The Sodium Pump Flashcards
what is the pump-leak hypothesis?
The cell will passively leak ions in opposing directions and therefore balances the extrusion of Na+ by the uptake of K+. The Na+ is pumped out against both chemical and potential gradients. On average the transmembrane potential is -65mV.
What evidence is there that the Na pump is an ATPase?
Oxidative metabolism serves as the main source of energy for cation transport. In the absence of oxygen, needed for mt oxidation, transport stops. In red blood cells there cant be oxidative metabolism so ATP must energise the pump. If ATP is given the RBCs, transport is reinstated.
what energises the pump?
The Na+ ion gradient provided the energy that fuels sodium coupled transporters. This in turn mediates other ions (Ca, Cl, h+) and substrates (protein, amino acids) to be transported.
What is the nature of the cation transport system?
Activity requires Mg2+, Na+,K+, ATP. Activity increases when ions NA+ and K+ are added. When the pump is inhibited by ouabain (digitalis glycoside) transport decreases/stops.
Describe the pump reaction mechanism?
Na+ binds from ICF (inside cell). ATP is converted to ADP and the phosphate binds to enzyme; enzyme is phosphorylated with the gamma phosphate of ATP joining the carboxyl group and causes protein to change conformation. Na+ affinity decreases and are released. K+ binds to sites and phosphate is hydrolysed at the aspartyl-phosphate bond. enzyme changes conformation, returning to the original shape. K+ affinity decreases and are release. Repeats.
List the five operational modes of the pump?
Some of the steps in the reaction are reversible so:
1) Normal mode (Na/ exchange)
2) Reverse mode
3) Na-Na exchange (1:1)
4) Uncoupled Na out
5) K-K exchange (1:1)
Describe evidence for the tissue distribution of pump isoforms.
There are isoforms of the pump in different tissues as the response to the same inhibitor ouabain varies; brain shows oscillations in activity decrease, kidneys do not.
Alpha 1 is ever present isoform whereas Alpha 4 is only in spermatozoa (testis). A2 and A3 are in excitable tissues.
Beta expressed in tissues dependant on the oxadative/contractile properties of muscle fibers
What are possible functions of the beta subunit?
1) Enzyme assembly
2) Intracellular transport of whole enzyme
3) stability of alpha subunit
What do electronic gradients do?
Maintain osmotic balance, the cell resting potential and muscle and nerve cell excitability. Maintaining the gradients requires energy.
What are examples of essential functions of the Na-K-ATPase?
1) In the kidney it helps to reabsorb water and Na+ and so maintains body fluid and electrolyte homeostasis.
2) pump maintains transmembrane electrochemical gradients of Na/K and so gives stable Membrane potential.
3) The sodium gradient allows for the translocation of some ions by providing energy to correct transporters.
How can ion gradients be removed?
When a cell dies, when glucose is withheld or when specific inhibitors are used.
What are the properties of the ATPase enzyme?
Optimal pH- 7-7.5, Km value for Na 5-10mM and for K 0.4-1.8mM, 3 Na+ out, 2K+ out per ATP hydrolysed.
What are the properties of the alpha subunit? CATALYTIC
1) The binding sites for Na, K, ATP, Mg and inhibitor ouabain, with intrinisic activity.
2) 4 isoforms -a1,a2,a3,a4
3) 112kDa in size
4) regulated like a tissue
What are the properties of the beta subunit? REGULATORY
1) Very Glycosylated (28%)
2) 3 isoforms- B1,B2,B3
3) 35kDa in size
4) B1 and B2 regulated like a fiber
5) B2 can be called AMOG
What are the properties of the gamma subunit?
1) Small axillary protein
2) 8-14kDa in size
3) FXYD protein family
4) single spanning membrane peptide
How do we know there are multiple isoforms of the ATPase?
Different ATPase activity sensitivity to inhibitor ouabain found in different tissues.
Antibodies also show different levels of affinity between tissues.
What effects does insulin have on the pump?
It stimulates translocation of the Na, K-ATPase subunits to the plasma membrane (via kinase phosphorylation pathways)
What effects does muscle contraction have on the pump?
It increases translocation; it is a good insulin replacement by electrostimulating the muscle.
Higher abundance of alpha subunits at surface.
ERK -dependent mechanism.
Give examples of long term regulation that usually involve altering gene expression and protein production.
Insulin, T3,aldosterone, catecholamines.
diseases such as diabetes, renal dysfunction, cardiovascular hypertension,thyroid, neurological disorders
Endurance training, electrical stimulation.
A diet of high fat feeding
List short term regulation of the pump?
change in pump affinity for substrates, surface abundance,or covalent modification.
Describe and explain the results from an experiment in which the frog calf muscle was immersed in ouabain, rinsed, then exposed to insulin before it was exposed to both.
Na+ inside cell is release and the associated radioactivity was measured.
Exposer to ouabain: Na+ released decreases
Exposer to only insulin: Na+ released increases. This is because additional pumps are TRANSLOCATED to the surface membrane from internal stores.
Exposer to both insulin and ouabain: decreases activity again as the ouabain inhibits the new pumps.
Describe the results when antibodies are used to identify which pump subunits are moving.
A control and an insulin treated muscle are compared
a1 and a2: more concentrated in the plasma membrane than the internal membrane.
B1 and B2 also more concentrated in PM than IM after insulin.
Explain what factors can cause pump translocation?
Phosphorylation switches enzyme.
In presence of calcium or insulin Kinase and a phosphate covalent modify enzyme to become ON/ACTIVE.
Phosphatase and removal of a phosphate switch enzyme OFF.
Can be caused by a signalling cascade; INSULIN RECEPTORS when active turn ON the cascade.
List the types of kinase that can phosphorylate
1) Protein Kinase B (PKB)
2) PKA
3) PKC
4) Phosphoinositide 3 kinase- PI3K
5) Mitogen activated protein kinase (MAPK)
