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BIO241 Lecture 3

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Jennifer Harris

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Tuesday, January 13, 2009 - Last lecture we covered different types of transporters & we covered both passive & active transport – looked at the glucose transporters as a type of passive transport & we looked at symporters & antiporters that carry out secondary active transport. And we also looked at ATP-driven pumps – the P-type ATP-driven pump (ATPase) as an example of primary active transport. - Today we’re also going to continue with primary active transport or ATP-driven pumps & look at 2 additional types – V-type ATPases & F- type ATPases. We’ll also look at how the different transporter proteins that we covered in the last lecture worked together in order to transport small molecules across a cell. In particular, we’re going to look at how a combination of transporters are used to transport glucose across the intestinal epithelial cells. - Last lecture we covered the glucose symporter that passively transports glucose down its concentration gradient. - We also covered the Na-glucose symporter – an example of secondary active transporter. - And we also looked at Na-K pump which is an example of an ATP- driven pump or transport ATPase that uses the energy from the hydrolysis of ATP to transport both Na & K against their electrochemical gradients. - Now these pumps don’t act on their own – in a cell there are many different types of transporters & they can act together in order to transport a molecule across/into a cell. - The example we’re going to go into is how these 3 pumps that we just looked at are used to transport glucose from the lumen of the intestine across the intestinal epithelial cell, into the extracellular fluid that will eventually end up in the bloodstream. - Diagram: This is the inside of the intestine (the cylinder) & we’re looking at the lumen of the intestine here & what’s wrapping the intestine would be these intestinal epithelial cells. So glucose needs to be transported from the intestinal lumen to the extracellular fluid outside the intestinal epithelial cells. On the right hand side: you can see what is the relative concentration of glucose in each of these different compartments. In the intestinal lumen, there is a relatively low glucose concentration – it becomes concentrated inside the intestinal epithelial cells so you have a relatively high glucose concentration. Then in the extracellular fluid, again the glucose concentration is relatively low. Can see that to transport st 1 from the intestinal lumen into the intestinal epithelial cells, you need to go from low glucose concentration to high glucose concentration which is against the glucose concentration gradient. - Then if you look at transport of glucose from inside the intestinal epithelial cells to the extracellular fluid, this is transport down the concentration gradient of glucose.  Restricted by tight junctions  Na /glucose symporter + +  GLUT2 uniporter & Na /K pump - What’s important is that the intestinal epithelial cells have different domains of the membrane that are separated by what are called tight junctions. So you have the apical plasma membrane as seen in the diagram which is distinct from the lateral & basal membrane – these tight junctions will occlude any proteins from traveling b/w these 2 membrane spaces – so a protein that is found in the apical plasma membrane will not localize down to the basolateral membrane & vice versa. So this allows for the asymmetric distribution of proteins in these intestinal epithelial cells. - So the transport proteins, the movement of these is restricted by the tight junctions so as we said proteins can be laterally in the membranes & since there are these tight junctions there, the lateral movement is limited to the region b/w these tight junctions here. - So in an intestinal epithelial cell the apical membrane contains a Na- glucose symporter. The basolateral membrane contains a glucose uniporter & a Na-K pump. - So if you recall the concentration gradient, glucose is very high inside the cell relatively speaking so when the 1 transport by a symporter where glucose will be transported against its concentration using the energy of driving Na down its concentration gradient. - So remember that Na is higher outside the cell than inside the cell. And this is maintained by the Na-K pump which is continuously pumping Na outside of the cell so this is carrying out primary active transport so any Na found in the cell will be pumped out using the energy from the hydrolysis of ATP to maintain the Na levels in the cell to a relatively low level. So you’ll have a low Na concentration inside the cell, a relatively high concentration of Na outside the cell & then this transport down the concentration gradient, that energy can be used to drive glucose into the cell against its concentration gradient through symport of Na & glucose. - Then you’ll recall that glucose is relatively high inside the cell & relatively low outside the cell so the glucose 2 uniporter can passively transport glucose down its concentration gradient from the inside of the intestinal epithelial cells to the extracellular fluid. - This diagram is basically showing the glucose concentration on right hand side. - So 1 of all you have the Na-K pump which is pumping Na outside of the cell against its concentration gradient & this is maintaining a low Na concentration inside of the cell. And the Na-K pump also pumps K against its concentration gradient so it’s pumping both of these against its concentration gradient using the energy from the hydrolysis of ATP. - So since there is a low Na concentration inside the cell relative to the outside of the cell, glucose can be transported against its concentration gradient by symporting Na down its concentration gradient so that energy drives the movement of glucose into the cell against its concentration gradient. - Now once glucose gets inside the intestinal epithelial cells, it builds up to relatively high concentrations relative to the extracellular fluid & then the glucose transporter (the uniporter) can passively transport glucose down its concentration gradient into the extracellular fluid. - So you can see how these 3 transporters work in concert in order to promote the transport of glucose from the intestinal lumen to the extracellular fluid. - Answer: B - GLUT Uniporter carrying out passive transport. - Answer: C - Answer: C - That’s how the transporters that we’ve covered work together in the intestinal epithelial cells. - Now we’ll move onto 2 different types of ATP-driven pumps – the V- type & F-type ATPase. - If you look at the structure of these 2 types of ATPases, you can see that they look very similar – see there is like a lollipop structure – there is a head of the lollipop & the stick is actually embedded in the membrane & this green part on both of these ATPases is actually able to rotate in the membrane & the orange part is actually static in the membrane & does not move. - What happens is the pumping of protons, in this case so both of these are involved in moving protons, causes this rotor (the green part) to rotate & when this rotor rotates, it actually induces conformational changes in the head of the lollipop (A & B or alpha & beta subunits) & this energy can be harnessed from the F-type ATPase so you get mechanical movement of this rotor which induces a conformational change & that energy can be harnessed in this case to synthesize ATP. So the movement induced in this rotor by the movement of protons, in this case, it would be down their electrochemical gradient, induces modifications in the protein & that mechanical energy can be harnessed to drive the synthesis of ATP - Now the V-type ATPase actually works in reverse in most cases, so it hydrolyzes ATP & then that induces a rotation in this part of the molecule which can then drive protons against their concentration gradient so one key feature of these (although they look very similar) is that most cases, they are working in opposite directions. - These molecules can work in reverse depending on the situation & the circumstances in the cell these molecules can actually work in the opposite direction where this one would pump protons out or this one would synthesize ATP. It depends on the cellular context but in most cases, the V-type ATPases are pumping protons using the hydrolysis of ATP as an energy source & the F-type ATPases are synthesizing ATP by harnessing the energy of moving protons down their concentration gradient. And they both have a similar overall protein structure.  Energy from ATP hydrolysis  No phosphorylation of the pump – unlike the P-type ATPases which are phosphorylated (P for phosphorylated) – these ones (V-type) are not phosphorylated. +  H pumped to inside of organelle - On the right hand side: the concentration of protons is not indicated but let’s assume it’s higher on the inside & then you would be pumping protons from a low concentration to a high concentration – this would be against the concentration & for a proton, it would have to be against the electrochemical gradient & the energy is provided by hydrolyzing ATP so this is a primary act of transport – ATP is hydrolyzed to move a proton against its electrochemical gradient. - In a number of compartments, protons contribute to the pH/acidity of a certain compartment – the more protons you have, the more acidic a compartment will be so by pumping protons into compartments, it can maintain a low pH (more acidic) in lysosomes & vacuoles, for example – continuously pumping protons into these compartments & makes them more acidic so it can regulate the pH of compartments & this is done by pumping protons inside the organelle using the energy from the hydrolysis of ATP. Now this sets up an H gradient in most cases – recall that a proton gradient can also produce a proton motive force – a proton motive force would be the energy that is harnessed from the movement of protons down their electrochemical gradient & that can drive the active transport by other transport proteins. Ex: symporter can use that energy to move another molecule against its concentration gradient.  V-type ATPase  PPi (pyrophosphate) powered pump  high [H ] inside – relative to the cytosol - A good example of where a V-type ATPase plays an important role is the plant vacuole (vacuole – V-type ATPase) – so this V-type ATPase plays an important role in the plant vacuole – it builds up or contributes to the proton motive force that is built up across the plant vacuole or membrane so there is a lot more protons inside the plant vacuole lumen than there is outside in the cytosol & this is generated by 2 pumps: the V- type ATPase (going to go into detail) & the PPi powered pump (not going to go into detail). So both of these are using energy to drive protons into the plant vacuole/lumen – 1 of them is hydrolyzing ATP to ADP & that is providing energy & the other one is hydrolyzing pyrophosphate to 2 molecules of inorganic phosphate & that provides the energy to pump protons into the vacuole also. - The V-type ATPase is contributing to the maintenance of proton motive force across the plant vacuole/membrane. This results in a high concentration of protons inside the lumen of the vacuole relative to the cytosol – so the pH is lower (actually quite a bit lower, it can be very, very low down to a pH of about 3 & that can contribute sometimes to when you bite into a fruit for example, can have a very acidic flavour & a lot of that is contributed by the proton or the acidity of the vacuole in those tissues). So the pH of the vacuole can be very low relative to the cytosol, down to 3 and the cytosol is about a pH of 7.5 which is more basic. - And then this gradient of protons actually sets up a membrane potential so that there is a difference in the electrical charge from one side of the membrane to the other & the difference in charge is about 20 millivolts so the inside of the vacuole is positively charged relative to the outside of the vacuole.  Down electrical gradient  (Against concentration gradient) - Now why set up this proton gradient? It is used by other transporters in order to maintain or bring molecules into the vacuole. - Proton antiporters will move one molecule down their concentration gradient & these are secondary active transport so the proton antiporter will move protons down their concentration gradient & harness that energy to move another molecule against its electrochemical gradient. So he might be using electrochemical & concentration gradient interchangeably but remember for a charged molecule we should be talking about electrochemical. - So you have Na, Ca & sucrose proton antiporters in the vacuole which will pump Na, Ca & sucrose into the vacuole & this can build up a very high concentration of these in the vacuole relative to the cytosol & the energy is provided by this prot
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