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BIOL 4315/6315 Neuroscience (18563/18591) - NEURO Lecture 8 Action Potential II Tue Th
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00:01 this is lecture eight of neurons and two lectures. We've been covering resting
00:07 potential and we talked about a couple important concepts and in particular we talked
00:13 how membrane doesn't respond to square wave stimulus in the square by like
00:21 but instead it has resisted the capacity properties. And so in reality plasma
00:28 can be represented as a membrane equivalent circuit like you would see in the
00:38 and the components of the circuit are the channels are described as resistors and
00:44 is a symbol for a resistor or . So conductance here G.
00:50 Conductance for potassium overall across the number potassium channel Sequels, total potassium conductance
00:58 number of potassium channels times that conductance an individual potassium channel across plasma
01:06 Of course in addition to the resistance and the resistor has high resistance and
01:13 conductance when the channel is closed. the channel is open the resistance goes
01:21 and the conductance goes up. Each of these ions have their own respective
01:26 . So this is a representation and symbol for batteries. This is a
01:31 for potash in its own electro motive . The charge build up with potassium
01:36 build up on the inside. So was built up on the outside.
01:41 put all of the major three channels really dictate main thighs, dynamics across
01:47 membrane and changing its potential or holding addressed, you have sodium potassium and
01:55 , each one of them have their resistor and this resistor or this conductor
02:01 variable. So channels closed, partially fully open, you vary how much
02:08 would be able to conduct through that . Each one of them have their
02:14 respective batteries. As you can see batteries here for sodium this plus on
02:20 larger plate on the side of plastic and negative on the societal aside it's
02:27 opposite for potassium because the potassium charge charges build up on the inside and
02:34 is the chloride. So if you again if you know what is the
02:44 . Okay first of all we have equals Ir that we reviewed as arms
02:51 here. Arms law is written in of the driving force. If you
02:56 the driving force is the difference between overall member and potential and the equilibrium
03:03 for a given iron. In this it's potassium so D M minus
03:08 K. In this case equals Heart. We know that conductance
03:14 Yama is equal one over R. if you plug in I is equal
03:21 over R. Or I because Is equal one over R. Or
03:27 is equal conductance, finds the driving . So the amplitude or the strike
03:34 the current depends on the overall conductance the driving force for that specific
03:42 And this is essentially what's written out . The overall conductance. Total conductance
03:48 on the number of channels potassium channels the conductance from these through these
03:54 So a number of channels is important the cell number and has a lot
03:58 potassium channels is going to be high value for potassium, if it has
04:03 lot of potassium channels but they're there isn't going to be conductance through
04:09 channels. So it's a number that's . And also whether the channels are
04:14 or close and what state they So overall, then again, these
04:23 properties for the cell membrane that we're . I'm gonna go back to this
04:31 that I was looking at here on exam and these are the three symbols
04:40 you should be able to recognize. is in seat the symbol for resistor
04:46 a conductor in B is another symbol we haven't discussed yet. This is
04:53 capacitor because cell membrane has resisted and properties. A capacitor and why plasma
05:02 is a good capacitor capacitors are good they have the two plates that store
05:09 charge close to each other, the starting charge plate and the negative plate
05:17 close to each other, physical and at these plates have large surface
05:26 the larger the extent and the surface of the membrane. And when you
05:30 into account that dendritic spines and then and other processes, they vastly increased
05:37 overall surface area off the plasma membrane it has to have a large surface
05:46 . And they have a good feature the capacity should be that one of
05:50 things that the plates are close it should redistribute the charge or discharge
05:57 that should do it in biological systems milliseconds. And the same goes for
06:06 systems and electronics. And it could even faster than milliseconds for certain
06:12 So this is a symbol for the and each one of these ions will
06:19 the respective batteries. Mhm. So go back this slide. Yeah.
06:32 let's discuss this again. What I've you before in the last lecture I
06:38 if you inject a little bit of then you are stimulating. In this
06:43 you're using electrophysiology using electrodes using You inject a little bit of current
06:50 you do polarize the cell. So is the current is being injected inside
06:56 cell outward current if you may. the cell d polarizes and you can
07:02 that on the left, you have recording that is from the electronics.
07:07 turn on the button to inject current turn off. This is representative of
07:12 stimulus. Could be representative of sensory of any other neuronal stimulus. You
07:18 see that for the cell number and reach this maximum value from -60 to
07:25 . Following this deep polarization. It some time to reach this plateau and
07:31 sometimes several milliseconds and that's because you're the charge across the capacitor which is
07:40 plasma membrane which is storing all of charge. And then when you end
07:45 stimulus here and release it. Then see a slow decline and redistribution of
07:53 across possible number. And again slow the sense of that. It's not
07:57 but it is fast in the biological that it's only taking a few milliseconds
08:01 something. So the more stimulus you and d polarizing stimulus you produce,
08:09 more you can see you have more this resistance of the capacity of
08:14 And that's why the response from the and from neurons. When you look
08:19 it, it never has a square look unless you're looking for unless you're
08:25 at single channel recordings which may have square wave like. And so now
08:31 test for you can test for individual and you can test for overall
08:37 How does the cell change? How the current change the voltage across plasma
08:45 of the south? And in this on the right what you have is
08:51 ivy plot. I stands for And the stands for voltage. In
08:56 case it's voltage of the membrane and balls. By convention positive current banana
09:03 pairs is outward current. Okay, positive charge from the cell moving to
09:10 outside his outward current. This deep means that the cell is d
09:18 That means that the positive charge is . Now if the N word by
09:24 is negative. All right. Another to look at it is this is
09:30 convention because you have a recording So current is rushing from electrode into
09:37 cell inwardly. Mhm versus into the . And causing deep polarisation into the
09:45 causing this following a stimulus. So is a linear I. V.
09:51 . Which means that for half a AM player positive, half nine full
09:57 and positive to nine and positive. always generate the same change in the
10:05 of potential incremental change. So as incrementally inject this positive currently do polarize
10:12 cell and what you're seeing is in opposite direction. Also half of the
10:19 empire will also cause a negative Middle of all the polarization, one
10:24 per negative 10. So this is linear I. V. Plot.
10:29 so there's multiple I. V. that one sound may have because one
10:35 subtype may express many different or several subtypes of ion channels. And for
10:42 discussions were really concerned with is both ion channels. Their gated by
10:50 So if you look at voltage, law, the E equals Ir.
10:57 input resistance into neurons depends on resting density and membrane surface area. And
11:05 this case the input resistance is equal the resistance of the membrane Over four
11:13 a square where a. is the of hysterical neuron. So the smaller
11:20 your on the smaller the A. you're dividing membrane resistance by the
11:26 The smaller the radius, the larger input resistance which means small neurons have
11:32 high input resistance and large, larger larger cells because they're not all the
11:38 size. They have lower input The capacitance another way to look at
11:44 change in voltage is really a change charge across the capacitor and change of
11:51 charge and the current this change of charge over time. Because what you're
11:58 , you're adding and subtracting charge from capacitor places you're pumping in either positive
12:04 negative current one or the other Yeah. Mhm. So the capacitance
12:11 the input, the capacity input capacitance neurons depends on the capacities of the
12:20 times for pie a square. This resistance it was over a. This
12:28 times a. So that means that larger the radius larger the radius,
12:33 more the square area you have the of the square area you have the
12:38 charge against or the more input capacitance capacities that cell has. So when
12:47 well again all of the components here can look at the circuit at the
12:56 and we can say, okay, our sodium with its respective battery.
13:01 is extra cellular side and this is cytoplasmic inside of the cell. Come
13:08 chloride has its own battery, potassium its own battery. And so we
13:14 see that sodium has a tendency addressing and potential to be moving inside from
13:22 to inside potassium and during action potential has a tendency from to move from
13:28 to outside chloride is not really flux much not much conductance in this particular
13:36 . In addition to these resistors you have the capacities of the member
13:43 here depicted cm with the capacities place charged on the outside, negatively
13:49 on the inside of the south addressed of course that will redistribute as a
13:55 of potential changes and then you generate action potential. And finally this is
14:01 A K pump, a symbol Finally pump which always works against concentration
14:07 meaning it always puts potassium more potassium and more potassium outside by using a
14:14 . P. Which we already So now you can basically understand that
14:19 membrane circuit can be represented in electrical manner if you made and it can
14:30 used for modeling the conductance is across plasma membrane for modeling conductance is across
14:38 . So you can do a lot computational neuroscience using some of the basic
14:44 understanding of these circuits as well as the dynamics of the action potential and
14:52 kinetics of the channels that produce action . Recall that the difference between Nerdist
15:02 and Goldman equation is that Goldman We use multiple ions potassium sodium chloride
15:10 calculate the overall numbering potential and the equation is used to calculate the neurons
15:18 , which I also call reversal which are also called equilibrium potential at
15:28 . We discussed that the dynamics of uh state of the cell is
15:34 The potassium channels are open, potassium leaking and the cell is most
15:39 Two potassium on and that's another important that the Goldman equation uses. The
15:46 changed during the action potential where the becomes mostly dominant by sodium conductance is
15:54 at the rising phase of the action . Mm The greater concentration on particular
16:03 and the greater is from the The greater its role in determining the
16:07 number of the country. But in you can use to relieve the same
16:14 but just change the permeability radically. 0.04 to 20. Uh That is
16:28 is that is going to change the and potential violent. Okay,
16:34 The action potential rising phase the overshoot falling phase. To undershoot this is
16:45 voltage clamp. And why you would the action potentials using voltage clown.
16:53 I will uh uh pause for a here. Uh huh. We outlined
17:04 equilibrium potentials for the four most important for calcium, sodium chloride and
17:12 This is the membrane potential VM and balls. This is zero. No
17:17 value. The resting member and potential about -65, negative 17 million bowls
17:26 this number and potential is doing this walk. D polarizes a little bit
17:31 polarizes. It receives excitatory inputs from analysis that deep polarizes, it receives
17:38 inputs and hyper polarizes local thermodynamics change the temperature heats up a little
17:44 It starts the polarizing a little it receives more deep polarizing inputs.
17:50 can reach the threshold for action potential . The value of that is -45
17:55 balls. If it reaches the it will generate all or non event
18:01 resting member and potential to sell is permissible to potassium because potassium channels are
18:06 and the cell is leaking potassium At same time addressing number and potential.
18:12 is quite a great difference between the potential or delivery um potential for sodium
18:17 positive 55 And the actual number of , the Millwall's which is about -65
18:26 . So as soon as there is deep polarization to start opening sodium
18:31 sodium goes into the positive feedback loop sodium more deep polarization, more sodium
18:36 deep polarization. The ultimate goal of sodium is to drive the number and
18:42 to its own equilibrium potential value. flux sing in is trying to do
18:48 and you can see a radical change the number of potential just over a
18:52 or so reaching the peak of the potential. The number of potential never
18:58 the equilibrium potential for sodium for two . One of these reasons is the
19:02 of the sodium channel. As soon sodium channels open the transit we opened
19:06 closed immediately will study that in the couple of slides. The second reason
19:12 the more the membrane is deep And the closer that membrane potential values
19:18 the equilibrium potential value for sodium, smaller is a driving force for
19:24 And at this point the driving force potassium is great because the number of
19:30 being a positive, 30, Positive million balls. And the equilibrium potential
19:36 potassium being at about -80 -90 million . Now you have a huge driving
19:42 for potassium and potassium is dominating the phase of the action potential. Its
19:50 is to drive the member and potential the equilibrium potential value for its for
19:55 for potassium. And it almost accomplishes . But with the help of an
20:00 pumps, Henry distribution of charge. rebuild this number and potential into this
20:05 -65 -17 resting membrane potential value during action print actually, you have the
20:13 refractory period and at the very tail of the following phase of the action
20:19 . And during the re polarization you in the relative refractory period which we
20:25 last time. Okay, so now understand the driving forces. Now we
20:32 the different components. But when you this recording and when you trace the
20:37 of potential, you don't get to individual currents for sodium or individual currents
20:43 precaution And so we have to wait about 1950s 1960s. When the voltage
20:50 technique was developed, in other before this one electrode would be inserted
20:55 stimulate to produce a stimulus. Another would be inserted to record the changes
21:01 the number of potential. And you pick up this beautiful action potential.
21:05 now everything that I've told you sounds . But how do you experimentally actually
21:11 and demonstrate The reversal potentials? Can demonstrate that sodium reverses at 55 or
21:19 ? Can you isolate sodium current and say that it is flexing during the
21:24 face of action potential. And the is you can but for that you
21:31 to use a technique that is called voltage clamp. And this is the
21:44 for the voltage clamp. And what you clamping in particular? You're clamping
21:50 potential. So that the value of 65 for minus 70. Now you
21:56 to be in charge of the number potential. Now you want to tell
21:59 I want you to sit at minus . I want you to go for
22:04 a millisecond to minus 40. I you to go for three milliseconds to
22:09 20. You want to manipulate the of potential value. So how would
22:15 do that? And to do You use voltage clamp your clamping the
22:20 in potential. You're looking at the of changes in the membrane on specific
22:26 conductance is using this technique, it's a negative feedback system. The way
22:33 the system works is that you have internal electrode and started here green inside
22:38 cellar. Inside the knocks on. it's measuring number and potential. And
22:43 is connected to the voltage clamp So you're measuring the connection. You're
22:49 here the VM number and potential as is referenced from inside electro to the
22:56 electrode to the ground electrode. And solution outside. Now that information that
23:03 goes to the voltage clan amplify which membrane potential to the desired command
23:11 So what is the command potential? is a command potential that gets placed
23:15 the amplifier also in the command potential I'm measuring VM are measuring that the
23:24 at -17. But as an I want the south to be at
23:32 . That's my command. I just that I wanted to be at
23:36 Okay, I wanted to be at . Sometimes sometimes the positive 20.
23:41 may not be physiological and self will live a positive 20 or positive 40
23:48 levels for second or two. But is the way that you can experimentally
23:53 out individual conductance is So who sets command potential? You do the
23:59 The person that commands the amplifiers and in the in the electorate. So
24:06 you're measuring this VM and this VM minus 70 minus 70. But you
24:12 it to minus 60. So when . When this member in potential is
24:18 from the command potential from the the clamp amplifier will inject current into
24:25 axon through the second electrode here. huh. This feedback arrangement causes the
24:33 and potential to become the same as command potential. So what is my
24:40 potential is -60. My VM is . What is the voltage clown going
24:47 do is going to inject 10 positive balls to keep it among the
24:57 And it can happen very quickly within , Within microseconds, the current flowing
25:04 into the axon and across the plasma can be measured here. So,
25:10 I told the south to be of 60 goes down to minus 70 The
25:15 10 passes through a registered positive So now this fluctuation that is
25:29 Now you can manipulate the member of . You can set it at different
25:35 . You can test for reversal potentials you can isolate individual ionic currents And
25:42 day voltage plan does not require two . It can be done with one
25:49 because the circuits are so so fast you can clamp and measure at the
25:54 time clamp and measure It's happening at , very high sampling frequencies of 10
26:02 , even 40 kilohertz. So this a voltage clamp technique for studying membrane
26:08 and the squid giant axon. It have to be in a square joint
26:12 can be neurons can be in different of neurons, different types of
26:17 different parts of the brain. I'm this voltage clan technique. This is
26:22 and Huxley and this is what they , he said okay let's record
26:28 let's inject the current and de polarize Sultan minus 26. What do we
26:35 , minus 26. We're seeing this blip. This negative current. Remember
26:41 by convention this negative current is an current, positive current moving inside and
26:48 current. This little prolonged signal here current moving outside followed by an outward
26:56 . What happens at zero millet This inward current becomes stronger and this
27:01 current is also stronger interesting thing is the inward current is transient. So
27:11 have transient producing an inward current and it is followed and dominated by outward
27:16 . The plasma numbering Centre, the 26. All of a sudden this
27:24 current is getting smaller When you set a positive 52. And what happened
27:31 your inward current? It's not And what is the inward current during
27:41 action potential? sodium, what is reversal potential or equilibrium potential value?
27:51 it is 55, Like I I'm not going to hold you down
27:56 the value but the principle of So at that value, that's what
28:00 call its reversal potential because if you go to more d polarized potentials like
28:05 65 here you see this little blip , this is sodium current. It's
28:18 and it became outward current. So the equilibrium potential potassium and sodium and
28:25 own equilibrium potentials they will reverse. what we also call it reversal
28:31 Uh huh. So what's happening to outward current? Well, first of
28:36 , as long as you sustain this polarization, the outward current is flowing
28:41 it's sustained and the more you do , the stronger the outboard current has
28:49 . And this is how Hodgkin and were able to dissect using voltage clamp
28:56 confirmed that the early current which is transient and fast activating and it's transient
29:04 it ends really fast. Even if just recorded an inward current it ends
29:09 it's followed by outward current which is . It's a late current and it's
29:14 and that's potassium potassium leaving the Al for this is the outward card
29:22 Nobel Prize in Physiology and Medicine for work on the action potential including the
29:30 physics model of the action potential and valuables and calculating and how the numbering
29:36 change and reproduced action potentials between the or electronic remembering circuits uh and voltage
29:50 in particular for electronics and mathematics. basically had the birth of the computational
30:00 , Hodgkin Huxley equation for the action and the experimental demonstration unfold isolated currents
30:08 the overall number of potential. Now can use these number and equivalent surface
30:14 build cells to pass current through them model neurons. We compute and to
30:21 have neurons interact with each other with conductance is from their art models that
30:27 available even in public domains to do of this. Mhm. So,
30:32 during the influx and during the rising of the action potential. Yes,
30:40 ahead. I missed your question. You did Yeah, it was a
30:49 back. I just remembered it. , I just like, okay,
30:53 , okay, that's fine. Go . The difference there any their
30:59 What for which previous line? The one. This one this is sodium
31:15 inside the saddle. So if you the potential, you'll see inward current
31:22 by a report card, inward current by ford car. Um And the
31:27 current. That's a good question. current is not just one sodium channel
31:32 , it's a number of sodium channels . And so this is 123.
31:36 an example sodium channels opening and the channel kinetics is that they close immediately
31:45 . So this is the overall sodium during the rising phase of the action
31:51 . And this is the awkward These are the potassium channels and you
31:55 see that the deep polarization starts But the potassium channels don't open
32:01 They open slower. So they're delayed opening. But once they open these
32:06 blue mountains they represent individual potassium current or channel conductance showing that they're open
32:13 a long time, they're opening a so they have slower and sustained conductance
32:21 that channel is compared to the voltage sodium channel. So if you put
32:26 two together, basically the initial phase that action potential is dominated by sodium
32:32 and channels opening not all at not all at the same time,
32:36 once they open the closed very quickly the late phase and this long for
32:42 re polarization and this this the sag all the way down to the equilibrium
32:49 for potassium is because of the sustained and sustained opening of the individual potassium
32:57 , which is the sum of it look pretty smooth if you basically average
33:02 the number of individual channels. You these nice smooth measure of photographs.
33:11 sodium channel. Let's understand why sodium closer some to understand why sodium channel
33:18 . We have to understand the anatomy . It's a four subunits channel
33:27 Each One of these subunits contains six , the trans numbering segments, labeled
33:35 one through S six Trans membrane segment four shown here in purple contains a
33:45 of positively charged amino acid residues and is the location of the voltage sensor
33:52 this protein. So each one Of sub units and S four will have
33:58 sensors. in addition to that between five and the six, you have
34:04 poor loop or hairpin loop. And four subunits will come together in each
34:09 of them will contribute this pen loop that coming from all four will form
34:15 selectivity filter of what is going to able to pass through that channel.
34:21 those ionic channel specific potassium potassium sodium selling and so on. Now as
34:28 can see this bolt of sensors is here, it's positively charged amino acid
34:34 and the channel addressed. It's sitting . Let's look at the following slide
34:42 following slide the two important components First of all, the channel of
34:49 is closed in part because the voltage which is positively charged Amino acids.
34:55 is this massive three dimensional structure. protein with each subunit is a three
35:01 structure coming together with three other So this negatively charged portion of the
35:08 is very much attracted to the negatively internal side of the south. So
35:16 inside of the south have a market . Mhm. Oh wow.
35:33 To the inside of this down is charged. Yeah. Mhm. Roll
35:42 membrane and so obviously this positively charged . Its sensor is actually attracted to
35:51 in place and by having this attraction is keeping the gates closed. This
35:59 have yes. Now if this negative on the inside slowly starts being replaced
36:12 positive charge, you have some deep . This positive charge is now going
36:21 ruutel positively charged. I mean a . And this repulsion will actually cause
36:33 confirmation will change in the pro And by causing a confirmation will change
36:40 sliding up its own structure. It for the opening of the sodium channel
36:49 . So this is why these channels called voltage gated channels. It's the
36:56 that is the key that opens And now in order for you to
37:05 this channel you have to move the sensor down again for you to move
37:11 ball to sunset down again. You to rebuild the negative charge on the
37:17 remembrance. Uh huh. This is four states of the sodium channel.
37:28 it turns out that sodium channel actually two gates bill gates two gates,
37:43 is activation gate, another one is activation. And I have it written
37:49 the slide here. So first of , what is depicted here is minus
37:54 million balls. And you d polarize south to minus 40 million volts.
37:58 this is about 20 milliseconds stimulus or this is a five millisecond bar.
38:04 this 20 millisecond, sustained deep And we're looking at individual sodium channel
38:10 . So it's a physiological traces of downward square way like deflections as a
38:16 ion channel opening. And you can that almost immediately with the start of
38:21 deep polarization. 123. The channels . And almost immediately after the
38:27 Within one millisecond of time or so close again. That's why you call
38:34 transient conductance or transient. We open educated sodium channel and sustained for potassium
38:42 the conductance is long and was sustained you saw in previous graph. So
38:47 happens? So first of all, you have a little bit of deep
38:50 and that voltage sensor slides up the dan. It slides up the pro
38:57 because of being repelled by the positive . Both games open activation gate opens
39:04 inactivation gate is this end of the that looks like kind of a ballroom
39:12 . So when the gate both gates , that ball and chain is
39:17 But the dynamics and the kinetics of the channel are that as soon as
39:22 ball comes out it swings back in millisecond later. So the confirmation will
39:29 of opening the gates also causes immediately that loose ball on the chain to
39:37 up the channel and to inactivate So you have fast inactivation. The
39:45 are open and active for one millisecond then its inactivation gate that closes
39:53 And the only way that you can into open channel again is if you
40:00 inactivation gate which is called d inactivation remove the inactivation gate. And the
40:09 way you can remove that inactivation And number three you see in this
40:14 three states and these traces the channel inactivated. So it doesn't matter that
40:21 is a positive stimulus. It doesn't that there's more positive stimulus. The
40:25 are inactivated sodium channels are not opening only way they go into this four
40:32 , number four, which is close . If you take away the deep
40:38 and the plasma number and hyper polarizes . And now you Dean activated and
40:46 allowed for the channel too close and the channel is closed, It can
40:50 into this one, # one position closed. It can get deep
40:56 The golden sensor will get activated Open the gates. An activation gate
41:03 swing and close it. You're going hyper polarize it. The voltage sensor
41:07 going to slide back down into this and the confirmation of the protein is
41:13 close the gates ready for the subsequent . So it has to be
41:19 It cannot be 1 to 1. has to go for 21 again,
41:26 has to go the entire sequence. other words, if you don't hyper
41:30 posit remembrance, do not Dean activate channel will not open again. These
41:36 the channel kinetics. So now you're the biophysics and biochemistry amino acid.
41:44 biophysics by the physics of the plasma and also the three dimensional structures of
41:51 channels that we discussed. That was why it was really important for roderick
41:56 . And for anybody to solve the dimensional structures of these channels and artificial
42:04 is doing a pretty good job solving of the proteins. So this is
42:18 Oh yes, the question is are any diseases that are associated with dysfunction
42:24 either sodium or potassium channel? So there's a whole slew of neurological disorders
42:31 have this functions of voltage gated sodium potassium channels, but it is very
42:37 altered. Especially well educated sodium And epilepsy also you have to realize
42:43 these voltage gated channels that we're talking , like sodium voltage gated channels,
42:48 have different subtypes and in the brain are certain subtypes that are expressed and
42:52 also some of the same cell types are expressed in the heart which is
42:57 with a lot of voltage gated sodium , it's that electrical center there,
43:04 that's very important. Um If you a mutation, remember on the shaker
43:14 that uh roderick Mackinnon, that was potassium channel mutation. Okay. Uh
43:21 . Um If you do, there's a number of diseases now, we're
43:29 exactly certain and in certain types of is there could be a genetic component
43:34 causes a genetic mutation and the voltage sodium channel which causes seizures. Uh
43:49 it's complicated because you have excited turning with their cells in the circuit.
43:54 then you have to say. so well if sodium channels are impaired
43:58 the excitatory cells, there isn't much . That's good. But seizures and
44:04 , it's the inhibit their into They also have appreciated sodium channel and
44:09 some of the diseases is certain some of cells that have the impairments of
44:14 channels. And that's enough to upset rhythm in the circuit and potentially cause
44:19 normal synchrony and seizures. So, we'll come back to when we talk
44:25 back propagation. You'll see we'll get the subtypes of both gated sodium
44:30 We won't be able to do it , we'll do it after the
44:34 But today I'm gonna wanna finish telling about the recording techniques which is we
44:39 it the patch clamp technique. And instead of using two electrodes now we
44:43 use one electrode and we can catch the cell and we can withdraw membrane
44:50 of the cell and record these individual . So we'll see the opening of
44:55 sodium currents and that's how we can up activity, not only clamping the
45:00 and would also pick up activity from molecules from single channels. Several patch
45:07 recording techniques. One of them is simple where you bring the electrode close
45:15 the plasma number and of the neuron you suction onto it. You essentially
45:20 hang on to this neuron and you're this neuron is a large antenna to
45:25 up the activity through the channels. if you go to electrophysiology lab RcN
45:33 set up these pipe paths are usually to little tubes and those little tubes
45:39 linked to syringe. And that syringe being held by a guy doing electrophysiology
45:44 a gal electrophysiology. And so literally suction onto these plasma membranes. You
45:51 the search to your mouth in your . Jump lit and you hold the
45:56 , you get attached to the Now if you attach to the cell
46:00 you suction harder, you actually can the plasma membrane and you go into
46:07 wholesale recording configuration. It is very that now of course the intracellular solution
46:13 your electorate is exact same chemical composition a side of Plaza of the
46:19 Because the side of Plaza becomes continuous the interior of the Pipat and you're
46:25 currents through the whole cell. That's it's recorded. It's called the whole
46:29 recording. If you attach to the membrane and instead of suctioning and breaking
46:37 plasma membrane, you kind of shake little bit and try to slowly withdraw
46:43 electrode. You may end up lucky withdrawing a patch of the membrane and
46:50 patch of the membrane will have the side cytoplasmic domain of this protein exposed
46:58 air but that's not air. It's to an experimental solution and any experimental
47:05 . So this kind of a recording called Inside out because the inside of
47:11 protein, the inside of the protein interest protein channel of interest is exposed
47:16 the outside world. The outside recording and the lower configuration. You can
47:25 two things you can actually attach to plasma number in shaken, slowly withdraw
47:33 of the number. Right? And break it. And you're lucky it
47:44 re Aneel itself as remembering in the with the extra cellular domain of this
47:52 protein will be exposed to the outside that's why this is called the outside
47:58 this domain of this protein is exposed the outside experimental environment. This is
48:05 important because you may want to study drugs, toxins, chemicals that don't
48:12 plasma number. And you want to how these toxins, whether they're spider
48:20 , other types of toxins, chemical , warfare toxins, how they affect
48:27 . Where do they buy into these ? It doesn't cross plasma membrane.
48:32 if I made his cross plasma Then attached it on the inside.
48:37 does it affect different substances when they're to you? The inside domain on
48:42 outside side of klasnic uh inside cytoplasmic Outside domain to the outside world for
48:51 . So, this is very, valuable techniques for neuro pharmacology. We're
48:56 single channel conductance is and also for different substances exposing different domains of the
49:04 to see how it affects the All right, the next we're gonna
49:10 about socio Narahashi and some mouthwatering tales these toxins that are actually specific channel
49:21 . Give me a second here. . Oh, a language shall be
49:39 . We have reason to believe you seen And then after the hospital I
49:50 back as many. Right. But work out your wife that I should
50:01 this trigger. Nobody. I can march like a book. All
50:07 Oh, that's good news, isn't ? If in fact you consumed the
50:13 and what is that? It's going be? It's quite probable You have
50:18 hours to wait 22. I'm I kept you waiting so long
50:28 Well, there's one consolation is that feel no pain at all until sometime
50:33 week suddenly explodes a little bit. expected to progress. I should leave
51:00 two alone. Perhaps this pamphlet. you're going to die. For
51:14 The poison's proper fish of which there about 100 species worldwide. You need
51:20 license to sell publication Japan. But a pilot you want to jeez Okamoto
51:32 a full restaurants and of course a . He's not here to buy the
51:37 popular toxic farm. Full room which been recognized by short events. Nor
51:43 he interested in the smaller species caught the wild in japanese waters. This
51:48 true concern is only looking for one toxic one as fresh as possible.
51:56 means you're a tiger pufferfish. Mhm Yeah. Uh huh. This
52:20 will cost 100 years. It was of the restaurants in the county that
52:39 are about 3000 restaurants specializing in Tokyo from the other side. Usually easy
52:47 recognize and that is highly specialist. is no antidote. The boy is
52:57 it's not The voice is tetrodotoxin. 1000 times more in Sinai and there
53:06 no antidote. The poison paralyzes its . But these little fully conscious proper
53:15 is critical. This in and entrance the fish are poisonous and they must
53:20 contaminate the non toxic meat on the . Mm hmm. High concentrations of
53:34 bracing. This etcetera toxin are found the in its especially the liver and
53:40 . Special disposal is necessary. The of water, sand takes about 10
54:01 to meet. You got to Most economy officials cut into thin slices
54:07 eaten raw as sashimi. In fact taste of the unprepared puffer fish is
54:13 . Rather land. The restaurant exclusively it's best for groom captured in the
54:21 . Uh huh. In small doses raising of the google fish triggers numbness
54:27 the mouth and is intoxicating. But they do not hate for guests who
54:34 be eager to try out tiny doses this poison. Mm hmm. Uh
54:46 more research there is 100% toxic if win this law were really like
54:55 A real will consist of at least courses. The ultimate in glory pleasures
55:09 mainly enjoyed in Japan when there is to celebrate nowadays. The chances of
55:14 poisoned at a google restaurants are practically as too high demands placed on the
55:28 50 years ago, more than 100 dying each year. Today there are
55:39 three here unlicensed amateur and recreational books afraid I was a child And it's
55:56 good to me, right reminds me uh that commercial tonight I'm meeting.
56:10 , no, I don't want to food. Uh so it's still three
56:15 a year die from unlicensed shafts consuming . Um um Really interesting market that
56:27 for it. I believe the chefs to have somewhere around seven year training
56:32 to be able to prepare this properly serve it to people and it's not
56:39 like he's saying we're not serving anything . And then they're saying well it's
56:45 . It's a very low levels of tetrodotoxin which is in the organs and
56:50 the scam of the fish and it not produced by the fish itself.
56:58 it's a bacteria. So it's that very low levels of it. And
57:07 can read, I'll send the material . Tetrodotoxin, it's attached in the
57:12 the class materials, low levels of I guess has a tingling sensation.
57:18 they still thrill seekers that seek out these kind of things. Um there's
57:25 people that like to live eat baby octopuses And uh that also causes several
57:34 a year in the world because they get lodged instead of the esophagus that
57:40 get large and track here and uh possible to take them out in time
57:46 there's a lot of different toxins, , a toxin Bracha brad track.
57:51 toxin. And Colombian frogs, taxi and clams mussels during red type.
57:56 fact sodium channels patrick talks in Colombian also has a toxin that over activates
58:04 channel and it plays with an activation . And so these different toxins will
58:09 different parts of the proteins. Some them may target inactivation, others may
58:14 a part that is necessary for the sensor to slide up and down
58:19 So these are the differences. But Narahashi in 1959 encounters this very specific
58:29 of TTX that blocks action potentials. he goes in the meetings and the
58:33 50s and early 60s and starts talking channels and biophysics and channel pharmacology.
58:40 if you read the book the story that there's in the room, there's
58:44 three or 400 about chemistry people. then he starts talking about channel from
58:49 and there's like three people that are that are interested to hear about.
58:54 are you talking about channels? Ion From a college of Italian channel.
59:00 1959, all of this is happening 60 years ago. But these different
59:08 and social Narahashi, he has a of tetrodotoxin takes it to the United
59:13 but he cannot do it just using electrophysiology recordings that he can do in
59:20 . He doesn't know how it blocks potential. He suspects it blocks action
59:25 because it blocks sodium channels. So blocks inward sodium conductance is but that's
59:30 what he suspects He needs to get voltage clamp that Hodgkin and Huxley and
59:35 are using in United States. And there's only, I think two functional
59:40 climbs in the United States at some in the late 50s or a few
59:46 them. And finally socio Narahashi is to use the toxin. Now this
59:53 really interesting because when we talked about toxins, we said that they help
59:57 deduce the three dimensional protein structure and because they will buy into different
60:02 Remember roderick Mackinnon used to genesis I directed me to genesis but he
60:07 used toxins that would buy into certain of the protein and he would say
60:12 part of the protein is the This part of the protein is to
60:17 because it changes the flux of irons the channel. So he was using
60:21 combination of electrophysiology, it allows you study effective channel blockade but also opening
60:27 the channel. So substances that block are called antagonists substances and open
60:33 So called agonists antagonists are also called . Nature is potent, quite specific
60:40 potent. So very minute amounts of natural toxins found in bacteria found carried
60:47 different animals were produced in the venom the snakes or spiders is something that
60:54 be very very potent. And so Toshio Narahashi finally uses the voltage clamp
60:59 he is able to record the inward and followed by the outward current and
61:04 step for the toxin. What you is is that it doesn't affect the
61:08 current but it specifically blocks voltage gated channel. Wow. What a
61:15 You have to tell people that there something out that that there is a
61:19 you have to have a substance. have to convince a few people in
61:22 audience. Go to the United States the vial around with the toxin for
61:28 year until you get to sit down rare opportunity with voltage clamp and show
61:34 . This is the substance that blocks gated sodium channels of course. Since
61:39 there's other blockers and antagonists and tho ethyl ammonium. As you can see
61:44 will specifically block voltage gated potassium channels it will not affect the inward conductance
61:50 or the sodium channels like tetrodotoxin So these toxins come in different
61:58 they're different sizes. Uh they have affinities. Tetrodotoxin, Saxon toxin,
62:05 , tetra ethyl ammonium too potassium all these three will affect sodium channels to
62:11 certain extent. And using them can you tease out different kinetics of these
62:18 and different important parts of the three structure of these channels. So finally
62:27 the I. V. Curves. is a simple onek linear resist a
62:32 that we've discussed. And I want show you this I. V.
62:35 . Just one more explanation. These the curves that some of them are
62:45 tv cards. Okay. And by way uh lidocaine also targets sodium channels
62:54 you can see that lidocaine here binds specific side on S six Trans membrane
63:01 six on the sodium channel. And is a local anesthetic. It is
63:06 used. So there's many different substances will bind to these channels and will
63:11 the channels. And lidocaine is used block the perception of pain locally.
63:17 dentist officer and minor surgeries. But is I. V. Curves and
63:22 I. V. Curves a lot times are rectifying, which means that
63:25 will prefer to be conducting ions in direction over the others. You can
63:30 this outward conductance here and the change voltage is much stronger with the same
63:36 of current here. The voltage changes larger here as it is supposed to
63:42 on this side. So what does tell you? That tells you that
63:47 that express multitude of channels will have of these. I've curves each one
63:53 them for each channel. And for , for example, if you have
64:01 linear the curve, you can also a situation where it's rectifying curve.
64:08 you can also have a situation where cell, one another channel another channel
64:21 channel, another channel another channel. channel. Another channel. Another
64:26 Another channel another channel another channel. this is one cell can have
64:34 20 different, both educated and other of channels. They all have their
64:40 five cards. And this is how would get the frequency in their ethnicity
64:45 get produced in neurons. If you when we looked at uh at the
64:54 the neurons in the cortex or when look at that different beats that neurons
65:01 and the reason why they speak different and why they have different frequencies and
65:05 potentials is because they express slightly different of these voltage gated sodium channels,
65:11 channels, calcium channels and they have different. I'd curse for each one
65:17 the channels which will influence the overall and potential, which will influence the
65:23 of the action potential firing as So now we're putting some of all
65:28 these things together and uh I think will end here because we're out of
65:36 . But as I mentioned at the of the class, we talked about
65:41 exam at the beginning of the I will upload a more extensive review
65:46 for some of you may not be . Some of you may just want
65:50 reward certain parts of certain lectures, I'll upload a more extensive review that
65:56 prepared on video points and I'll have of your electricity for you available
66:03 I know that I have Tuesday's lecture is still, uh, in the
66:08 mode. It failed a couple of that I have to do it
66:12 but it should be there. So just a technical glitch on my
66:17 So prepare yourself well. Take your over the weekend studying. Make sure
66:22 have everything squared away with CASA in tech preparation. Good luck on the
66:28 . And then I will see you week from today. But Tuesday you're
66:33 the exam and there's no class on
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