Structure, Function and Regulation of the Na Pump - 1 &2 Flashcards
(51 cards)
What is the Pump leak hypothesis?
mechanism for the reabsorption of sodium in the kidney nephrons, in order to achieve and maintain the fluid-balanced amount of fluids within the human body.
Give evidence that the Na Pump is an ATPase ?
Na Pump is transporter and an enzyme (ATPase - an enzyme that breaks down ATP)
What is the Na-K-ATPase help with ?
-helps establish and maintain the high internal K+ and low internal Na+ concentrations ( typical of most animal cells )
Why is the electrochemical gradient critical ?
- maintaining Osmotic balance and a stable resting membrane potential in most tissues,
- and the excitable properties of muscle and nerve cells.
What is the Na gradient useful for?
- provides energy which fuels Na-coupled transporters mediating the translocation of ions (H+, ca2+,cl-,PO3-4,SO2-4), substrates ( glucose and amino acids) across the plasma membrane.
What is the enzyme Na-K- ATPase useful for?
in the kidney , the Na-K- ATPase plays a primary role in driving the reabsorption of Na+ and Water
-the enzyme is essential in the maintenance of body fluid and electrolyte homeostasis
What is the resting membrane potential ?
-65mV
Why do cells have gradients ?
- every cell has gradients within cells - so that they can be taken in or out of the cell
- a large amount of energy required to push charged ions through the hydrophobic core !
there is a concentration gradient and an electrical gradient and an electrochemical gradient
how can gradients be abolished ?
- cell death
- withholding glucose
- use of specific imhibitors
( this implies that maintianing these ion gradients requires metabolic energy
what is the pump- Leak hypothesis ?
- the membrane barier has holes
the pump leak model is a mechanism for the reabsorption of sodium in the kidney nephrons, in order to achieve and maintain the fluid-balanced euvolemic state within the human body.
The pump-leak model explains the relationship between the transport of ions and cellular metabolism.
What are the experiments used to prove the pump - leak hypothesis?
- cells were incubated with radioisotopically labelled Na or K indicated that under steady state conditions there is a positive influx of Na and a passive efflux of K, This has led to the so - called ‘pump leak’ hypothesis!!
SUMMARY:
active extrusion of Na and upatke of K ions is balanced by a passive leak of these ions in opposing directions
when cell takes in radioactive sodium - there is a leak of Na+ into the cell and so there are holes in the membrane barrier
( in the case of K+ the leak is outwards , in the case of Na+ the leak is inwards )
(n.b. the pump is 2Kin and 3 NAout !)
a Transmembrane potential also exists - therefore extrusion of Na not only takes place against a chemical gradient but a potential gradient.
Does the movement of Na and K across the membrane requires metabolic energy ?
Yes !
Oxidative metabolism occurs in many cells , why is this?
- it serves as a major source of energy for active Cation transport.
when oxygen is withdrawn from the cells or inhibitors of the mitochondrial oxidation inhibit cation transport
is oxidative metabolism present in red blood cells ?
No ! - since red blood cells do not contain mitochondira , however inhibitors of glycolysis and removal of glucose still inhibits active cation transport.
this suggests that ATP rather than glycolysis events energise the Na Pump !!!
What is the nature of the cation transport system?
ATPase activity is linked to Na+ pump.
What is an inhibitor of the Na pump?
-Ouabain it inhibits the ATPase activity
are the propeties of an enzyme similar in different animal cells?
- Yes !
they require : Mg ( cofactor!), ATP, NA and K for activity - the pump splits the ATP and transports 3 Na out and 2 K in per ATP hydrolysed.
-it is always inhibited by ouabain !
-optimum pH is 7.0-7.5
What are the Km values for Na and K?
Na- 5-10mM
K- 0.4-1.8mM
What does electrogenic mean?
- the net movement of 1 positive charge out of the cell (e.g. 3 Na+ out and 2K+ in!)
What does the Na/K- ATPase functional cycle look like?
( refer to diagram on p5 of Na pump notes 1 )
steps:
1. 3Na+ from the intracellular fluid bind
2. ATPase is phosphorylated with Pi from ATP
3. ( the protein changes conformation) , Na+ released into extracellular fluid
4. 2 K+ from extracellular fluid binds
5. ( protein changes conformation) 2K+ released into intracellular fluid ( increased affinity for sodium occurs now )
and cycle repeats
What is the reaction mechanism based on the Albers prost scheme ?
- substrate binding - step 1 - binding on one molecule of ATP accompanied by cooperative binding of 3 Na ions
- phosphorylation of the enzyme - step 2- gamma phosphate of ATP is transferred to an Aspartyl B Carboxyl group with the release of ADP
- transformation of the phosphoenzyme - step 3 - transistion from an ADP sensitive to an ADP insensitive phosphointermediate which undergoes spontaneous hydrolysis that is stimulated by the presence of K ( ste 3-5)
transisition involves a reduced affinitiy for ADP and a drastic change in Na and K affinities. ( this step is called the transport step!)
The ratio of Na release and K binding by the enzyme corresponds to the ratio of 3 Na ions expelled for 2 K ions taken up, ( just the transistion step is the transport step!!!)
hydrolysis of the phosphoenzyme - step 5- phosphate is released into intracellular space. the hydrolysis of the aspartyl = phosphate bond is OUABAIN SENSITIVE !!!
Return to the native enzyme form - steps 6 and 7 - the enzyme undergoes a confromational change from the occluded E2-K from ( step 5) to the non- occulated E1-K form, K is released into the cytosol and the protein is ready to begin another cycle.
What is an integral part of the enzyme?
-the K - stimulated phosphotase activity !!!
the pump and the ATpase are the same !
What can hydrolyse ATP and synthetic substrates?
purified proteins reconstituted into liposomes can hydrolyse ATP in a K- dependent manner
this process can be visualised using synthetic substrates such as P- nitrophenylphosphate ( potassium stimulates is and increases its hydrolysis ) (can be given instead of ATP and since the enzyme cant tell the difference between ATP and this - it breaks it up and changes it to yellow)
- this is hydrolysed by the enzyme to yield - P- nitrophenol which is yellow !
- this is OUABAIN sensitive ! ( prevents yellow colour being produced - which demonstrates the indicator must work)
What happens when the pump is taken out of the cell?
- it is found that the protein can work in different ways when taken out of the pump. ( when using a radiotracer labelled Na and K ).
when looking at the :
- Na-Na exchange (1:1) - if there is no Potassium outside the cell - you can force it to do NA exchange
- uncoupled Na efflux - all Na will get ‘locked in the outward face’ of the membrane.
- K-K exchnage (1:1) - K exchange !
