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BIOL 4315 & 6315 Neuroscience Mon-Wed 1-2.30 PM - NEUROSCIENCE Lecture 07 Action Potential II
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00:01 This is lecture seven action potential. so if we look at the at
00:10 previous lecture notes, we talked about changes in the concentrations of ions.
00:18 discussed two formulas, we discussed neurons and we discussed the Goldman equation and
00:25 is the equation that allows us to the equilibrium potential value for each ion
00:33 li ion the ionic and the number potential value is a calculation that incorporates
00:44 than one ionic species. So sodium potassium and it can include chloride and
00:51 also includes the premier ability term for specific ions as the premier ability for
00:58 ions will change. We discussed the membrane potential. The membranes are built
01:04 such a way that we have these potassium channels under leaking potassium A to
01:12 membrane potential. The membrane is most to potassium. But when you start
01:19 the action potential, the membrane becomes permeable to sodium, way more permeable
01:25 sodium potassium. And so just by the premier ability for either potassium or
01:31 in this formula here you can see it would alter very much the overall
01:38 for the membrane which is VM. ahead. We talked about how extra
01:47 increases in potassium concentrations Okay, on outside can lead to significant deep polarization
01:55 the member and potential. And so these concentrations are pretty tightly regulated outside
02:00 inside of the cell. And we the spatial buffering by astrocytes. Then
02:06 talked about roderick mackinnon as a person very interesting from the career perspective,
02:12 also from the drive and also from scientific discoveries and how he used multitudes
02:18 techniques to reveal the structure of the channel. And we talked about how
02:28 used simple animal systems such as Uh he used genetic mutations that would
02:38 mutations in these potassium channels that would to certain symptomology is in these
02:43 He discovered the hairpin loop, which the selectivity poor off the off the
02:50 . He studied a lot of very parts of this channel using electrophysiology side
02:56 me to genesis as well as as as uh alright as well as
03:08 So it's important to note that it's to use a multiple techniques like
03:14 A combination of these techniques in order solve the structure of the channel,
03:19 to visualize the structure of the he needed to use X ray
03:24 So yet another technique talked about how lot of the amino acid sequences are
03:30 and therefore understanding the function of these acid sequences within the more primitive systems
03:38 such fruit flies, for example, mean that it's really important for humans
03:43 well. Okay, so then we venturing sort of into the beginning of
03:50 action potential. But here I would for you to switch to lecture
03:55 it says lecture notes six and seven we'll proceed from here because before we
04:01 in and talk about that action potential greater detail, we have to discuss
04:06 very important properties as the brand. in particular. When we talked about
04:15 responses that sells produce. We discussed things already pointed out, for example
04:23 whenever you look at these kind of wave like images here that you see
04:30 the left and a one this is images and the figures scientific figures and
04:37 for electrophysiology that denote instrumentation or denote actual stimulation protocol in the middle of
04:47 to what you're seeing is you're seeing response which is membrane potential recording.
04:54 the left Visa current changes and it outward versus M. Words. And
05:00 convention positive current that is injected is . And the cover current that is
05:07 this direction is inward. And it actually be on a negative term scale
05:15 . So in the b what you is you have a relationship between current
05:21 voltage which we call ivy. So Y axis you have current in nano
05:30 and on the X axis you have balls and in this case you are
05:37 membrane potential of GM. So the that you can think about this,
05:43 that okay? So these are this instrumentation basically. And then I'm using
05:51 this is my protocol forgiving inward or currents to the south. This is
05:59 response that I record as traces in of potential and this is that
06:07 V. Relationship voltage current relationship or . V. Plot that I get
06:12 I simulate the cell with Albert or current and then measure the change for
06:19 step of this current for each change this current. And measuring how much
06:25 the membrane change when I injected outward versus inward card. And I measure
06:33 amplitude of the change in the membrane or deep polarization. The amplitude of
06:38 polarization and the amplitude of hyper And because I am an experimental so
06:45 know the amount of current which I measure. I can inject -19 and
06:52 ampere of current. I can inject emperors of current. And then I'm
06:58 see where the membrane potential is with one of these manipulations. Now it's
07:05 stark difference here. When you look the two images left to the
07:08 right. Otherwise did they start recording ? I did when you look to
07:18 lap again it all looks very like and in the middle it looks smoother
07:26 more rounded. And these are the uh the plasma membrane that are resistant
07:33 capacity. The properties of the membrane the way that you can envision
07:39 Is that when you use instrumentation or and you push a button, the
07:45 is immediate. The click of a . It's really fast circuits are very
07:51 be translated into neuronal circuits are very fast. If you translate it into
07:56 an analogy of turning on the light the room. So the light,
08:03 way that you instrumentation works is light on. And let's switch off the
08:11 that the cell and this light switch . This card flowing right recurrent rejection
08:17 no current flowing. The way that cell responds. It's not the same
08:23 way that the cell responses can be analogy of a dimmer. So electronics
08:31 the switch on but the lights take time. Even actually all of the
08:36 bulbs still do take some time to up until it reaches the maximum brightness
08:43 . And so it takes time for charge to load up across the non
08:51 and to discharge again across the so cell has resisted and capacity of
09:02 The way that we think about neurons that they're very small and they have
09:07 resistance. So if you want to the current inside, you're going to
09:14 the membrane. So you have to open channels. The cell is gonna
09:18 numb brain resistance and it's gonna have cell resistance. This resistance depends on
09:24 and channel density. So if the has no channels at all, nothing
09:31 current can flux inside and outside. you have to have channels. And
09:37 you have a few channels there's gonna little current flux. And if you
09:41 a lot of channels it's gonna be lot of current flux instead it depends
09:46 the density for a lot of current in that means a lot of
09:50 The resistance also is decreasing resistance Equals ir. Or change in voltage
10:01 current times resistance. That's arms law resistance in this time and in this
10:07 the self input resistance. So input is dependent on the number and surface
10:16 small neurons and the smaller the neurons the higher the input resistance. The
10:23 the neurons the lower the resistance. input resistance is the resistance of the
10:31 brain over 45 A squared. Where . Is the radius of the sparkle
10:41 . So if A. Is small . N. Is large the small
10:49 small radius large input resistance. Because dividing here RM is over four pi
10:57 two at the same time. A in voltage can also be viewed as
11:08 change across the two plates of the . In this case the two plates
11:13 the capacitor is the possibility it by . So it has an intracellular cytoplasmic
11:21 with one charge and has extra cellular with another charge. This is where
11:27 have charge separation right? It's at level of the plasma membrane have an
11:32 distribution of charge and we have charge at the level of a plasma
11:37 That way to you change the voltage a change in charge queues charge over
11:45 capacitor. So capacitor can have more or more negative charge. And you're
11:52 that charge over the capacitor. So change voltage charge has to be added
11:58 removed from capacitor. And if we at the capacitance properties in the input
12:05 in particular, we're going to focus this. In contrast to input
12:10 the input capacitance is the capacities of membrane. In this case times four
12:19 a squared. Which means the larger radius, the larger the neuron,
12:26 more capacitance and capacity input that neuron in this case the smaller small girls
12:37 input resistance if you had small neuron would have small capacities. But if
12:45 have a large mirror it will have lot of capacities. Um Is the
12:52 membrane like the membrane membrane are a . Uh No, because resting channel
13:09 may change. The opening of the may change, the channel expression may
13:13 . So it's somewhat fluctuating within a dynamic range. And it also depends
13:19 you're talking, resting membrane potential is to be fluctuating within like 2030 million
13:25 dynamic range. But once uh action happens or the secular release happens,
13:31 learn there are certain moments where the surface area in neurons increases. So
13:38 example, an external terminal when vesicles to the membrane before they release
13:44 all of a sudden those pre synaptic just gained an extra patch of membrane
13:52 is extra surface area and increase So there will be some transient changes
14:02 are more abrupt that are related to potential firing or the secular release.
14:07 addressing number in potential will be fluctuating a certain dynamic range where it's uh
14:15 some minor changes but not very noticeable unless you have to sell it very
14:22 activity. All of the currents of and resistance changes in capacitance changes or
14:29 releasing the vesicles and binding the vesicles the number and capacity goes up.
14:36 when we talk about neurons in we can view membranes as numb brain
14:46 circuits. And so if you look what is illustrated as each one of
14:53 channels, remember the channels are selected potassium sodium chloride in this case potassium
15:01 . Each one of these channels is resistant. It's a channel because the
15:06 can be closed and resistance to pass , that channel is very high.
15:11 channel can be partially open and the is lower, channel can be fully
15:19 and the resistance is the lowest it be to pass through that channel.
15:25 the symbol for resistance is this right and a lot of times if you
15:35 G. Or conductance is inverse of . So you can view this as
15:41 symbol for conductor or a symbol for . Either way resist or a conductor
15:49 sometimes you will see something like like an arrow going through the
15:53 which means it's a variable resistor, variable conductor. So that means that
15:59 it's open it can conduct a lot it's half open and conduct a little
16:04 it's closed, it doesn't conduct its amount of conductance. Each one also
16:11 the ions has a battery and battery as this. And it's important to
16:17 these symbols because this is some of basic physics circuits and electronic circuits that
16:26 is essentially built on and carry these and then they become really sophisticated
16:32 It's like so but you have uh , right conductors, variable conductors,
16:40 is the battery. And then in to to these two is the
16:47 And you can see that each one these images, each one of the
16:52 will be depicted as having their own or conductor and their own battery.
17:00 battery, the sign of the battery philosophers is minus is inverted here because
17:07 the concentrations and the separation of charge is different potassium inside dominating sodium dominating
17:16 the outside. So the other way we can think about Homes Law and
17:25 will come into play is with respect electrochemical driving forces and the driving
17:34 In this case we define as the between the membrane potential D.
17:41 And the Librium potential for a given on U. K. This is
17:46 same most law vehicles I. But here are the driving force is
17:51 difference how much of a driving force ir the difference in the number of
18:00 and the political potential conductance is inverse resistance. Therefore I. Is equal
18:07 over R. Or I. Is conductance times of driving for us the
18:17 . Is equal G. V. sometimes gamma. Sometimes it's depicted like
18:24 for individual uh islands and individual channels is I. Is equal G.
18:30 . In this case the conductance finds driving force dM minus C.
18:37 Remember that equilibrium potential values for single . The number of potential value is
18:46 from at least two different islands. in the potassium you can also chloride
18:54 a lesser degree. So now if want to look at the current calculation
19:02 is equal conducting stands the driving force equal conductance times V conductance minus conducting
19:13 of liberal potential. So current generated chemical gradient of potential difference. And
19:21 would be for like an individual conductors potassium channel. And if you wanted
19:28 know for example what's the total If you determine the individual conductors of
19:34 channels. And also if you know total number of potassium channels and individual
19:40 is you can calculate the overall conductors potassium. So you can use
19:47 You can use sophisticated techniques, you block all the channels. You can
19:51 what we learn later technique called voltage and you can detect single channel activity
19:58 then you can detect activity through all the potassium channels if you want all
20:02 the sodium channels, all of the flexing through the self positive and
20:07 So there's different ways that you can these conductors is but if you want
20:12 of the potassium conductance is we need no, hopefully no. The individual
20:19 to potassium channels and the number of if you're recording from. And some
20:24 these things have been standardized already in corporations. So the last symbol
20:30 that's important to add that we already also is a capacitor And the symbol
20:38 the capacitor is this? So it's plate that stores a lot of charge
20:46 there are certain features that make capacitors . So we talked about how good
20:53 should have a lot of surface area the more membrane or surface area it
20:58 , the more charger can store good should have two plates that are located
21:05 to each other. And in this we're talking about possibility by layer.
21:10 the separation is only basically the possible . Yeah, the capacitor should charge
21:17 quickly and also discharge quickly. So you put positive charge and take away
21:26 charge that has to be done This is a whole circuit that kind
21:34 a depicts next to sell you Uh huh. And the cytoplasmic side
21:45 have batteries for sodium chloride potassium you movement of sodium current. So this
21:53 an active numbering circuit. sodium is inside from outside. Like this would
22:01 is moving this direction. So maybe is actually something happening like during the
22:06 potential and this symbol here are the . Remember that N A K A
22:14 . P. S will always work the concentration gradient. So this is
22:18 blocks of sodium inside the pounds will sodium to the outside of the cell
22:27 cm which is the capacity. So capacitance properties and remembering. And so
22:34 why when we talk about cellular we talk about this gradual build up
22:41 charge. And is it a good ? It's very good capacitor because the
22:47 build up takes only a few So we're not talking about stimulating neuron
22:53 reaching its peak deep polarization seconds later tens of milliseconds later, it's typically
23:01 few milliseconds to reach the peak charge polarization or to reach the peak hyper
23:09 . And once the stimulus stops, takes also just a few milliseconds for
23:15 stimulus to re polarize with a number potential to go back to its previous
23:21 values. So this is more about membrane properties. It's important to understand
23:31 the statements here is that uh capacitor leaky because channels are open and they're
23:47 and they're using potassium. So it's like a really tight historic charge
23:56 So somewhat of a legal capacity. when we talk about the two formulas
24:04 , when we come back we discussed equation. The nurse equation was using
24:12 a qua Librium potential calculation for a ion. So e ionic or potassium
24:17 on the facility and so on this . Goldman equation, which literally is
24:22 , Hodgkin and Cats equation steady state . It talks about permeability with different
24:32 and it uses the same R. . ZF. Here you just I
24:40 take the surveillance out if you have one and you calculated with chloride if
24:49 want to. But here you basically talking about permeability as probably some of
24:57 most important variables because the concentrations on outside and the inside again, they
25:03 change much. They will fluctuate but certain dynamic range. So you have
25:08 30 million miller of ion on the of the south. It's not going
25:12 drop the $20 million will go 1 32 1 33 1 31 28.
25:19 know. But it will fluctuate if goes outside that range. We saw
25:24 potassium builds up on the outside the will become d polarized and will become
25:31 . So the concentrations of the science the outside of inside are pretty tightly
25:36 within a certain dynamic range. But permeability is can very drastically change.
25:43 that that resting membrane potential or before activation of the action potential, potassium
25:52 the highest premier ability. This the ratios of potassium versus sodium versus chloride
25:59 the rising phase of the action potential is sodium sodium. The plasma membrane
26:07 the highest permeability to sodium during the phase of the action potential. And
26:13 by changing the permeability values. But keeping these concentration values pretty constant,
26:21 can alter the membrane potential significantly the concentration of particular island and the greatest
26:31 and permeability. The greater role in the overall number and potential. So
26:37 the membrane is most preferable to potassium is probably going to influence the
26:42 potential to be closer to its own potential value. If the plasma membrane
26:49 most profitable, the sodium sodium is to try to drive the number of
26:56 to very deep polarized values to its equilibrium potential values. And we'll see
27:02 as it plays out during the action . So the action potential recordings and
27:10 I had this uh diagram will be so far. We understood what's happening
27:17 the wrestling number of potential, unequal of charge and a leaking membrane leaking
27:27 that has the biophysical properties that we with resistance capacities. Now from the
27:36 changes and a lot of other things during the action potential. When we
27:41 voltage gated sodium and potassium channels and understand a lot about how these
27:49 voltage gated sodium channels are different from gated potassium channels and how they play
27:56 differently in dominating different parts of the potential, sodium dominating rising phase,
28:04 dominating the falling phase as well as resting membrane potential phase. So let's
28:15 a video. The careful applauds, plans and habits are so very different
28:46 those of humans that there might almost aliens from another world. So perhaps
28:52 not surprising that it took a long for scientists to discover that there are
28:56 similarities between the nervous systems of pods vertebrates. Yet it was the recognition
29:07 a useful difference in their nervous which enabled scientists to undertake research that
29:12 led to a growing understanding of the controlling our own nervous system. The
29:19 concerned the nerves that control the contraction the mantle, muscles used in jet
29:26 , as this archive film shows by contracting its mental muscles. Even a
29:32 sized squid can inject a huge amount water with great force. In the
29:42 19 thirties, the british zoologist Professor . Young was engaged in a study
29:47 the squid's anatomy. Young observed an of large tubular structures, each as
29:54 as a millimeter in diameter in the mantle as these structures were never filled
29:59 blood. They could not have been vessels from their similarity to surrounding nerve
30:05 . Young thought they must be single . Giant axons, they're transmitted nerve
30:11 from the concentration of nervous tissue called ganglion to the mantle muscles using
30:22 he stimulated the surrounding nerve fibers and that he could only produce large muscle
30:28 in the mantle when the large tubular remained intact. So these were
30:39 giant axons. Scientists quickly appreciated the of young's finding. For here at
30:49 was an axon, large and robust to investigate with the techniques available at
30:53 time and one that survived for several when isolated from the nucleus, the
31:04 contents of the giant axon could be and analyzed, leading to the discovery
31:09 sodium ions were more concentrated outside the cell and potassium ions more concentrated
31:18 By refilling the empty axons with solutions precisely known chemical composition. Experimenters were
31:24 to unravel the mechanisms of iron transport the membrane. The giant axons are
31:35 enough and robust enough for fine electrodes be inserted through the cell membrane and
31:40 the axa plasm. In these early , a fine glass tube was first
31:51 into the axon and secured with Then the tube was used to introduce
32:17 fine wire electrode from which the voltage the inside and the outside could be
32:24 . But the formation of the Nerve was far too rapid for detailed study
32:29 any of the electrical measuring devices of late 1930s, It wasn't until the
32:36 following the wartime improvement of electronic equipment as the cathode ray Oscilloscope that major
32:42 was made. Scientists found that the impulse was transmitted as a characteristic wave
32:51 electrical potential and that this all or action potential was generated mainly by transient
32:58 of sodium and potassium ions across the membrane. Research on the squid giant
33:07 unravel the mechanisms of the formation and of the nerve action potential. This
33:13 led directly to the development of drugs block action potential formation and so act
33:19 local anesthetics now used routinely as painkillers dentistry and minor surgery. So all
33:26 these techniques were absolutely necessary to discover basic rudimentary things about neuronal signaling and
33:35 potentials. And this is think about , this is 1930s. Put it
33:39 the historical perspective a little bit. what professor young is doing, he's
33:46 the squid that he finds the And he remember we talked about how
33:53 can study slow and fast acts of transport or external transport. So the
33:59 slow external transport studies were essentially injecting stained dye molecule with something else of
34:09 and looking how long it takes for to travel, measuring the distance over
34:15 . Uh he also stimulates the axons he sees the contraction of the mantle
34:23 that that's where in 1930s, he doesn't record the action potential. So
34:31 with the developments that come about uh the late thirties and later in the
34:39 and fifties people start on a regular recording action potentials when they first start
34:46 action potentials from these giant axons, one millimeter in diameter. So you
34:53 see them with the naked eye. And of course later fifties, sixties
35:00 eighties people start recording from individual neurons in the brain from much smaller axons
35:07 dendrites and and so on. So Hodgkin and Huxley that are responsible for
35:16 the action potential. These two giants and they received the Nobel prize in
35:23 and medicine for their work on the potential. And the best way for
35:29 to kind of start putting everything back is if we open this diagram that
35:36 prepared for you on the action potential equilibrium potentials and this is what I
35:42 like for you to study for the . And in fact I recommend that
35:47 you look about three lectures of this and dedicated to the action potential we
35:54 about and we will continue talking for next lecture again about the action
36:01 So sometimes students ask me about, what are the good ways I can
36:06 or prepare for the exam? What of tools I can use? These
36:10 of diagrams and slides are your great to prepare and study for the
36:17 for example have a digital copy of or printed copy and define every single
36:25 that you're seeing on the slide. can you do it? What is
36:29 . K. Equilibrium potential for What about E. N.
36:43 In other words there will be questions the action potential. You can help
36:49 answering a lot of these questions if actually went through this diagram and pointed
36:56 everything and written down everything that you've on this particular diagram. So for
37:03 RMP is resting member in the The resting membrane potential is about -70
37:13 . It doesn't mean it will always here. The cell receives excited their
37:18 . The number of potential will be as a synaptic excited to really intimate
37:23 . The cell will be polarized if cell receives inhibitory inputs, the number
37:27 potential will hide for politics If from membrane potential, the cell receives enough
37:35 the excitatory input and deep all arises the action potential threshold value, which
37:41 -45 million balls that will produce all event. So these are synaptic inputs
37:48 are graded. Some of them can larger polarization, smaller, deep
37:54 larger, smaller hyper polarization. But you reach the threshold it's an all
38:00 event. And the altitude of these potentials in the same south is also
38:06 constant. So now what happens is is the equilibrium potential for potassium.
38:15 is for chloride in this direction. have deep realization in this direction and
38:21 . What other else can you take ? How do you calculate the liberal
38:25 learns the equation? So for the equation, what are important terms in
38:29 equation are TCF? Is there a of potassium on the outside or inside
38:36 ratio. This you covered all of two or three slides of potassium.
38:40 need to know what else can you here? Outside potassium goes up,
38:44 get deep polarization so you can group of it just one corner here
38:51 what else Leakey potassium channels. The membrane potential is dominated by potassium at
38:58 rest. It's leaky. Therefore the of the number of potential value is
39:03 to potassium, right? It's not preferable to chloride. It's similar to
39:08 Bowser but not much of florida's Its mostly dominated by passing. So
39:15 of course it's influenced by islands there's flux of chloride, there is small
39:19 of sodium going on and that's why not exactly the equilibrium potential for potassium
39:25 other ions are flexing at the same . Now, if it reaches this
39:31 here of action potential threshold, what here is the sodium channels both educated
39:38 channels which will start today. dedicated sodium channels that open and more
39:44 opens more deep polarization, multipolarization, sodium or globalization, more sodium multiple
39:51 , it's a positive feedback loop. , once in this situation here at
39:58 , the number is most vulnerable to . Once you reach the threshold and
40:06 channels open, the plasma membrane becomes permeable to sodium. What sodium is
40:13 try to do is going to now drive the VM. This is VM
40:21 . So sodium will drive the VM , which is interplay of other islands
40:27 to the equilibrium potential value for sodium it will fall short of reaching that
40:36 . The reason also is the When we talked about driving forces which
40:41 the difference between DM. And K. Addressing member and potential.
40:46 difference between E. K. And . And P. Is very
40:52 That's not what's driving this potassium is the driving force that's driving potassium.
40:57 the leak. It's just potassium leaking a trusting member in potential. The
41:05 force, the difference between E. . A. And resting membrane potential
41:11 value here is huge. The sodium huge driving force but as the member
41:19 potential becomes more and more and more polarized. The difference between this green
41:26 which is equilibrium potential for sodium and the number and potential value decreases.
41:34 the driving force for sodium decreases. that's one of the reasons why sodium
41:40 manage to drive the number of potential the way to its equilibrium value.
41:45 the second reason is we'll learn is kinetics of sodium channel the sodium channels
41:51 and they also close very quickly. that's the second reason. Otherwise,
41:56 it was positive feedback loop, the the more the more the more the
42:00 it should reach the equilibrium potential and there. But it doesn't. So
42:05 channels close At the peak of the potential and the sodium channels close now
42:12 at the number of potential here, the peak of action potential, let's
42:17 , positive 30 million balls And the potential for potassium of -90. So
42:26 this point potassium has the highest driving and potassium channels they delayed and they
42:35 and delayed fashion and then they take . Now potassium says, okay,
42:41 had your chance. I'm gonna now this membrane potential to equilibrium value for
42:49 . It actually goes below the resting potential. It doesn't quite reach the
42:56 potential because at that point the sodium start recovering this flux is of other
43:03 and and the cape pumps are also in working against concentration gradient in trying
43:10 restore this membrane potential back to the valley. So now you understand how
43:18 driving force, which is VM. difference between membrane potential and the delivery
43:23 potential plays into influencing the flux of ions and how the driving force has
43:32 as the number of potential changes and relationship to equilibrium potential values. And
43:39 you see how you have a sodium influx ng during the rising phase of
43:48 potential of potassium dominating the flux ng the following phase of the action.
43:58 , during this portion, when the reaches the action potential threshold value during
44:07 portion here, in pink. If were to inject more current or the
44:13 received more excited for example. So question is, can I even produce
44:18 larger amplitude action. Can I produce action potential on top of this.
44:22 cannot and that has to do with kinetics of the channels that involves sodium
44:28 potassium channels. And how do we that sodium is dominating here? We'll
44:34 about this in a second, but cannot produce another action potential. But
44:39 , for example, once the number potential re polarizes again close its resting
44:45 potential value, the stimulus was strong year, it would evoke another action
44:52 . So this is called this absolute period and this is relative refractory
44:58 And if you recall some cells will able to produce very fast patterns and
45:04 of action potentials, 600 action potentials second. And other cells will produce
45:10 23 action potentials a second and a of it has to do with their
45:16 to in the duration of the relative period, their ability to conduct different
45:23 and the expression of different ironic channels is slightly different. So these voltage
45:29 channels, sodium and potassium will also their own slightly different subtypes of these
45:37 and different subtypes of neurons that we about. They will express these different
45:42 of sodium and potassium channels. So of them will have very fast sodium
45:46 and therefore very fast recovering sodium channels therefore being able to produce very fast
45:54 of action potentials and others may have subtypes of voltage gated sodium channels of
46:00 gated potassium channels that are slower. takes longer time for the number and
46:05 recover for the channels to recover to again in their full abilities. Um
46:16 need to explain again why? So are able to doesn't reach the 55
46:26 okay. It doesn't it still but it comes closer because it's a
46:39 membrane that prefers to conduct us of . Yeah. And also because potassium
46:47 channels open and close very quickly. want potassium channels open and actually stay
46:53 longer too. So it gives longer for more potassium conductance is to come
46:57 . It doesn't quite reach the but it gets closer. Yeah,
47:01 almost there. Uh And maybe some it will be a little bit more
47:06 on a few slides too. This is what's on your test. It's
47:23 action potential. Well, I a lot of things that people
47:32 Yeah, let's say it's ubiquitous that going to talk about it as neuronal
47:37 potential. Not for any self but neurons. Yeah. When you talk
47:41 muscular action potential, talk a little about muscular action potential, discuss neuro
47:47 junction, you're gonna have a lot calcium and bowling. The action potential
47:53 will be longer. The same with muscle action potential too. So this
47:59 neuronal action potentials. But yeah, cells will produce these action potentials,
48:06 internals will produce them for kIM ji of the cerebellum bill all that's the
48:12 . Remember? They'll have different dialects that's the language we will not Andrea
48:18 have these very slow calcium ways of waves of transferring information and transferring to
48:26 and ions across the networks. But is what what I would recommend is
48:35 on the slide. Take notes, down everything you can if you don't
48:41 back look at that. The I think it's it's a good way
48:43 think about the dynamics of action potential a good way to digitalize the driving
48:49 of the size of that driving Um And yeah you will not need
48:55 do any calculations but you need to the different terms and the differences between
49:03 equation and Goldman equation and you need know the relative ratios of the ions
49:10 versus inside either milli molar ratios and do need to know the equilibrium potential
49:17 . Action potential threshold value, wrestling of potential value. So there's a
49:22 of information on the slide that's contracted two or 3 lectures. Good study
49:32 . So Washington and Huxley recorded action but how do we know how different
49:42 is are really taking place? We to record them. Remember roderick Mackinnon
49:49 was not satisfied to calculate and to the structure of the channel. Used
49:55 crystallography. So you can visualize the . So the same way if you
50:01 Professor Young and you had the concentrations ions and squeezed out of the axon
50:08 calculated Yeah the outside inside nursed he Professor nursed and plugged into his equation
50:17 the time. Then you would calculate equilibrium potentials. But have you seen
50:29 ? You just calculated them you've seen you know the concentrations of violence that
50:35 calculated, you know our T You know that right? We got
50:43 -19 for potassium positive 55 for From these calculations prove it, prove
50:53 that it is self in a real . And how is proving down?
51:01 you have to invent a new technique you have to show something in a
51:05 way. So if you calculate the potential, women would be nice to
51:10 an experimental technique where you can measure equilibrium potential. And so in order
51:16 measure the equilibrium potential, we had invent not us but scientists in general
51:22 me had to invent a voltage clamp . This is an older technique where
51:30 clamping and your commanding the voltage of number. Why is that? Because
51:37 I told you that. Oh well you have a cell right. And
51:42 put an electrode inside the hotel and gonna record minus 65 mil a bowl
51:48 here. Huh? All right. here you are son of a like
51:56 -65. And then the positive stimulus in it's -55 provide an action
52:03 So, right. You're recording changes voltage mila vaults. But How do
52:16 get to the equilibrium potential value for ? Which is -80. And have
52:23 there. How do you get to equilibrium potential value for sodium of positive
52:29 ? Experimental. So you use this clamp technique and you want to be
52:35 to clamp or command the voltage You don't want to just sit there
52:42 the electorate passively. Oh minus 65 50 action potential. Fire grate and
52:47 current. Oh, nice, But you want to be able to
52:51 out individual currents. The action potential we're studying is a combination of sodium
53:02 coming in and dominating during the rising and then potassium influx sing during the
53:11 things. There's some overlap between these . We didn't know that sodium dominates
53:17 and potassium dominates here. Until we the voltage clamp and what voltage client
53:24 . This voltage clamp allows you to that I want to keep this member
53:28 potential. This is again the square like a parent. So, this
53:33 the instrumentation. I want to clamp potential at negative 26 At zero positive
53:42 positive 52 positive 65. I want see what happens to membrane potential as
53:52 doing that. I wanna see what are flexing. I want to see
53:57 direction they're flexing. I want to one. The sodium active one is
54:02 active. So this technique is invented clamp where you have an electorate.
54:08 green measuring the membrane potential Gm And connected to voltage clamp amplifier here.
54:16 it's measuring this difference with respect to reference electorate or the ground zero
54:22 So let's take -65 here and it's the voltage here. But here you
54:28 , you know what I want to . Use this voltage clamp amplifier and
54:33 want to command and clamp this voltage of -40. So you are in
54:40 experimenter is the one that is clamping commanding a -42 -41. Does't matter
54:49 -50 positive point. You're gonna put command now, when the membrane
54:58 Deanna is different from the command the clamp amplifier inject parent into the
55:06 through the second electorate. So you this injection elector that will pass the
55:13 . This feedback arrangement causes the membrane to become same as the command
55:19 And in number four, the current is flowing and is measured here back
55:25 the axon can be measured. And is the membrane comment. So,
55:31 try to make sense of this a bit. So Effects of changes in
55:38 potential depends on the conductance is of islands. We just discussed that this
55:45 like a negative feedback system. So think about air conditioner. You said
55:51 at 66. Really spoiled and the goes up to 67 A.
55:58 Kick Sim was called there brings it to 66, it was up to
56:02 kicks in brings it down to 66 feedback system. Okay so the same
56:09 here you set the command potential of 40 and it goes to some value
56:15 negative town. So the feedback negative system comes in and injects the difference
56:22 measures that differences, injects the And now you know that everything coming
56:26 here is coming in from like synaptic . Modern voltage clamp does not require
56:34 sophisticated multi electorate setup. It uses single electorate because the circuits are super
56:40 and they can inject and record currents the very, very fast fashion.
56:43 you don't need two electrodes voltage clamp still being used to this day.
56:49 don't worry about the actual diagram here you're seeing that you will have to
56:54 a label on the exam. But should understand that voltage clan is necessary
57:01 order for you to command or clamp number of potential that the desired
57:07 And you would want to do that you wanted to study individual ionic conductance
57:12 so do conductance is versus potassium versus and others. And so Hawkman and
57:21 uses the voltage climb and then. minus 26. And remember we talked
57:27 the currents inward currents, negative nano . milli amperes in this case.
57:34 so there's inward current and this inward , this bump here is followed by
57:40 outward current. The more deep the more inward current but also followed
57:46 more outward current. So what's happening here we are at -26 mil
57:52 Also we had passed the action potential . This inward current, the sodium
57:57 coming in and then it switches sodium coming in and then switches to potassium
58:04 going on. This is this is deep polarization. So despite the sustained
58:11 polarization the inward current is short and and it gets taken over a couple
58:20 milliseconds later it gets taken over by current. So now you have isolated
58:27 inward and the outward current the inward is the sodium. The outward current
58:31 the potassium You d polarized it to 52 mil evolve value and the inward
58:42 has disappeared. You have just reached collaboration potential value for sodium because if
58:50 deliberate potential there is no net flux sodium And you still have this really
58:59 output current because the liberal potential value potassium is actually wait, wait,
59:05 negative potentials. What happens if you to positive 65. So you go
59:11 the other side of the equilibrium potential a lot of times the equilibrium potentials
59:17 also referred to as reversal potentials. if you can see this little blip
59:24 , this little blip on the this little blip is inward current.
59:28 used to be inward current that now outward current. So that the equilibrium
59:34 value. If you cross these values one or the other direction, the
59:39 direction reverses. So sodium current can outward current if it's on the other
59:45 of its equilibrium potential value, otherwise inward current inward current until it reaches
59:53 potential value where there is no current if it crosses equilibrium potential value,
59:59 is positive 52, positive 55, actually becomes an outward current. So
60:05 what this is why we refer to political potential value sources reversal potential values
60:11 general. The early phase of this polarization is dominated by inward current and
60:18 transient and the late phase is dominated the potassium current which is prolonged and
60:26 . If you look at these traces , each one of these lines,
60:30 one of these traces is a single this case sodium channel in this case
60:37 single potassium channel. This is during rising phase of the action potential
60:44 This is also during the rising phase the action. So using voltage clamp
60:49 , non Hodgkin and Huxley and others able to identify and measure not only
60:56 and outward currents, but using patch and voltage clamp techniques were able to
61:02 individual channel currents to individual channels and for example, this inward current of
61:12 is a reflection of multiple sodium channels thousands of channels opening up during the
61:19 phase of action potential. And it just like in the previous diagram that
61:25 the sustained deep polarization here, inward is fast activating but it's transient,
61:32 no more inward current, 1.5 to milliseconds later. And that's because you
61:38 see that individual sodium channels open and open and close open and close.
61:47 this is average or some sodium current all of the channels would have this
61:55 smoother looking inward current graph for outward . You can see that during the
62:03 potassium channel opening here individual traces are blue and during this initial phase of
62:10 deep polarization rising phase of the action potassium channels are not opened. So
62:18 member of potential has to be d for a while in order for the
62:23 channels to open. Once they do all of these channels, they never
62:29 at the same time. This slightly in thermodynamics and in individual channels surrounding
62:39 . But once they open they close sodium channels, potassium channels take time
62:45 open so they're delayed in opening. once they open their opening is sustained
62:51 prolonged. So the outward current, can see the curve of this outward
62:57 , the influx of potassium and blue much longer and this is what you
63:03 say, a net trans membrane It doesn't mean that during this phase
63:10 of the potassium channels are closed. one all time when I was starting
63:16 open but it is totally dominated and by sodium. So the net is
63:24 the sodium influx. The falling the nut is the potassium influx.
63:34 this maybe will answer some of the that you had a little earlier about
63:41 not reaching the equilibrium potential sodium or number of potential not reaching equilibrium potential
63:46 sodium, sodium channels are consisting of sub units 1234 each one these are
64:00 gated sodium channels are so neat. one of the sub units has six
64:05 member excitements. S 123456. This the poor loop that was discovered originally
64:14 roderick Mackinnon. So we have the filter selectivity for potassium channels. We
64:21 sodium channels and all selected channels. can see that subunits will come together
64:30 put these loops for the selectivity for trans membrane segment. S four has
64:39 lot of positively charged amino acid residues as the protest the voltage sensor.
64:48 these channels are gated by voltage. what it exactly means. Is that
64:54 is going to either open these channels close these channels in this case,
65:00 by voltage and the sodium channel has gates that keep it closed and this
65:09 charged Resendues that are located and positively residues and s. four. This
65:21 the channel and it's closed these positively residues that are sitting here in
65:33 Four. They're attracted by negatively charged of the number and and they are
65:45 in part by positively charged outside of moment. So they're sitting in this
65:50 but now as the number of potential and the cell receives these synaptic
65:57 excitatory synaptic inputs and there's deep what happens is now there is positive
66:04 build up on the inside of the and guess what happens to outside becomes
66:12 negative respective inside inside becomes more positive positive charge and starts repelling the voltage
66:22 . So these voltage sensors are the dimensional amino acid structures. All the
66:30 that are sitting within the sub units the channels and as they get repelled
66:35 the build up of positive charge, literally start moving and changing the confirmation
66:43 the entire protein channel as they change confirmation the protein channel gates open.
66:51 sodium contains two gates, It has activation gates and that is the gate
66:59 opens for sodium to be conducted. as this moves up as this voltage
67:09 moves up and changes the confirmation for gate to open activation gate. The
67:17 confirmation will change will influence the closure this ball and chain mechanism through inactivation
67:30 that's the reason why sodium channels they . You have the sustained deep polarization
67:36 sodium channels open and close Open and . Open and close. If they
67:42 open and sodium was flocks ng and inactivation gate wasn't closing then they would
67:47 open for a long time more like channels for long. But they have
67:51 inactivation gate which swings and closes and you have to move the sensor down
67:59 . You have to reposition the sensor up and down. And the only
68:04 you can reposition the sensor back into position and have the gates closed again
68:09 if you go from d polarized potentials minus 40 back to hyper polarized.
68:16 this is the second reason why the sodium minds don't reach the liberal potential
68:21 because they're fast activating but they're also and activated. And the only way
68:28 you can d inactivate them and close is if you hyper polarize the plasma
68:37 . So this is great stuff and slightly over time. I appreciate everybody
68:42 here. I will see everyone on for one more impersonal lecture and then
68:53 Wednesday we'll have our resumes on monday will have the zoom link for you
68:58 Wednesday. Okay thank
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