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Biomembranes > Structure, Function and Regulation of the Na Pump - 1 &2 > Flashcards

Structure, Function and Regulation of the Na Pump - 1 &2 Flashcards

1
Q

What is the Pump leak hypothesis?

A

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.

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2
Q

Give evidence that the Na Pump is an ATPase ?

A

Na Pump is transporter and an enzyme (ATPase - an enzyme that breaks down ATP)

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3
Q

What is the Na-K-ATPase help with ?

A

-helps establish and maintain the high internal K+ and low internal Na+ concentrations ( typical of most animal cells )

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4
Q

Why is the electrochemical gradient critical ?

A
  • maintaining Osmotic balance and a stable resting membrane potential in most tissues,
  • and the excitable properties of muscle and nerve cells.
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5
Q

What is the Na gradient useful for?

A
  • 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.
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6
Q

What is the enzyme Na-K- ATPase useful for?

A

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

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7
Q

What is the resting membrane potential ?

A

-65mV

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8
Q

Why do cells have gradients ?

A
  • 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

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9
Q

how can gradients be abolished ?

A
  • cell death
  • withholding glucose
  • use of specific imhibitors

( this implies that maintianing these ion gradients requires metabolic energy

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10
Q

what is the pump- Leak hypothesis ?

A
  • 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.

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11
Q

What are the experiments used to prove the pump - leak hypothesis?

A
  • 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.

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12
Q

Does the movement of Na and K across the membrane requires metabolic energy ?

A

Yes !

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13
Q

Oxidative metabolism occurs in many cells , why is this?

A
  • 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

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14
Q

is oxidative metabolism present in red blood cells ?

A

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 !!!

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15
Q

What is the nature of the cation transport system?

A

ATPase activity is linked to Na+ pump.

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16
Q

What is an inhibitor of the Na pump?

A

-Ouabain it inhibits the ATPase activity

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17
Q

are the propeties of an enzyme similar in different animal cells?

A
  • 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
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18
Q

What are the Km values for Na and K?

A

Na- 5-10mM

K- 0.4-1.8mM

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19
Q

What does electrogenic mean?

A
  • the net movement of 1 positive charge out of the cell (e.g. 3 Na+ out and 2K+ in!)
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20
Q

What does the Na/K- ATPase functional cycle look like?

A

( 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

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21
Q

What is the reaction mechanism based on the Albers prost scheme ?

A
  • 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.

22
Q

What is an integral part of the enzyme?

A

-the K - stimulated phosphotase activity !!!

the pump and the ATpase are the same !

23
Q

What can hydrolyse ATP and synthetic substrates?

A

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)
24
Q

What happens when the pump is taken out of the cell?

A
  • 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 !
25
Q

What do you know about the Na Pump?

A
  • helps maintain normal transmembrane electrochemical gradients of Na and K in association with the membrane permeability properties.
  • through the maintainance of these gradients helps maintain a stable membrane potential.
  • energy in the Na gradient is used to drive the secondary active transport systems for sugars and amino acids !
26
Q

What are the subunits making up the sodium -potassium pump ?

A
  • alpha
  • beta
  • gamma
27
Q

what do you know about the alpha subunit?

A
  • catalytic subunit - possesses binding sites for - Na, K , ATP, Mg and cardiac glycosides (e.g. ouabain )
  • intrisnic ATPase activity (allows phosphate to bind)
  • 4 isoforms - (A1,A2,A3 and A4)
  • molecular size = 112kDa ( large protein!)

developmentally regulated and expressed in a tissue specific manner - some tissues express different isoforms.

linked to expression in different times and development

28
Q

What do you know about the beta subunit ?

A
  • regulatory subunit
  • three isoforms ( B1,B2, B3)
  • heavily glycosylated ( has a sugar attached )
  • protein moiety (35kDA) crucially required for full enzyme activity - but also for:
    enzyme assembly , intracellular transport of complete enzyme molecule, stability of the alpha subunit
  • in plasma membrane
  • required for intracellular transport and stability of alpha subunit

ALPHA NEEDS BETA SUBUNIT ! WITHOUT BETA - ALPHA WILL DEGRADE ! BETA IMPORTANT FOR ITS STABILITY !

29
Q

What do you know about the gamma subunit?

A
  • small auxillary protein (8-14kDa) belonding to the FXYD protein family
  • thought to be involved in stabilising the whole thing !!
30
Q

what is the evidence for multiple isoforms of Na K- ATPase ?

A
  • sensitivity to cardiac glycoside different in different tissues ( e.g. Brain and Kidney ) ( refer to diagram on lecture 1 of Na pump - page10)

tissue specific antibodies against purified enzume antibodies against one tissue only partially inhibited – Na , K-ATPase activitiy of other tissue. ( antibodies generated that would bind to the alpha subunits )

( refer to lecture 1 of Na pump page 11 diagram)

31
Q

What is a difference in alpha subunits in brain and kidney ?

A

brain may have 2 or more alpha subunits in comparison to kidney

32
Q

What unique functions does the Beta subunit have in skeletal muscle ?

A
  • the B1 and B2 subunits are expressed differentially in skeletal muscle depending on the oxidative or contractile properties of the mucle fibers
    e. g. Na/Kpump can be fine tuned depending on which tissues it is being used in !
33
Q

What can Oubain be used for?

A
  • it can be used as a means to identify high and low affinity binding components - e.g.ligand binding assays
34
Q

What are the two types of regulation ?

A
  • long term regulation

- short term regulation

35
Q

What is long term regulation?

A
  • induced over hours/ months
  • can induce changes in gene expression - and can take hours !

HORMONES: insulin , T3, aldosterone , catecholamines

DISEASE: Diabetes , renal dysfunction , cardiovascular hypertension , thyroid and neurological disorders

  • these not being able to react to changes ( e.g. in insulin)
  • result of hormonal imbalance.

EXERCISE: endurance training , electrical stimulation (in vitro) - mimics exercise you can see effects of chronic exercise
- training able to do change in gene expression!

DIET: high fat feeding
- induces alot of changes , increases onset of diabetes etc. can cause changes in the Na/K pump

USUALLY LONG TERM INVOLVES CHANGES IN THE Na , K- ATPase GENE EXPRESSION/PROTEIN SYNTHESIS
- e.g is mRNA levels drop , less protein is made!

36
Q

What are short term regulation?

A

can be induced over matter of minutes.

  • changes in pump affinities for substrates ( e.g. adrenalin with increased affinities)
  • changes in pump activity/ surface abundance ( regulating the number of sodium/ potassium pumps e.g. translocation
  • covalent modification ( rapid mechanism to modify the Na/K pump ; phosphorylating / dephosphorylating a protein is a type of covalent modification.

such changes can be mediated acutely in response to , for example insulin and C- peptide.

37
Q

list five different regulation types of the NA/K pump ?

A

1.insulin
2. edurance training
.3. high fat feeding
4. changes in pump activity
5. covalent modification.

38
Q

Is the concept of the Na pump translocation to the plasma membrane recent?

A

NO !

an experiment done by M.Omatsu - Kanbe and H kitasato:
- the muscle ( from a frog) was incubated in radioactive Na
- Na pump releases the radioactive Na to surroundings
- then the muscle is added to Oabain ( inhibitor)
- so the amount of Na released decreases
- therefore no mechanism to allow Na out
- then the excess Oabain was washed off
- then muscle soaked in insulin
- then Na started to come out again
( N.B. since additional Na Pumps are inserted when insulin added.)

39
Q

What is Insulins role in Na/K pump regulation?

A
  • Insulin stimulates the Na/K pump ( and allows the addition of more pumps )
  • insulin promotes the translocation of Na, K - ATPase subunits to the plasma membrane in ( rat ) skeletal muslce.

NB. if alpha goes up , Beta must go up too!.

40
Q

What is translocation?

A
  • transporters ( e.g. the Na/K Pump) moving from internal compartments to the plasma membrane on stimulation of insulin.

skeletal muscle tells transporters to move from inside to plasma membrane , when insulin is detected.

41
Q

What is the difference between a kinase and phsphatase?

A
  • kinase = adds a phosphate
  • phosphotase= removes a phosphate

( refer to page 4 notes on Na pump lecture 2)

42
Q

What are the extracellular stimuli involved in Na/K pump stiumulation?

A
  • Growth factors
  • Insulin

( refer to diagram on page 4 of NA pump lecture 2 notes)

43
Q

What is the inrtacellular stimuli involved in Na/K pump stimulation?

A
  • calcium
44
Q

What happens if there is an increase in insulin?

A
  • if insulin goes up , blood sugar goes up ,
  • this triggers protein kinase to go up
  • activating the protein
45
Q

What is the effect on the Na/K pump of insulin ?

A

when insulin is bound to inslin receptors on the plasma membrane , it triggers a cascase of events which triggers the NA/K pump to move up to the plasma membrane from inside the cell.

N.B if there is no insulin - very little phosphate is stimulated in the cell!!!

Insulin - stimulared recruitment and phosphorylation of Na/K ATPase alpha subunits

46
Q

What happens when the Na/K pump is phosphorylated?

A
  • the Na/K pump is made inactive and so prevents the stimultion of Na/K pump!
47
Q

give some examples of Protein kinases in the cell capable of phosphoylating multiple targets?

A
  • phosphoinositide 3 - kinase - PI3K
  • Protein Kinase B - PKB
  • Protein KIinase A-PKA
  • Protein kinase C- PKC
    Mitogen activated Protein Kinase - MAPK
48
Q

What does the Na/K ATPase alpha subunit have?

A
  • it harbours putative kinase phosphorylation sites

when insulin is added , there is an increase in the amount of alpha 1 and alpha 2 within the cells.

49
Q

What is found by exercise and translocation correltion?

A
  • exercise ( and muscle contraction) increases translocation in the cell ( just like insulin)

( refer to page 6 of the notes on Na pump lecure 2 - for diagprams and graphs of experimental examples)

50
Q

What are the effects of C- peptide on Na, K ATPase?

A
  • C - peptide increases Na/K pump amount on this membrane ( increasing the driving force - which allows more nutrients to be reabsorbed , since more NA is thrown out.
  • C- peptide allows translocation of Na/K pumps from renal cells to basolateral cells
  • peptide increases alpha subunit in baso-lateeral membrane.
  • C-peptide promotes phosphorylation of Na , K-ATPase and is Increased.

N.B.alpha and beta subunits work as heterodimers - if one goes up so does the other !

51
Q

What are the main conclusions of the two Na pump lectures?

A
  • INSULIN - stimulates the Naa pump in skeletal muscle cells by phosphorylation of Alpha 1 and alpha 2 subunits and their translocation to the plasma membrane- this requiers PKC and ERK signalling
  • C-PEPTIDE - induces phosphorylation of alpha 1 subunits in HRTC via a ERK- dependent mechanism and translocation of alpha 1 and beta 1 subunits to the BLM from an endosomal compartment
  • CONTRACTION- increases surface Na pump alpha subunits by an ERK- dependent mechanism in rat skeletal muscle.
  • HIGH FAT FEEDING - modifies Na, K- ATPase expression in rat skeletal muscle , which is antagonised by endurance training
  • MAPK SIGNALLING - serves as a major mechanism for regulating the Na, K-ATPase

Biomembranes (15 decks)

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