62:08 in. So you have this deflection in a single channel, inward current
62:13 coming in through the sodium channel. this ball and chain is not just
62:21 there. You change the conformational change confirmation structure of the protein. You
62:27 the gates here and this ball and because it says this conformational change actually
62:34 and plugs up the channel. So closes the challenge. So as soon
62:39 you open the activation gate, the gets inactivated with the second gate.
62:50 kind of just happened. It's it's a little bit of a charge,
62:53 it also has to do with the three dimensional structure. Like when you
62:58 the gate, something else may So in this case you open the
63:02 and this other gate comes up and and and and plugs it up.
63:08 just a complex movement of this three structure. Uh Now you have this
63:16 inactivated and this is the reason why doesn't reach its equilibrium potential value because
63:25 soon as it opens, it as soon as it opens, it
63:30 . As soon as there is a in the channel opens the gate,
63:34 same conformational shift encourages the second mechanical in the channel closure of that
63:42 So how do you deign activate? do you remove the inactivation gate or
63:50 activate this channel? The only way do it is still hyper polarized the
63:55 again to -65 million bowls. Because when you do that, this
64:04 sensor, we just slid up open the gates and plugged up the
64:07 . Now, if you re polarize cell and make the inside of the
64:12 , negative voltage sensor will be attracted slide down to the negative voltage back
64:18 its position and as it slides down the position you have a confirmation will
64:25 again, which kicks out the ball change Dean activates the channel and closes
64:31 gates. And so the sodium channel to go through 12341 and four is
64:38 same. But you cannot leap 13 you cannot leap 124. You have
64:46 have this re polarization Dean activation, sliding again of that voltage sensor Dean
64:54 and closure of the channel. So funny squiggles that I drew on the
65:01 and I said some of them are . Some of them are nonlinear.
65:05 that's because channels will have slightly Vietnam and different subtypes of voltage gated serving
65:15 will have slightly different channel kinetics, that maybe they're inactivated faster or maybe
65:22 takes longer time for that ball to because they have slightly different structures or
65:29 and it may take away that linear and make it more nonlinear. And
65:36 kinetics of these sodium channels and potassium are not the voltage gated channels and
65:42 not only influence the ivy plots that talked about, but also the overall
65:47 and properties, passive number and properties their ability to produce the active firing
65:55 the action potentials during the stimulation. lecture we will discuss this voltage clamp
66:04 and wholesale patch clamp technique and brief , you shouldn't worry about it if
66:09 not into biophysics too much. Just that voltage clamp is really important to
66:17 out these individual currents and without it couldn't do it. We could just
66:22 the voltage forever, not knowing what are contributing to the changes in that
66:28 . We'll talk about these different We'll talk about tetrodotoxin. What Some
66:35 review. Some of the things that already talked about. So some of
66:39 things will come up. Talk about anesthetics briefly and the back propagation of
66:46 action potential. So this is the for next week. And then if
66:51 recall on thursday, a week from , we have our midterm exam review
66:57 then you have your midterm exam coming uh the following Tuesday. All
67:06 great. Thank you very much for here. Thank you. Everybody on
67:11 . I took these pictures. Is gonna be helpful if I upload these
67:16 ? Okay. Thank you very I appreciate you appreciating my artistic
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