The Sodium Pump

Question 1

what is the pump-leak hypothesis?

Answer 1

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.

Question 2

What evidence is there that the Na pump is an ATPase?

Answer 2

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.

Question 3

what energises the pump?

Answer 3

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.

Question 4

What is the nature of the cation transport system?

Answer 4

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.

Question 5

Describe the pump reaction mechanism?

Answer 5

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.

Question 6

List the five operational modes of the pump?

Answer 6

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)

Question 7

Describe evidence for the tissue distribution of pump isoforms.

Answer 7

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

Question 8

What are possible functions of the beta subunit?

Answer 8

1) Enzyme assembly
2) Intracellular transport of whole enzyme
3) stability of alpha subunit

Question 9

What do electronic gradients do?

Answer 9

Maintain osmotic balance, the cell resting potential and muscle and nerve cell excitability. Maintaining the gradients requires energy.

Question 10

What are examples of essential functions of the Na-K-ATPase?

Answer 10

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.

Question 11

How can ion gradients be removed?

Answer 11

When a cell dies, when glucose is withheld or when specific inhibitors are used.

Question 12

What are the properties of the ATPase enzyme?

Answer 12

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.

Question 13

What are the properties of the alpha subunit? CATALYTIC

Answer 13

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

Question 14

What are the properties of the beta subunit? REGULATORY

Answer 14

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

Question 15

What are the properties of the gamma subunit?

Answer 15

1) Small axillary protein
2) 8-14kDa in size
3) FXYD protein family
4) single spanning membrane peptide

Question 16

How do we know there are multiple isoforms of the ATPase?

Answer 16

Different ATPase activity sensitivity to inhibitor ouabain found in different tissues.

Antibodies also show different levels of affinity between tissues.

Question 17

What effects does insulin have on the pump?

Answer 17

It stimulates translocation of the Na, K-ATPase subunits to the plasma membrane (via kinase phosphorylation pathways)

Question 18

What effects does muscle contraction have on the pump?

Answer 18

It increases translocation; it is a good insulin replacement by electrostimulating the muscle.

Higher abundance of alpha subunits at surface.

ERK -dependent mechanism.

Question 19

Give examples of long term regulation that usually involve altering gene expression and protein production.

Answer 19

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

Question 20

List short term regulation of the pump?

Answer 20

change in pump affinity for substrates, surface abundance,or covalent modification.

Question 21

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.

Answer 21

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.

Question 22

Describe the results when antibodies are used to identify which pump subunits are moving.

Answer 22

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.

Question 23

Explain what factors can cause pump translocation?

Answer 23

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.

Question 24

List the types of kinase that can phosphorylate

Answer 24

1) Protein Kinase B (PKB)
2) PKA
3) PKC
4) Phosphoinositide 3 kinase- PI3K
5) Mitogen activated protein kinase (MAPK)

Question 25

Name some kinase inhibits and where they interfere in the signalling cascade.

Answer 25

Along the pathway from insulin recptor: PI3K is inhibited by WORTMANNIN so it the pathway cant stimulate the pump to translocate.

PI3K IS NEEDED FOR TRANSLOCATION as in the presence of its inhibitor, translocation is completely stopped.

Along from the growth factor receptor MEK1/2 is inhibited by PD.

The kinase PKC (independant of a pathway/on its own) is inhibited by GFX so cannot directly phosphorylate.

PKC HOWEVER, IS NOT NEEDED FOR TRANSLOCAION, as in the presence of its inhibitor, translocaion is not effected.

Question 26

Explain the alpha subunit of the pumps role in kinase influence on traslocation?

Answer 26

There are specific residue sites on the subunit that allow for direct modification.

Amino acid TYR10 is a site for kinase Scr.

SER11 and SER18 for PKC

THR81 for ERK

THR414 for PKB

SER943 For PKA

Question 27

What does biotinylation detect?

Answer 27

Change in membrane alpha subunits

Question 28

What are the effects of high fat feeding?

Answer 28

The subunits were found at different levels of abundance at the surface, ie, a1 was more abundant than a2 and b1 was less abundant than b2; modifies expression levels.

The pump only functions as a dimer so requires all the subunits to translocate.

Question 29

What affect does endurance training have on pump expression in the membrane?

Answer 29

It antagonizes the effects for high fat feeding and increases subunit translocation.

Question 30

What effect does the C-peptide, (cleaves as part of proinsulin) have on the pump?

Answer 30

It stimulates the PHOSPHORYLATION of a1 subunits in an ERK dependant mechanisms

It stimulates translocation of a1 and b1 to the basolateral membranes (BLM) from an endosomal compartment.

Question 31

Why is MAPK signalling important?

Answer 31

MAPK signalling serves as a major mechanism for regulating the Na,K-ATPase and is translocation.

Its inhibitor PD decreased phosphorylation of the pump, it is needed.