Lodish 4th edition: Chapter 21 pages 921 - 924
Moyes: Chapter 3.
ATPase pumps use the energy from ATP to transport ions against their
A lot of energy in the cell (25% of the ATP) is used up by the ATPase pumps. Used for many different ions. Essential to maintain the Na+, K+ and Ca+2 concentration gradients that we will be talking about when we discuss cotransport, action potentials, and muscle contraction.
We will discuss mostly P-class pumps a class of ATPase pump that is phosphorylated to drive transport. There are a number of other classes diagrammed above and listed in Table 15-2 in your text book.
pumps 2 K+ in and 3 Na+ out
- important for many cellular functions (osmotic balance of cells)
- 3 Na+ out for every 2 K+ ions in
- uses ATP as energy source
- binding of phosphate from ATP drives conformation change that allows ions to be transported to appropriate sides
- an asparate residue becomes phosphorylated and the energy transfer changes the proteins conformational shape
- Na+ binding sites switch from high affinity on inside to low affinity on outside to allow for binding of Na+ on inside and release of Na+ ions on outside.
- K+ binding sites switch from high affinity on outside to low affinity on inside for the same reason
- can be blocked with poisons like ouabain or digitalis
- blocked pump can't transport ions across membrane and the chemical gradients for Na and K slowly disappear due to continuous action of the leak channels, resting potential removed.
- the potential built up in the Na+ ions will be used by many different processes i.e. cotransporters, neuronal signaling etc.
- pumps 2 Ca+2 ions out for every 1 ATP molecule used
- again uses ATP to drive Ca+2 out against a very large concentration gradient
- internal Ca+2 binding sites have a very high affinity (in order to overcome extremely low Ca+2 concentrations inside cell)
- energy transfer from ATP to the aspartate of the Ca+ ATPase causes a protein conformational change and Ca+2 transported across membrane
- Ca+2 binding sites on outside are low affinity and Ca+2 is released
- the transfer of eneragy from the ATP to the pump triggers a conformational change that moves the protein and allows the translocation of Ca+2 across the membrane. At the same time the Ca+2 binding sites change from high to low affinity.
- in muscle cells the Ca+2 ATPase is the major protein found in the membrane of the sacrcoplasmic reticulum (SR). As we will see the SR is a storage site for Ca+2 that is release to drive muscle contraction. The Ca+2 ATPase will remove excess Ca+2 from the cytoplasm and pump it into the lumen of the SR.
80% of the protein in the SR is the Ca+2 ATPase. Therefore this made it easy to purify and study in liposomes as we discussed in class.
Figure 15-12 is a schematic diagram of a potential model of the Ca+2 ATPase pump.
- 10 transmembrane regions are shown (these are predicted based on an alpha-helical like structure and perhaps hydrophobic properties, - those helices that interact with the Ca+2 of course will not be so hydrophobic)
- the large intracellular loops were thought to be important for pump function.
How could you go about testing which regions are important for function?
One way possible way is to carry out "structure/function" experiments.
- this is possible if you have cloned the pump (or the protein you are interested in)
- to test its function you can make mRNA from the full-length clone in the test tube and then express the mRNA by injecting into a cell or translating in an in vitro system
- the cell could be a tissue culture cell or a Xenopus oocyte for instance if the protein is not normally expressed in these membranes
- or the protein could be translated and introduced into liposomes which are "balls" or spheres of pure lipids (outlined in Figure 15-4 below).
- once the protein is is the membrane you can now test the function of the expressed protein
- using a Ca+2 tracer, radioactive Ca+2 or a Ca+2 sensitive dye for instance you can now test the function of the pump (some researchers follow the current generated by the Ca+2 crossing the membrane)
- the next step is to make changes in the pump by altering the DNA (remove regions that you think are important by deleting or changing their coding sequence) and then express the mRNA from these mutant pumps, inject this into your cells or liposomes and test the function of the pump
- in this way you can define the regions that are important, like the ATP binding region, the asparatate, and the energy transduction regions outlined in the figure above.
These pumps transport H+ only.
- found in lysosomes, endosomes and plant vacuoles.
- transport H+ ions to make the lumen or inside of the lysosome acidic (pH 4.5 - 5.0)
- many of these pumps are paired with Cl- channels to offset the electrical gradient that is produced by pumping H+ across the membrane.
As H+ is transported into the lysosome Cl- flows in to keep a balance. If Cl- doesn't flow in then there is rapid build up of potential (charge) across the membrane which would block the further transport of H+. This would occur long before the lumen becomes acidic because not that many ions need to be transported to produce the voltage potential.
Other pumps like the H+/K+ ATPase pump (a P-class pump) transport H+ out and at the same time K+ in to make sure there is no voltage potential created.