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BIOL 4315/6315 Neuroscience (18564/17642) - NEURO Lecture 08 Action Potential II Mo Wed
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00:02 Welcome Back. This is neuroscience. eight. We're going to be finishing
00:09 talking about member and properties of the and also finish up talking about the
00:19 potential uh channel kinetics. Um some the antagonists for voltage gated sodium
00:30 And uh oops folks, worse world , yeah. So before we go
00:42 finish talking about the action potential, is an important feature here that is
00:50 uh following lecture materials that you That actually shows you that plasma membrane
00:58 be approximated into a physical circuit where channel, for example, potassium channel
01:07 be a conductor or resistor either way want to take it. And so
01:15 symbol war the resistor is this symbol here, Trying to find the 10
01:29 person at all in the classroom. interesting. A single mark or
01:36 and I don't have one in my . Um So I'll just show you
01:41 right here. But this symbol right , that looks like sort of a
01:47 repeated lightning bolt of lightning is a for resistor. In physics or conductor
01:55 which department you're. And so these are actually variable resistors are variable
02:02 Uh They depends how open they or how much of the driving force
02:08 risk, that's how much the conductance going to be. Each one of
02:13 channels, potassium channel sodium channel also their own respective electro motive for battery
02:21 , which is created by unequal distribution charge. The cross flies my
02:27 So, if you were to approximate plasma membrane into a electrical circuit,
02:35 would essentially have uh conductor resistor. sodium conductor resistor. For potassium conductor
02:45 resistor for chloride. And we're showing three main bionic channels because the main
02:51 that contribute the rusting number and potential the signaling dynamics overall across plasma membrane
02:59 influence the member and potential to change the greatest degree. So once again
03:05 that the um minus E. Equals IR. Which is essentially the
03:12 IR. But the driving force which the difference between the number and potential
03:17 the equilibrium potential value for a given on. Then you know that the
03:24 gam or G. is in verse over R. Therefore I current is
03:32 V over R. And other expression this is that current Because G.
03:41 equal one over our current instead of over R. The over our current
03:47 equal conductance. That was the driving via minus E. K. This
03:55 the strength of the current, is much of driving force there is how
04:00 conductance. There's through that channel. one of these channels is their resistance
04:04 a conductor and each one of these has the respective battery and the symbol
04:10 battery is this plus and minus right plus and minus bars Plus and minus
04:20 you can see for sodium it's in direction, the battery for potassium,
04:24 in another and for chloride, it's another. That's because of the uneven
04:29 of the science, potassium being dominant the inside, sodium being dominant on
04:34 outside. So you have total conductance on the number of all open
04:42 You can calculate conductance for an individual but the plasma member and they contain
04:49 of potassium channels and you want to the overall potassium conductance which would be
04:55 to do if you calculated potassium current you knew a single channel recording and
05:02 uh conductance measures through that single You can do that using electrophysiology.
05:09 you can calculate the overall potassium conductance will depend on the number of potassium
05:14 and okay France the conductance through each of these individual potassium channels. And
05:22 not all. When we talked last said that when you inject current into
05:30 or you stimulate neurons electro physiological e obviously use electronics and circuits and equipment
05:39 you can turn on and produce what called square wave like pulses. So
05:44 can produce the deep polarizing pulses which excitatory impulses or you can produce hyper
05:51 pulses which are inhibitory. In other you can either inject positive current into
05:57 south and cause them to de polarize negative current into the south and cause
06:02 to hyper polarized. And I also that the south never respond just by
06:10 a square wave like response across plasma because plasma membrane has resistance resistance and
06:21 capacitance properties, R. C. . So it takes time when you
06:28 the stimulus and you incrementally produce higher of stimulus. You can see that
06:34 cell will incrementally d polarized from minus to minus 55 minus 15 minus 45
06:41 it may reach the threshold for action generation. And at which point this
06:47 or the strength of the symbol of strength of the stimulus, the stronger
06:51 stimulus, the larger number of the potentials have yet produced. The size
06:57 the action potential is always the But the frequency or the number of
07:01 potentials maybe larger. So we talked how the number of action potentials also
07:08 with the strength of the input or strength of the stimulus. But this
07:13 why you have this rounded response from south and when you release the current
07:18 you hear your charging up The Plasma membranes are very good. Capacitors
07:25 uh a sign for capacitor actually, not illustrated here. I should produce
07:31 slide and I don't have a pen show you. But it is two
07:38 instead of uh two unequal lines how have here. Plus and minus.
07:43 would have to equal lines and one them will have positive charge on one
07:48 and then it will have negative So this is just a symbol for
07:52 capacity. I wish. I could draw some help now but I will
07:57 this uh here remember an equivalent circuit will include what the resistor the capacity
08:05 uh the battery uh conventionally depicted in circuits. So the resistance of the
08:19 or the inside are in depends on resting channel density. So the more
08:26 there are and if the channels are the least the less resistance there
08:31 It also depends on the number in area. Small neurons have high resistance
08:38 the input resistance or the neuronal resistance two membrane resistance around over four pi
08:48 . Which is the radius of hysterical squared. So the smaller the
08:58 the smaller the a value, the of that neuron, the smaller than
09:03 on, the larger is the input into that south. And of course
09:10 will vary now depending on how many that cell has or how many of
09:15 are open. Of course. Another to look at the change of voltage
09:20 plasma membrane is to think about a of charge over a capacitor. So
09:29 have to add charge on the capacitor when you're putting a positive current here
09:35 the cell, you're adding positive charge that positive charge. Slowly building up
09:40 the inside of the south when it the plateau the maximum and that's because
09:47 membranes are actually very good capacitors and are some of the features of a
09:52 capacitor. The two capacitor plates have the charge of positive and the negative
09:58 , they should be very close to other physically, they should have a
10:03 surface area because the larger the the more charge you can store.
10:09 the larger the surface area and having dries and having dendritic spines and having
10:17 of the south tremendously increases the surface compared to just what the soma would
10:24 provide for the surface area of the . So the larger the surface
10:28 the better more charge you can And really good capacitors can charge up
10:35 discharge really quickly. And that's one the features while you want them in
10:40 proximity, the two plates starring the and the negative charge you want them
10:44 to each other. So this current the charge would flow over time.
10:50 the capacitance of the cell. Or input capacitance into neuron is the capacitance
10:56 the membrane. But now, instead divided by four pi radius alpha uh
11:04 squared. You're multiplying it. So this case the larger the a and
11:12 larger the combatants capacitance, the smaller radius of the cell, the smaller
11:17 capacitance itself. So these two features resistance and the capacitance plays in to
11:25 storage of the charge two discharging into this curve like response across plasma
11:36 And as opposed to the square wave stimulus that you're producing with electronics because
11:42 you're conveying that electronic charge stimulus onto plasma membrane which plays it by its
11:53 rules, It resists and it stores . And then when you release the
11:59 it resists and it's Recharges re accumulates charge again. So once you stop
12:05 stimulus, you also don't see an drop back to the -60 middle of
12:10 value, but rather relatively slow And it is fast. So we're
12:17 about a few milliseconds in delay for capacity to fully charge out plasma membrane
12:23 to discharge. That's that's fast. So you have these ivy plots which
12:34 called basically current voltage plots. And is an example of a current voltage
12:42 . That is linear. That means for the same amount of input,
12:48 it's positive input or negative input, same amount of Positive charge that is
12:55 pumped in here. And then you it you go 112-2-3. You have
13:02 linear response from the cell. That that it responds linearly to the change
13:11 the current that is being injected. you inject one negative milli ampere of
13:19 . And by convention negative nana answers this case of milli amperes. But
13:27 amperes of current is an inward This is just my convention. You
13:36 think about it as injecting a negative . Uh huh is an inward current
13:42 a negative deflection. Yeah, an current would be positive nana empire
13:52 But the most important message here is inward and outward will repeat some of
13:58 again is that this particular cell membrane a linear uh line. It has
14:06 linear curve. It responds the same for negative one nana bear 10 million
14:15 change to 2015 million volts changed about , 10, 15 million volt change
14:21 each of these gradations and it does in the opposite direction. Also,
14:28 is also the curves by which you the channels. The I've curves and
14:33 of the channels don't have linear Actually they have curves that are bent
14:38 we call rectifying curves. But it's that you understand that the cell has
14:45 resistance and capacity of properties that can the signaling. Okay, so now
14:56 don't need to draw it actually. forgot I have this other slide that
15:01 a full, so to speak, membrane circuit representation here you have
15:09 variable conductors of resistance for sodium For potassium we have batteries. You
15:18 a capacitor cm capacitance of the membrane to overall charge. Okay. Because
15:26 charge, sodium and potassium are both but it depends which direction it's
15:32 So obviously the cytoplasmic side of the side of the capacitor plate is negatively
15:40 . And so this is the symbol the capacity. And the last symbol
15:44 you're seeing here is the pump which always working against the concentration gradient.
15:51 you can see that the batteries and driving forces for sodium dr sodium from
15:59 the direction of the arrow for sodium positive charge going from outside to inside
16:05 for potassium, the driving force is opposite from inside to outside. So
16:12 you have a full understanding of this equivalent circuit uh reminding you that the
16:22 membrane potential is determined by this goldman , Hodgkin and cats equation, steady
16:32 equation, which depends on the permeability and what we talked about when we
16:37 about resting number and potential, we that at rest the cell is dominated
16:43 potassium ion conductance, is potassium is . But at the rising phase of
16:54 action potential, the permeability ratios would where sodium we become the most
17:02 the most permeable i onto the plasma . So if you just take and
17:08 the permeability ratios without changing the ionic , you already see a massive change
17:16 the number of potential value overall. this VR values and this equation is
17:23 from nonstick were asian, because nuanced is an equilibrium potential for just one
17:28 on. It doesn't take into consideration other ionic species and does not use
17:35 variable action potential for action potential to properly recorded and analyzed. There was
17:46 technique that was used and it's a clamp technique in this diagram may seem
17:52 uh complicated to you. I'll explain in a second and maybe I'll explain
17:58 to you. Uh just uh let's . No, I'll use this slide
18:08 . So now voltage clown comes about a very important technique because all of
18:13 calculations for equilibrium potentials, forgiven Our calculations. Remember you squeezed out
18:21 internal solution and you said that there's many islands, you have the Nerdist
18:28 , you calculated equation, reversal you can calculate number and potential.
18:33 can record number in potential, so can sink an electorate and we'll give
18:38 this action potential. But now what want to do, you don't just
18:43 to passively record the currents you actually to manipulate the charge and how the
18:51 membrane of this neuron is charged. negatively charged or it's positively charged.
18:55 experimental conditions, you want to alter charge and hold that charge and we
19:01 it a holding potential or a clamping . And that's why this slide is
19:06 a technique called the voltage clown. you're clamping is a membrane, you're
19:13 numbering and you're affecting changes on specific conductance is. And this technique was
19:23 most important to experimentally demonstrate the reversals individual ions such as sodium and potassium
19:31 to actually dissect that individual ionic and is during the action potential because if
19:37 record the member and potential, it tell you about the activity of individual
19:42 or potassium channels to do that. need to use the voltage clamp
19:48 It's essentially a negative feedback system in you have a set up experimental setup
19:54 one internal electrode is inserted inside the and of course, you have a
20:02 electrode or the ground electorate which is on the outside of the south.
20:07 this electorate is connected to the voltage amplifier. Mhm. So this is
20:15 is amplifier and the voltage clamp What it does. You set the
20:22 potential on that amplifier and the command is basically telling The cell to stay
20:31 a certain membrane potential value -70. to -50. Stay there for 30
20:38 , Go to -40. Stay there a second, Go to -90.
20:43 clamping the voltage across plasma membrane. you have the command potential. This
20:51 clamp amplifier, you have a second here. The second lectured here.
20:58 , it's recording what current is flexing voltage clamp amplifier compares membrane potential to
21:05 desired command potential. So, it's , It's recording and it's saying the
21:13 is -60. And your command You as an experimenter says, I
21:18 command potential at -70. So it the command potential versus the actual number
21:25 potential and it produces an equivalent change voltage that produces an equivalent charge across
21:34 membrane. So, when the membrane is different, like I was just
21:40 minus 60 vs minus 70 from the potential, the potential is minus
21:45 The plasma membrane is of minus You're doing an experiment. We clamp
21:51 now injects current into the axon onto through a second electorate. This is
21:56 second electorate. This feedback arrangement causes number and potential to become the same
22:03 the command potential. So what is electorate and Jack? If my command
22:09 is minus 17, which I said I'm recording minus 60 measuring membrane potential
22:16 from this green electorate, what is to be my output here 10 mil
22:26 difference. That's what is going to . The current flowing back into the
22:30 and thus across its number and can measured here. So any fluctuation,
22:36 have the south set to -70 the . Any fluctuation to sell guns to
22:43 60. Your voltage clamp injects 10 millet balls, keeps it at minus
22:49 goes to minus 80. Injects positive million to keep it minus 70.
22:54 locking it, you're clamping it at command potential. And that's very important
23:01 just that you sink an electrode, like a a radar that's picking up
23:09 action potentials. You can inject current record action potentials but you have to
23:15 voltage clamp And you have a specific , the whole cell voltage clamp recording
23:21 that allows you to cramp number and given command potential. And so Hodgkin
23:29 Huxley used this voltage clamp technique that perfect that voltage clamp technique and they
23:37 the currents during the action potential. what they do is they dip polarize
23:42 cell To -26. And what they're is that they're seeing this inward current
23:49 relatively short followed by a small outward . And that this outward current is
23:57 and sustained. So they polarize the membrane, they clamp it at zero
24:03 balls and they see what happens To inward or outward current. Now this
24:09 value of zero millet balls, they a stronger inward cards zero million
24:16 But they also see a stronger outward . It's positive 26. That inward
24:23 is actually reducing but the outward current increasing one second. I need to
24:39 the right slide. Yeah. so remember what we talked about last
24:49 that sodium will influx, this is inward current and the potassium will be
24:59 . So you have to demonstrate this this is the challenge. How do
25:04 do it experimentally? You use voltage Now you can de polarize the membrane
25:13 Deposit of 25 and hold it there see what currents are still coming in
25:18 out. And so what's happening at rising face of the sodium, It's
25:23 huge driving force for sodium sodium channels up deep polarization sodium coming in deep
25:29 . Trying to reach the equilibrium potential sodium. It does not because of
25:35 channel kinetics the sodium channels inactivate and the driving force reduces for sodium
25:45 So this is what you would see you want to go more positive
25:49 That inward current is what's happening to inward current. It's reducing what's happening
25:54 this outward current. And these deep values, potassium has this huge driving
26:03 . And I told you these ions selfish sodium when you open sodium channels
26:07 drives it membrane potential which doesn't belong just sodium but sodium wants to drive
26:13 to its own equilibrium potential diarrhea and the following phase of action potential with
26:20 e flux sing and it's driving the number and potential to its all new
26:25 potential value for potassium. So at the polarized potentials the inward current which
26:34 sodium would be decreasing just like in experimental charts that I was showing in
26:40 ago, the outward current will be . The other thing that's demonstrated.
26:57 other thing that's demonstrated is that this current which is sodium current is
27:05 It's fast activating and it's transient. the fact that you have sustained deep
27:11 here at minus 26 or zero millet or positive 26 positive 52. This
27:19 just a blip. And that's because the sodium channel dynamics that we'll discuss
27:24 the second, outward current becomes stronger outward currently sustained. So potassium outward
27:32 or sustained currents, Where's the inward of positive 52. Is there any
27:39 work on the positive 52? why not? What I on is
27:51 for this inward current. What I is going inside the south during the
28:00 for control sodium, what happens with 52 that there is no sodium
28:10 The potential at which the equilibrium potential net ionic movement, electrical. And
28:24 chemical gradient forces are equal and opposite each other. What's happening with potassium
28:30 is still flexing. You see this blip over here, You know what
28:37 to to to to sodium current positive . It starts flowing in the opposite
28:45 . That's why it's also called reversal non time. So if you d
28:50 the cell even further, the current of coming in and reducing coming in
28:55 actually now start coming out even for which is physiologically not possible. This
29:03 the death and the cell never reaches potential and the membrane doesn't sit at
29:07 potential. But it was imperative to the voltage clamp that technique and to
29:13 member and potential at 0 -20 of 20 positive 40 to demonstrate.
29:20 indeed, That sodium reversal potential is 52 or 55. Now start understanding
29:30 overlap of the two. So, early current here, the inward current
29:35 transient, fast activating sodium channels and channels closing very quickly and you have
29:43 late current which is outward current, potassium current and it's sustained meaning that
29:50 long as there is deep polarization. long as there is this positive
29:56 potassium channels have a different dynamic and potassium channel parent will be sustained on
30:02 matter. So if you were to essentially and say okay, during the
30:08 phase we have an influx of sodium the following phase. The flights of
30:15 you have all of these individual sodium . Each one of these red Mountains
30:21 downward is an inward sodium current produced one channel. So during the rising
30:28 you de polarize the cell seven channels 10 2080 100, however many on
30:36 patch of the number in and if some across all of these individual ion
30:42 and their activities who get this really inward current recording that looks rather smoothed
30:51 on the outward side is the same the same thing. You will see
30:54 nice openings and blew up the potassium , their outward current instead of inward
31:03 . And the striking difference here is duration but the opening of an individual
31:09 channel, The stimulus is the sodium opens and closes with potassium channels
31:15 open and sustainable. So, you this e flux of potassium. This
31:21 the rising phase of sodium inward sodium . The following phase is potassium.
31:30 following phase of the action potential is potassium the flux. So sodium channels
31:38 comprised of four subunits. These proteins we talked about building blocks these four
31:47 . Individual each one of these sub is comprised of six trance member and
31:53 S one through S six. As the trans member and segment is a
32:00 sensor. So we're talking about voltage channels. This ivy plot is everything
32:08 voltage dependent or voltage gated Between segment and segment trans membrane segments six.
32:16 have the poor loop. Remember? is the selectivity filter. And so
32:21 one of these subunits will contribute an of this poor loop and four of
32:26 from each subunit will come into the the lumen of the channel serving.
32:32 that selectivity filter? Keeping the ions . The problem wants to going in
32:38 let us go out and when sodium is closed. You have this voltage
32:44 here that's positively charged. Okay, is that voltage sensor? It's positively
32:52 amino acid residues. All right, , you have a number of these
32:58 positively charged that are gathered gathered up the sequence and segment four. Why
33:06 this important? Because proteins move channels and close. There's three dimensional
33:18 Canterbury ordinary structures they move around. change their shape. The channel opens
33:25 gate. That means that something That means that there is confirmation will
33:30 in this program. And that confirmation change can be significant. And in
33:36 case, what happens is the voltage and voltage gated sodium channel reacts to
33:43 change across the plasma membrane. Did charge change across plasma membrane.
33:50 When the charge on the inside of member in his -65. These positively
33:57 amino acid residues and s. They are drawn toward the negative
34:02 There's an attraction there from the positive its sensor toward the negative charge which
34:08 accumulated on the side of plasma inside the south. But once positive
34:18 once there's deep polarization, positive charge accumulate on the inside of the membrane
34:25 that positive charge will repel the positively amino acid residues and the voltage sensor
34:34 repelling positive charge. Now on the of the num Bryant, repelling these
34:40 acid residues, changed the confirmation of sodium channel protein and caused the opening
34:45 both gates of these voltage gated sodium have to gates. So what opened
34:52 channel voltage? It's both educated both . It moved the portion of this
35:02 dimensional structure reacted and moved it changed confirmation of the channel causing the channel
35:08 open. So sodium channel kinetics and is the reason why, as I
35:19 before why sodium, when it when in flux, sing, it never
35:26 the equilibrium potential for uh global potential for sodium. It's because they're fast
35:36 but they're also fast inactivating. So the sodium channels open up. They
35:44 inactivate. And in order for you reset these channels and opened up
35:50 you have to d and activate And you do that only by hyper
35:55 , the overall plasma number. So zoom in in this portion right here
36:00 the slide right first. What we is on the top. You have
36:07 line that shows you deep polarization. is our square wave like pulse.
36:13 stimulating the cell and we're locking it Voltage clamping it from -65 millibars resting
36:21 and potential to our command potential of million balls. Now we d polarized
36:28 plasma membrane and what we see is see individual sodium channel openings. So
36:35 also simultaneously recording single channel activity from south. This Channel one Channel 2
36:42 three. It opens and closes It opens and closes. So it's
36:47 open for a millisecond an individual sodium . So you can see that this
36:53 polarization here, this is sustained. last thing here, it's still at
37:00 but the channel is already closed. what is happening first the channel is
37:07 . Then you have to gates. two arms that are shown like
37:12 those are activation gates. And this with a chain hanging out is in
37:20 gate. This is just a piece the protein hanging out there in a
37:24 dimensional space. So as the positive accumulates on the inside of the plasma
37:32 . As four voltage sensor slides it causes confirmation will change and it
37:39 both gates, activation and inactivation But as it opens up activation gate
37:48 removes this ball and chain and activation . The inactivation gave due to the
37:55 confirmation will change, swings and plugs the sodium channel n activates the sodium
38:03 . So sensor slides up deep polarization slides up gates open and then one
38:10 later inactivation gave closes the channel. the only way, the only way
38:20 reset this channel so that it is and can open again. The only
38:25 to do it is by releasing the here and hyper polarizing the south.
38:33 you release the stimulus and you hyper the cell. Huh? And now
38:41 you hyper polarized the cell you deign . That means that the inactivation gave
38:49 removed from the poor And the activation right here it gets removed from the
38:56 number four and activation gave us closed the channel was closed. So what
39:03 to happen for that channel to open another D. Polarizing stimulus? And
39:09 you go 12341234. You can never 1 to 1 1231. You have
39:18 hyper polarized or otherwise these channels are going to open again because you have
39:24 reshuffle that voltage sensor back into its place. It takes some time.
39:30 takes the milliseconds of time and you repeat the activation of the same
39:35 But these are the kinetics of these fast sodium transient sodium. Okay,
39:47 potassium sustain potassium conductance is these are kinetics and these are the mechanics behind
39:53 kinetics of these channels. Why does close and open so fast? And
39:59 channel? Which we don't have the to discuss it in greater detail.
40:04 have different kinetics obviously slow and So to perform these voltage clamp experiments
40:11 have to use voltage clamp technique and voltage clamp you essentially put the pipe
40:19 right onto the surface of the You suction onto a piece of this
40:24 membrane and you can record activity from channels you can replace remain attached to
40:31 south. So the major techniques that should know this exam of these
40:37 First of all, you attached to salads called cell attached recording. So
40:42 just bring your electrode and attached to solemn you have a tight contact between
40:47 pie pad and the membrane. When sitting there attached to your sort of
40:51 the cell almost like an antenna to up activity from other cells. And
40:58 you get lucky. And you have channels in that patch of the membrane
41:03 of course we will that you are in hopefully. And you can record
41:08 activity. So if you go to lab you will see that these glass
41:15 have gone underneath the microscope and sent the microscope. Actually connected to little
41:21 and those little tubes are connected to syringes. And the syringes are held
41:26 people like myself and other electro And so when you form a contact
41:33 the cell, you literally produce very suction through the syringe and the tube
41:40 helps this micro electro to basically suction the sound. Now if you do
41:48 you pop your lips like like that on the syringe you can break the
41:53 number and then you can start making is called the whole cell recordings.
41:59 inside of the electrode solution has to exact same as the inside of the
42:05 of plaza because there's gonna be a opening now between this electorate and the
42:12 side of plasmas continues with a pipette . And so you're going to be
42:16 whole selectivity of all of the current that is traversing through the plasma membrane
42:22 that self. Now, if you suction hard and you just instead try
42:32 slowly kind of a shake and lift electrode out, you may be lucky
42:38 you may withdraw a patch of the and you will have the cytoplasmic side
42:45 this protein exposed to the outside environment an experimentalist environment. You can change
42:52 as you want it. You can drugs and see how the drugs affect
42:56 cytoplasmic side of this protean channel. why these recordings are called Inside out
43:02 . The inside part of the channel exposed to the outside world. There's
43:09 technique, it's called outside out In this case you do both.
43:15 actually withdraw the patch of the membrane and then you suction to break
43:24 And then with some luck and a of practice and pain you hope that
43:29 plasma membrane will re Aneel and if lucky it will re Aneel in the
43:35 where the outside of this plasma membrane will be exposed to the outside a
43:42 experimental environment. And these are outside recordings are very important because you may
43:49 have substances to cross through plasma membranes as drugs such as chemicals. And
43:53 may want to know whether these substances the outside of the channel if they
44:00 . Well what if you could get in the cell, would they affect
44:04 inside of the channel? You can these types of techniques to really determine
44:10 different molecules agonists antagonists, what part the channel on the inside or outside
44:15 bind and how they affect the kinetics the current flow through these potassium and
44:22 channels that we're studying. This is of my favorite stories And uh patch
44:30 electrophysiology in the early ion channel pharmacology uh for that we have to watch
44:42 Simpsons in this episode. If you the Simpsons are offensive to everybody.
44:50 may be offensive to some in this , But I believe it's PU 13
44:59 uh they will offend everybody at some in some episodes and make fun of
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45:12 that they want to go and try different food because they just tried everything
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45:33 And so this is where we weep cup. Yeah. Uh Yes.
45:52 huh. Yeah. A mess. are in the kitchen food but we
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46:22 huh concentrate, concentrate chesty. A is a we have reason to believe
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48:12 as soon as your progress is found you two alone, perhaps this
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49:30 courses. The ultimate in Gori pleasures mainly enjoyed in Japan when there's something
49:41 celebrate nowadays, the chances of being at a google restaurants are practically
49:48 As to the high demands placed on shelves a 50 years ago, more
50:04 100 Japanese guy teaching today. There just three here victims of unlicensed counter
50:12 recreational books is a delicacy still eat Japan despite earthquakes, tsunamis and nuclear
50:42 and in the future to it's likely remain quite exhausting. The stories of
50:51 Amara Hashi. He was a professor this is happening in the 60s and
50:59 start talking about these things spreading in . So you? Re take it
51:04 granted. But after the meeting, only maybe three or four people in
51:10 big biochemistry meeting that are interested in about toxins and agonists and pharmacology of
51:19 channels. So there were three guys the audience in Japan that cared about
51:25 ion channels in the late sixties, sixties and Tasha Narahashi is one of
51:31 . And what he has, he's has this tetrodotoxin and he isolates the
51:39 from two blue fish puffer fish and why it's called mouthwatering Tales of toxins
51:47 obviously it's used this delicacy and this . He has a little of Iowa
51:54 but he thinks he demonstrates using electrophysiology it blocks sodium that it blocks action
52:04 . But he doesn't know exactly how blocks action potential. So he takes
52:08 little vial of that with him to United States In the 60s and finds
52:15 voltage climb set up in Hodgkin and Lane and isolates the currents and demonstrates
52:22 tetrodotoxin is a specific blocker or antagonist both educated sodium channels. And this
52:30 how it blocks the formation of the potentials. You also have different other
52:40 in nature, sacks of toxins, and mussels during red tide when it's
52:45 warm Batre coe toxin Colombian frog will these animals, little frogs, little
52:54 and spiders will have toxins and they interact with channels, a lot of
53:01 are very potent specific binding sides. like when we talked about roderick
53:07 we said that it's important that he these toxins to identify the specific sequences
53:13 the function of these sequences in the channel in the potassium channel structure.
53:20 these toxins are very important because they us to study their facts on specific
53:26 to understand the mechanisms of action of toxins that we can uh use electrophysiology
53:34 understand that some of them may over the channel. Some of them may
53:39 inactivation of the channel. So there's toxins that may target the same channel
53:44 they do different things. Some of will block the channel, others will
53:48 it, others will make it open but will not open them. Nature
53:54 very important. Uh And we use both. You know we suffer from
54:00 . We suffer from viruses and we take advantage of nature. Take advantage
54:04 viruses. Take advantage of these poisonous to understand exact function and to apply
54:11 for therapeutic purposes as wrong. So you repeat it that same experiment,
54:17 Huxley experiment and you d polarize the zero millet balls and clamp the potential
54:23 voltage clamp zero millet balls. you would see a very fast transient
54:28 sodium current followed by slow activating sustained potassium cards. If you were to
54:36 to through the toxin. And that's the experiment that he did. What
54:42 saw is that it's objectively blocked sodium but it doesn't block potassium current.
54:50 so sodium current is necessary and deep is necessary in order to generate the
54:56 potential. But the definitively answer what substance does. You have to have
55:02 isolated current and you have to isolate current by using voltage clamp. So
55:07 have inward versus outward current then you oh it only affects inward current is
55:13 to voltage gated sodium channels. And course there are other substances and
55:19 agonists and antagonists. This is an of tetra cell ammonium A.
55:24 A. Which does not affect inward channels at all. But instead of
55:31 potassium channels. So you would use same voltage clamp technique and you would
55:35 that this substance D. A. specific to blocking potassium channels and it
55:42 block of course the following phase of of the action potential. So this
55:48 TtX. This is an example of toxin. They all have different
55:55 We can target different parts of the . Um uh but the most important
56:04 for us is the tetrodotoxin Ttx for blockade of the sodium channels and tetra
56:10 ammonium T. A. For the of potassium currents. These are the
56:17 . V. Curves again that we and the V. Curves would be
56:23 is a simple onek linear curve. means that for the same amount of
56:28 current deep polarization you would see the negative current inward current or the same
56:34 of positive current produced proportionately. In mm a lot of the channel conductance
56:47 and kinetics conductance is depend on the motive electrochemical driving forces and a lot
56:55 the channels that are in the plasma . And they will not have these
57:00 I. Ve. Cars. But they would prefer to conduct. In
57:04 case it's an outwardly conducting channel. produces outward current. So it prefers
57:15 conduct outward. Because you can see for the same change in voltage is
57:20 a few million balls here. You a pretty massive change in current in
57:25 . Well, the change in this current is not as strong.
57:28 a lot of channels are rectifying channels rectifying currents because who pauses for?
57:43 lot of channels Can be expressed by neuron. So one neuron can express
57:50 different voltage gated channels sodium channels potassium calcium channels variations of those channels.
57:59 there's receptor channels and so on. each one of these channels will have
58:04 and some of them can prefer to outward and be outwardly rectifying. Some
58:10 them can conduct inward and can be rectifying rectifying. So if one cell
58:24 20 channels. All right, these the curves that each individual channel.
58:50 one of these red lines is an car for a different stuff type of
58:57 . So now you have this wonderful in channel kinetics and their I.
59:03 . Curves where their linear where they're with their rectifying or not.
59:17 Local and aesthetics lighter came Well Uh Both educated sodium channels and it
59:24 discussed in the movie that we watched B. It's used cocaine also blocks
59:30 of the pain pie place. Uh There is a variety of these
59:37 gated sodium channels. When we come actually after this exam we will have
59:43 to review the back propagation of the potential. Review different voltage gated sodium
59:48 there but there is different voltage gated channels expressing different neurons, expressing heart
59:55 express different parts of the body isolating studying the function of these channels are
60:03 important. And a lot of times you're trying to isolate something small or
60:08 understand the function of that channel electrophysiology that substance is very small. Single
60:15 . You may resort to larger systems you sides and over express that channel
60:21 record activity on that channel, understand gross, how many of these?
60:27 lot of channels are strong activity, the kinetics at a gross level so
60:32 you can compare and find the same and analyze them and systems that are
60:36 lot more complicated and a lot more and sensitive. So I was going
60:43 start talking about the generation of the propagating spike but we won't have time
60:50 actually get into it. Uh Um So let me check for more
61:02 and had we discussed uh neurons have dialogues. So this funny crazy drawing
61:12 I just did here. This is neuron. This is hypothetical representation.
61:22 an artistic rendering of what may the of one sub fabric neuron may look
61:31 and the dialect of another neuron may slightly different. And that's because the
61:35 properties and the I. D. of the channels that those cells express
61:43 the cortex or in the hippocampus is . Therefore they speak different language.
61:49 all speak different dialects. Uh So this really concludes our lecture.
62:00 the material that is going to be
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