School of Life and Environmental Sciences

Cell movement Lecture video 1 Ion

Transport
[Phil Poronnik]

—
[Beginning of material]
OK, so Phil again, surprisingly. In the last session, we learned about the
organelles inside the cells and the membrane and how things permeate- how it's
not particularly permeable. So the question is, how do things get actually inside
and out of the cells? And now we'll talk about something involving ion transport
and transport across the cells. OK, so in the last session, we spoke about the
balloon and stuff like water and carbon dioxide, oxygen get across; pretty much
can't- nothing else can. So is there anything we know about the membrane that
we should- that can help us? OK, so obviously we know that it's got an outside
and an inside section. And it's got proteins that can go- span across the
membrane. OK, and the important point, this is really important, the cell
membrane contains trans membrane or integral membrane proteins that span the
membrane and have both a cytosolic facing side and a face facing the outside of
the cell. OK. And you can see from- this is a typical diagram out of any
textbook you can find. You can see that these various things that interact with
the cell on the outside, so the extracellular matrix and things like that, the cells
can stick to, there's carbohydrate stuck out there, doing things. And of course,
inside the cell, the trans membrane proteins interact with the filaments of the
cytoskeleton and everything else. So very, very complex, very, very beautiful.
And we'll look at this in a bit more detail. So some of these trans membrane
proteins can form pores through the membranes, which like a straw through
some ice cream, or something, you can suck fluid through it. And there are two
broad categories, one are channels, and these undergo so-called facilitated
diffusion and the other are transporter's that are also facilitated, but can also be
active transport. OK. And through these pore, we can get ion and fluid
movement to occur. OK, so for example, here, this is a really exciting lecture
because we'll learn about three Nobel Prizes, I think, and there's one, and you
can see here that here you've got an ion channel sitting in the membrane, OK,
and sodium can move from outside to the inside depending on its concentration
 Cell movement Lecture video 1 Ion Transport
[Phil Poronnik]
gradient. So wherever the sodium is more concentrated, it'll flow in the opposite
direction into the cell. OK, you can see these pores can be quite complicated, so
the right hand picture, you can see that permeation part of that channel is not a
simple pore, it's a very complex pathway. OK, and this is a very important field
of study. So important that Rod MacKinnon got the Nobel Prize some years ago
for discovering the structure of ion channels. OK. Very, very critical discovery.
So in other words, these trans membrane proteins can form holes or
passageways for things to move through, OK, down their concentration
gradients typically. Here's nice example, this is actually the glycine receptor, it's
a chloride channel in the brain, and it's responsible for the excitation of things.
It's- when you get strychnine poisoning, it binds to this channel and you can see
the purple bits are the strychnine binding sites in the channel. This is actually
molecules derived from a protein database that we put together. You can play a
video and you can see the structure of it, right. So it's five pentamers. And if you
look down the barrel, you can actually fly through the channel and think about
what's going on there so you can say it's obviously a pore, through which you
can move and you come out the other side. OK, so. We can look at it from this
angle here, looking down the barrel, and you can see obviously it's a beautiful
example of- this thing spans a membrane and it makes a big hole down which
chloride can flow, OK. You can see those orange and purple things, they're
actually the constriction pore, so when glycine binds, that pore gets bigger to
allow the passage of chloride. OK, so that's actually the way this thing regulates.
The pore gets smaller and it gets bigger when it's activated. When it's activated,
you can fly through and start to appreciate the true beauty of the structures. OK,
so this is this concept of pores, so that ions and other things can get across.
Now, remember, we spoke about in the previous talks about all that water in the
human body and how much water gets absorbed in the guts and kidneys, and all
for what? Just to remind ourselves, in the gut, about nine liters of water gets
reabsorbed every day. OK, so most of the water that you drink and ingest gets
reabsorbed. And as you'll learn from Tim, the kidney is pretty amazing. It filters
about 180 litres of water a day. And although the plasma membrane is
permeable to water, the question is, can that much water get across? So for
many years, people were looking for water channels. And at some stage,
eventually, these things called aquaporins or water channels were discovered.
And here's the structure on the right hand side here. You can see it's showing all
those little blue things- the water molecules- so it's showing the water
permeation pathway through the channel. OK, so these are called aquaporins,
water channels that were discovered by Peter Agre, who got the Nobel Prize for
chemistry in 2004 for the discovery of these water channels. And these guys are
everywhere in the human body and they really do significantly increase the

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 Cell movement Lecture video 1 Ion Transport
[Phil Poronnik]
amount of water taken up. And because they're membrane proteins, they can be
taken in and out of the membrane, so they dynamically regulate it. So when
your body needs to absorb more water or get rid of water, you can insert the
channels, when it doesn't want to it takes them away. And you learn about that
in thirst responses and stuff like that, in the kidney. Very, very important
discovery. So if you think about, we've used this word facilitated diffusion and
here's an example, there's those two railway tracks, it's the membrane, it's
impermeable to those little blue things, they can't get across, OK? So what do
you do? What about if you put a pore in there, what's going to happen? So it
can't get across there, if you put the pore in, like a channel, lo and behold, they
move across to the contrary- until the gradient's equilibrated. So that's all
facilitated diffusion in something that allows ions or whatever, glucose, to get
across the cell membrane. Down it's concentration gradient. Now, what about if
you want to move things in the other direction, so perhaps you've got a lot more
on one side and you want to move even more against that concentration
gradient? Active transport is the use of a pore, a specialized protein that can use
ATP- the energy in the cells- to pump things out against their concentration
gradient. OK, so once again, these are two very important concepts of facilitated
diffusion and active transport. And here's another typical picture you'll see in a
textbook passive is typically down a concentration gradient active can be
against a concentration gradient. So you can see diffusion occurring there across
the membrane, very slow for water, facilitated diffusion under different kinds of
conditions, different kinds of proteins. But move things across the cell, transfer
the membrane into the cell or out of the cell and ATP pumps. OK, so these are
the important- and you see these over and over again. Now, one important thing
to remember about movement across the membrane, if you put some sort of a
pore or a hole across the membrane, they have particular behavioral
characteristics, they saturate. Ok, so, if there was nothing there, right, as the
concentration of the ion increased, the rate of movement across the membrane
would increase also linearly. And that's pretty much what happened with things
like oxygen and everything else, because there's no real barrier there. OK, but
because this is moving through a pore, what happens? It's like sucking water
through a straw, isn't it? And if you suck very slowly, some water will come up.
If you suck fast more water will come pretty quickly, as you start to suck harder
and harder no more water can come through because you've saturated the rate at
which the channel can conduct the substance. OK, so in other words, channels
saturate. OK, this is what happens. And that's a very important feature to think
about. An example is glucose in the kidney. OK, so what's a sign of diabetes?
It's increased glucose in the urine, isn't it? And the reason is because in the
kidney, the protein responsible for taking up glucose is working pretty much on

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 Cell movement Lecture video 1 Ion Transport
[Phil Poronnik]
maximum efficiency under the normal conditions. So when you saturate the
carrier with too much glucose you end up excreting more glucose, they get more
glucose in your urine. So it's a very good example of that kind of principle. OK,
the next thing is also a very, very important, fundamental concept. If you're
moving ions, these have charge, they're either positively or negatively charged.
If you move them across the membrane, you'll move charge. Okay, so if we put
a channel here and move some positive charge across, what happens? You start
to generate an electrochemical potential difference across the membrane,
depending on how many charge- how much the charge is spaced across. So this
sets up a difference in potential between the outside and inside of the
membrane, OK? Very, very important fundamental principle. And because ion
channels conduct charge, guess what? These guys, Burt Sakmann and Erwin
Neher came up with a really, really clever, elegant method for measuring these
things, they actually discovered that if you took a glass micropipette and pushed
it against the membrane, and you can see on top right- top left hand corner,
there's a neuron being patch clamped with this glass pipette. You can often
apply some suction and the suction suddenly forms a seal between the glass and
membrane of such high electrical resistance, you can actually measure the
current flowing through a single ion channel, the so-called giga-ohm seals. And
you can see the top right hand picture is a single channel inside that patch of
membrane. And you can see these are real-time tracings. So you can see with
nothing active in C, when you activate the channels in D, you can see all these
channels opening and closing in synchrony- that's just a single channel in a
patch of membrane opening and closing. Burt and Erwin won the Nobel Prize in
1991 for this. At Sydney Uni we were the second group in Australia doing this
kind of work in 1986. So it's a really, really amazing technique, it's still one of
the few techniques in the world where you can still watch the action of a single
protein in real time. It's really mesmerizing and beautiful. But this is all because
these things conduct charge. OK, OK, so we've spoken about how things can get
across the membrane through these pores, they move down diffusion- they can
conduct charge if they're moving ions and set up electrochemical gradients. But
the next missing bit of the equation is what is the concentration of the major
ions inside the cell? In this case, we'll think mainly about potassium and
sodium. OK, so we know from previous- the last the talk we discussed blood
concentrations. You know, if you look at your blood charts, you can see that
sodium is about 136-144 millimoles and potassium is very low, 3.65, "over
seven you're in heaven", that's the clinicians say- very tightly controlled. OK, so
what about inside the cell? So inside the cells, it's remarkable because it's
reversed, so if you've got high sodium on the outside, you've got low sodium
inside the cells, you've got low potassium outside the cells, high potassium

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 Cell movement Lecture video 1 Ion Transport
[Phil Poronnik]
inside the cells. OK, that's pretty amazing. And why? Because this actually sets
up these electrochemical gradients to allow us to use that gradient and drive
things and cause charge differences and everything else. So the way this
gradient works is it's set up by this active transport process by the ubiquitous
sodium potassium pump. You also hear it as the sodium potassium ATP-ase or
the sodium pump. OK, but this is a ubiquitous protein that actually generates the
gradients that help life to occur. And how it works is like this, right, the pump
uses ATP to pump sodiums out and bring potassium in. So if you're pumping
sodium out the sodium stays low, bring potassium in it becomes higher. OK.
And this is an old but very really quite excellent example. You can see the three
sodiums bind to the pump. It becomes phosphorylated by ATP. Undergoes a
conformational change, where it's open to the outside and potassium can come
in. It get's dephosphorylated and undergoes the original conformational change,
at the same time brings potassium back out of the cell, OK? That's so good I'll
let it play one more time. Sodium binds to specific sites there, becomes
phosphorylated, conformational change. These are all important words to
remember. Extracellular face, potassium comes in and binds.
Dephosphorylation occurs. And we're going through that cycle and that cycle
happens tens of thousands of times per second inside a cell, it's quite amazing.
Now. What does this mean? We're moving ions in opposite directions, OK, and
we're moving, remember, 3 sodiums out of the cell and only 2 potassiums in, so
3 positive charges out of the cell and 2 potassium- 2 positive charges into the
cell. That means the inside of the cell is more negative to the outside. Yes?
There's more positive charge outside that on the inside. So it's a negative
membrane potential. So all cells have these negative membrane potential and
this negative membrane potential is the socalled electrical driving force that
allows transport to occur inside cells. And if you think about it from a simple
perspective. Here's a graph, forget the things at the top. Just look at that red line.
So if you think about a cell, it's got a negative membrane potential of about -70
millivolts. And that's kind of what neurons have, fairly high negative membrane
potential so do muscle cells often. And nothing much is happening. But all of a
sudden, if you allow the sodium channels to open, right. So something allows-
triggers the sodium channel to open what will happen? Sodium will flow down
the gradient because there's more sodium outside than inside the cell. It'll start to
rush into the cell. As more positive charge comes into the cell membrane- into
the cell, the membrane will depolarize, become more positive. Yes? And at
some stage, all those sodium channels might switch off. So you've got a positive
membrane potential, lots of sodium moving inside the cells. If they shut, it'll
stay high. But what happens in return? Potassium channels open and potassium
starts to leave the cell. OK? As potassium leaves the cell rapidly down its

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 Cell movement Lecture video 1 Ion Transport
[Phil Poronnik]
concentration gradient, the cell becomes more negative and you get this
repolarisation phase. Okay, so what have you got here? It's an action potential,
isn't it? So this is how neurons work, effectively. Their voltage gated sodium
and potassium channels are open at certain voltages, when they get triggered,
this is your action potential, it gets fired. Okay, so it's a really important concept
to understand, and it just shows the elegance of these relatively simple
arrangements of proteins and channels. OK, so that's the active transport
between sodium-potassium ATP-ase, and membrane potential. If we look at
facilitated diffusion, things like glucose, OK, so glucose can move down it's
concentration gradient into the cells, and that's done by various transporters like
GLUT transporters as shown there. And these basically open and bind the
glucose on the outside of the membrane. And in response to the binding of
glucose, then they undergo a conformational change that might involve some
sort of phosphorylation event and they enter the cells. So this is the way that
glucose and amino acids enter the cells by these diffusion potential, facilitated
diffusion pathways, and as you'll learn later, these often use a sodium gradient
as a driving force to help. So the glucose hitches a ride on the sodium and
comes into the cells. Now, just OK, I recommend you look at this video, here's
we'll set it up at about ten minutes. It's about five minutes long, but it is
absolutely excellent. And it shows dancing proteins. So if we just skip to this bit
here. I think it's just- "What we do is we start with the initial structure of the
protein and then we push the protein along different pathways to go from the
initial state to the final state. And then along each pathway, we can measure
how hard it is for the protein and compare this pathway to see what pathway
might be the most relevant pathway. So in order to act as a pump, you need to-"
Okay, so we'll put the link to the video under the lecture. But as you saw just
then, this is using molecular data- X-ray crystal data of the real proteins to show
how the actual- these transporters can dance by opening and closing, and
moving things from the outside inside in response to things like
phosphorylation. So this is a really good video to watch. So we recommend that
you watch that video in its entirety, or at least for the bits around that structure,
it gives you a really good idea about how researchers can work on how proteins
work in real time and also gives you a lovely idea of the dynamic nature of how
proteins work. So I hope this is giving you an idea of how ion transport works.
The basic principles around active and passive transport in the generation of
membrane potential. And the all-important sodium-potassium ATP-ase OK, so
see you in the next lecture.

[End of recorded material]

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