Fundamentals Transcript

9/15/08 (Dr. McNicholas-Bevensee)

10 – 11

I will be following along with the slides emailed to us that contain the notes. The reason for this is that there are extra slides that are in this packet than the original one posted.

Membrane Transport Mechanisms II and the Nerve Action Potential –slide 1

Epithelia – slide 2

Diagram – slide 3

Please refer to notes for slides 2 and 3, the recording did not start until middle of slide 4.

Models of Ion Transport in Mammalian Cells – slide 4

  • Basically this protein is there to set up the gradient, it only contributes a small amount to the potential of the membrane.
  • If the K channel is present, its going to allow movement of K out of the cell.
  • In the basolateral membrane, there is a transporter known as the Na, K, Cl co-transporter.
  • What it does, is moves Na, K, and Cl in co-transport in the same direction.
  • So this is a symport mechanism, but instead of having 2 ions move, there are 3 moving.
  • In this case, what this transporter is utilizing is the Na gradient produce by the Na-K ATPase. Generating a high Na gradient from outside of the cell to the inside of the cell.
  • This allows Na to be co-transported and take with it K and Cl.
  • This transport would stop working all together if there wasn’t any K around, so the movement of K out of the cell not only alters the membrane potential, but it also feeds this transported of K.
  • This is important for example in the lumen in the nephron (sp?) apical, membrane, where you need that K reabsorption. So this transport would actually be on the opposite side. So there not always on the same side of the cell, it depends on the role of the cell.
  • For example, if you block these channels you won’t have enough K to actually feed that so that Na, K, Cl co-transporter, and co-transport would stop.
  • In this case what this transporter is doing is moving Cl against its electro-chemical gradient. Because of the high intracellular negative potential, Cl is a negative charged ion and doesn’t want to go inside the cell. So it utilizes the Na gradient; Na is moving down its electro-chemical gradient into the cell. The movement of Na enhances the chemical gradient, but also the electrical gradient. Remember the inside is negative, so the positive charged ion wants to go inside the cell. That moves Cl into the cell and then on the apical membrane there are Cl channels present. That when open, they allow Cl to move into the mucosal side and Cl secretes into the apical or the luminal side of this epithelial shades.
  • The movement of Cl is moving small substance, so water being permeable through the tight junctions in this region will allow the osmotically active solute will attract the water molecules to it. So water moves with Cl; but if you just moving a net negative charge you have to have that charge balanced. In this case we have a epithelial layer that allows for paracellular Na transport/movement through the tight junctions. So to balance out the negative charge going into the lumen side, a positive charged ion is moving with it. That allows Na, Cl secretion and it takes water with it. So you have a watery solute secreted into the lumen.
  • Now one thing you will see time and time again especially in the renal material, is the transepithelial potential difference. Because of the differences between the proteins, the ion channels present in the apical vs. basolateral membrane; what happens is you have a different electrical potential on the basolateral side vs. the apical side. That generates a transepithelial potential difference. The point of this is to state that there is a difference of potential between the apical and the basolateral side.
  • The way this model is set up, there is a negative potential on the apical membrane, and a positive potential on the basolateral membrane. So, the negative luminal potential difference will actually help in the movement of Na across that tight junction and into the luminal side. Because it is negative potential and will attract that positive charged ion.

Absorptive Epithelia – e.g. Villus cell of the small intestine – slide 5

  • Ok, so now we are going to talk about an absorptive epithelium. In this case we are using an example of a cell of the villus of the small intestine in the GI section.
  • But the villus is the part of the structure that faces out into the gut lumen, and that’s involved in the absorption of solutes.
  • In this case we are going to look at two ways at looking at cell models. You will not always see the same diagrams.
  • Lets concentrate on the left hand side to begin with:
  • In the small intestine you want to be able to absorb glucose. But glucose is moving against its chemical gradient into the cell.
  • What you have to do is enable the movement of glucose and you use the secondary active transport mechanism, known as the Na-glucose co-transporter.
  • Again, you have a gradient of high concentration of Na on the outside of the cell compared to the inside of the cell. So, the Na is going to move down its electro-chemical gradient into the cell and its also going to take with it glucose.
  • Glucose moves into the cell and then it has to get out on the other side in order for you to utilize, you have to get it into the blood. In the basolateral side of the cell, epithelial layer, there is a different type of transporter, its not a glucose dependent transporter, it’s a simple transport mechanism that just allows for glucose movement out of the cell and into the blood side so you can absorb the glucose into the blood.
  • Again, the Na gradient is set up by the Na-K ATPase on the basolateral side of the cell membrane. So in epithelial cell, you’ll always find the Na-K ATPase on the basolateral side. It’s setting up the gradient of the movement of Na.
  • Again, you have a high concentration in cell and low concentration on the outside, so you need some help to get that solute against its chemical gradient. It’s not a charged molecule, so all you have to take into account is its movement against its chemical gradient.
  • Ok, so this is showing the same thing in a different way (talking about right side of the slide). You have the sodium-glucose symport that is present in the apical membrane. Na is transported out of the cell by the Na pump and then glucose is transported out of the cell by the glucose transporter. In this case it is giving the name of the transporter, GLUT 2. There are different kinds, but in this type of cell it is GLUT 2. That allows of glucose absorption. Again, the Na gradient is set up by the presence of the Na-K ATPase.
  • So this is an absorptive mechanism vs. the secretive mechanism.

ARS question is given.

Electrophysiological Technique: Patch Clamp – slide 6

  • Ok so what I’m going to talk about is, you’re going to hear about a technique called patch clamp technique. I’m just going to introduce a few physiological techniques that have been used.
  • Basically, this is a technique that I have done for way more years than I care to remember.
  • What you use is a glass pipette and an electrode, and because of the electro properties of the membrane, it attaches itself to the cell membrane and forms a high resistance seal.
  • So you can actually measure the ions moving between a channel.
  • But what you see on the screen are these blips. I’m going to show you how you sort of read these blips just in case someone in other lectures shows you these recordings you will know what you are looking at.
  • So basically, the channels are gated. So you have openings and closings. Some channels stay open all the time, some channels flickering away. In this case its open about ½ the time. So closed state is here, every time the line blips downward it means there is one channel opening, and because it is going down to a second level again a second time, there is a second channel opening, because it has reached two levels.
  • This is what’s known as the cell-attached patch. That means where you have only access to the extra-cellular side of the membrane.
  • If you pull that pipette away from the membrane, you have what’s known as the inside-out patch. Where you can now add substances that will mimic what was in the cytosol and look at regulatory processes, for example.
  • There is also, the whole-cell patch. You attach this glass electrode to the membrane; they’re very very small, tiny, tiny electrodes that you attach to the cell membrane. When you are looking at a whole-cell patch, you are looking at all the channels, all the activity of all the channels of the cell.
  • Here you can look at individual transitions in electrical activity through an ion, a single down to one ion channel. And in the whole cell you are adding them all together and you are looking at all of the ion channels present in the membrane.

Measuring Membrane Potentials (new slide) – slide 7

  • When looking at a whole cell recording when you measure a membrane potential. What you’ll do is stick it with an electrode, one electrode inside the cell and another electrode reaches the outside of the cell.
  • In this case you are measuring the potential that is present in the axon cell. So remember, this is also to illustrate most cells at rest have a negative intracellular potential.
  • The recordings show there is a -70 potential inside the cell with reference to the outside of the cell.
  • If that potential changes, the voltmeter will record the change. That is what will occur if you are recording an action potential along that axon.
  • So in the case where you are measuring single channel movements, you can tell it to be at -70 or at +50. The ion moving through that channel is determined by the electronics. In this case, its telling you what the overall intracellular potential is that is generated by all the channels that are active.

Terminology and Electrophysiological Conventions – slide 8

  • Ok, so here is some of the terminology that you will hear and some of the conventions that you will hear in lecture when talking about electrophysiological activity.
  • So if you look at the left hand diagram to start with: this is looking at the membrane potential. This is going to be important as we go through the nerve action potential to understand what these conventions are.
  • So when you hyperpolarize a membrane, you are making it very negative; and when you depolarize a membrane, you are making it very positive.
  • When there’s 0 potential the cell is at 0, there is no depolarization it is neither positive nor negative. So if you go more positive than 0, you are depolarizing and if you go more negative than 0, you are hyperpolarizing.
  • So what you might see and you will see in my lecture today, a current – voltage relationship. The current is on the y axis and the voltage is on the x axis.
  • When you are doing a technique such as patch clamp, you dictate what that potential is and you can measure the current that is moved through a channel, for example. And the current is plotted against the holding potential, the voltage that you are holding this cell membrane at.
  • Where there is no current movement in the positive or negative direction it is known as the reversal potential.
  • Now, if you’re talking about the movement of a single ion through a specific ion channel, that reversal potential will be the equilibrium potential for that channel. So you can tell what kind of channel you are looking at based on the reversal channel.
  • So if I had a channel and saw a positive reversal potential, the line went on the right hand side, in the positive direction = positive equilibrium potential, it will be Na. Remember Na is a positive, and equilibrium potential to stop Na from moving into the cell, down its equilibrium potential, so it’s to stop the net movement of Na.

Diffusion of electrolytes through membrane channels – slide 9

  • Other things that you will hear are the open probability channel. That is the probability that the channel is open.
  • Again, you have transitions between open and closed, and the open probability is characteristic of certain channels.
  • Conductance is basically the inverse of the resistance and it will tell you what kind of channels. So if it has a conductance, in this case its 200pS, it’s a high conductance channel. If it has say, 5pS, it’s a low conductance channel. And that means those upward and downward blips are greater for the high conductance vs. the low conductance.
  • We’ve already talked about the selectivity. Don’t worry too much about all the details, this is to just introduce these terms incase you see this later on in other lectures.
  • Basically, right now it’s a characteristic of a channel that the open probability can be changed. For example, a voltage gated channel, such as Na channel in the action potential; if there is a membrane potential change in that channel, there’s a configuration change in the protein and that channel opens up. It’s going to increase its open probability. If you block that channel its going to decrease its open probability.
  • There are other ways to do this, for example, some ligand gated channels. If the ligand binds to a channel, that binding causes the conformational change and that will increase its open probability. So if you increase its open probability it forms a conjugate that allows for more ions to move through, and so you’ll get more ionic movement and a current. For example, if you’re looking at all the channels in a cell it will increase the conductance in all those channels and will increase the current movement across the membrane.
  • I introduce the term selectivity. That’s determined by the structure of the ion channel. For example, in a K channel it’s a very different structure in the pore vs. the Na channel. That gives the feature and the selectivity in this specificity of the channels to do their job.

Potassium Channel – slide 10

  • Ok, so this just shows how some people might illustrate channels.
  • This is a ribbon diagram of a K channel, showing the selectivity filter of the pore, of a K channel.
  • Where you see ion movement through the pore.
  • It shows a different way of illustrating ion channels, where the K wants to move out of the cell.
  • So, the K is sitting and waiting to move from the intracellular side to the extracellular side. This illustrates the same helical part of the structure present. And the selectivity filter is just shown as this hole in the membrane.
  • Its just 2 ways of looking at K channels.
  • And in this case the K channel is open all the time.

Common Gating Modes of Ion Channels

  • There are different gating modes, different things that make the ion channels open and close.
  • For example, voltage gated channels, if there is a change in the membrane potential, it will cause a conformational change in that protein that allows the channel to open, and that allows ionic movement through the pore that is created by the channel opening.
  • If you have a ligand gated channel, for example, if it’s an extracellular ligand that binds. The binding causes a conformational change and opens up the pore and the ions move through. You can also have ligand gated from an intracellular substance. So, instead of a ligand that binds from the outside of the cell, a ligand can bind from the inside of the cell and does the same thing, in which causes a conformational change in which ion movement can occur through the pore.
  • There are also, mechanically gated channels. And these channels open up as you stretch the membrane, is causes an opening of that channel. These are found for example, in certain kinds of receptors in the heart; where they are non-selective and they open up in times of when you got hypertophy and is stretching on the membrane. That is a bad thing for the cell to have happen. Basically, these require the mechanical movement, the pull on the membrane that allows for those channels to open up. And so, they’re mechanically gated. They can also be regulated by intracellular substances, but the initial opening requires a change in the environment in that they are in that causes a pulling of the membrane.

Picture – slide 12

  • This is illustrating a voltage gated channel. If you look here this looks like a pair of gloves, I guess.
  • But as you cause a conformational change, a change in the potential across the cell membrane. This causes a conformational change that opens up the pore and there is a voltage sensor that senses that change in potential that causes the actual movement through the membrane. That movement then causes the movement of the opening of another part of the protein that allows of for extrusion through that pore.
  • For example this is a K voltage gated channel, where there is a change in membrane potential that causes a conformational change in the channel protein that opens up the pore and allows K movement.
  • So this is to illustrate how you can change the amount of current that can flow through an ion channel.

How the behavior of an ion channels can be modified to permit an increased ion flux – slide 13