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
- 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
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
- 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
- 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
- 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
- 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
- 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
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.
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
- 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
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