Lecture 4 and 5

Question 1

What is the concentration of K+ inside and outside the membrane

Answer 1

4 mM inside

140 mM outside

Question 2

What is the concentration of Na+ inside and outside the membrane?

Answer 2

10 mM inside

140 mM outside

Question 3

What are the three situations in which the electrochemical gradients of K and Na are abolished? What do they suggest?

Answer 3

1) Cell death
2) Withholding glucose
3) Use of specific inhibitors
This suggests that maintaining these concentration gradients requires metabolic energy.

Question 4

Describe what is the pump-leak hypothesis:

Answer 4

Experiments in which cells were incubated with radioisotopically labeled Na or K indicated that under steady state conditions there is a passive influx of Na and a passive efflux of K. Active extrusion of Na and uptake of K is balanced by a passive leak of both these ions in opposite directions.

Question 5

Where does the proof that oxidative metabolism serves as a major energy source for active cation transport come from?

Answer 5

Withdrawal of oxygen in these cells or inhibitor of mitochondrial oxidation (dinitriphenol) inhibits cation transport. In red blood cells, inhibitor of glycolysis and removal of glucose have the same effect.

Question 6

What did Skou (1957) demonstrated?

Answer 6

That he could measure ATP activity in a crab nerve particulate fraction and this was Mg2+ activated.
The activity increased considerably by adding Na and K to the assay mixture.
The digitalis glycoside, ouabain completely inhibited the additional ATPase activity.

Question 7

What are the four properties of Na-K ATPase?

Answer 7

Requires Mg, ATP, Na and K for activity
Optimal pH 7-7.5
Km values for Na (5-10 mM) for K (0.4-1.8 mM)
Always inhibited by ouabain

Question 8

What does the Na-K ATPase pumps?

Answer 8

3 Na+ out for 2 K+ ions taken up per ATP hydrolised.

Question 9

Describe STEP 1 of Na-K ATPase cycle:

Answer 9

Substrate binding: binding of one molecule of ATP accompained by co-operative binding of 3 Na ions.

Question 10

Describe STEP 2 of Na-K ATPase cycle:

Answer 10

Phosphorylation of enzyme: gamma phosphate of ATP is transferred to apartyl-B-carboyxl group with release of ADP.

Question 11

Describe STEP 3 of Na-K ATPase cycle:

Answer 11

Transformation of the phosphoenzyme: Transition from ADP-sensitive to an ADP-insensitive phospointermediate which undergoes spontaneous hydrolysis stimulated by the presence of K.

Question 12

What does the transition from E1 to E2 of ATPase involves?

Answer 12

It involves a reduced affinity for ADP and a drastic change in Na and K affinities.

Question 13

Describe STEP 5 of Na-K ATPase cycle:

Answer 13

Hydrolysis of the phosphoenzyme: phosphate is release into intracellular space.

Question 14

Describe STEP 6 and 7 of Na-K ATPase cycle:

Answer 14

Return of the native enzyme form: the enzyme undergoes a conformational change from occluded E2-K form to the non-occluded E1-K form. K is then released into the cytosol and the protein is ready to begin another cycle.

Question 15

How do we demonstrate that the ATPase and the pump are one and the same?

Answer 15

The hydrolisis of ATP and synthetic substrates is ouabain sensitive as well as the active movement of Na nd K.

Question 16

List the five modes in which the pump can operate:

Answer 16

Normal mode (Na/K exchange)
Reverse mode
Na-Na exchange (1:1)
Uncoupled Na efflux
K-K exchange (1:1)

Question 17

What are the two molecular contribution of the Na/K pump?

Answer 17

By maintaining the normal transmembrane electrochemical gradients for Na and K it maintains a stable membrane potential.
It generates the energy in the Na gradient used to drive a secondary active transport system for sugars and amino acids.

Question 18

Describe the catalytic subunit of the pump (alpha):

Answer 18

It possesses binding sites for Na, K, ATP, Mg an cardiac glycosides (ouabain)
Has an intrinsic ATPase activity
4 isoform (α1, α2, α3, α4)
Developmentally regulated and expressed in a tissue specific manner

Question 19

What is the molecular size of the catalytic subunit (alpha)?

Answer 19

112 KDa

Question 20

Describe the regulatory subunit (beta):

Answer 20

3 isoforms (β1, β2 and β3)
Heavily glycosilated (28% w/w)
Crucially required for enzyme activity but also for enzyme assembly, intracellular transport of complete enzyme molecules, stability of alpha subunit.

Question 21

Describe small auxillary protein

Answer 21

8-14 KDa, belong to the FXYD protein family named after an invariant motif. It is a single spanning membrane peptide.

Question 22

Define the tissue specific expression of subunit mRNA’s:

Answer 22

α1 has ubiquitous expression
α2 found only in excitable tissue or insulin responsive tissue
α3 found in excitable tissue
α4 only expressed in spermatozoa

Question 23

What are the four types of long-term regulation of Na/K pump?

Answer 23

Hormones
Disease
Exercise
Diet
It usually involves changes in Na/K pump gene expression and protein synthesis

Question 24

What are the three types of short-term regulation of Na/K pump?

Answer 24

Changes in pump affinities for substrates
Change in pump activity/surface abundance
Covalent modification
Such changes can be mediated acutely in response to for example insulin and C-peptide.

Question 25

What is the effect of insulin on skeletal muscle cells?

Answer 25

Insulin stimulates Na/K pump by phosphorylation of α1 and α2 subunits and their translocation to the plasma membrane, this requires PKC and Erk signalling.

Question 26

What does C-Peptide induces in Basolateral Membrane (BLM)?

Answer 26

C-peptide induces phosphorylation of α1 subunits in Human Renal Tubular Cells via Erk-dependent mechanism and translocation fo α1 and β1 subunits to the BLM from and endosomal compartment.

Question 27

What effects does contraction have on Na/K pump?

Answer 27

Contraction increases surface Na/K pump alpha subunits by and Erk-dependent mechanism in rat muscle cells.

Question 28

What effect does fat feeding have on Na/K-ATPase?

Answer 28

Fat feeding modifies Na,K-ATPase expression in rat skeletal muscle, which is antagonised by endurance training

Question 29

What are the two factors that contribute to the formation of the characteristic transmembrane distribution of Na+ and K+?

Answer 29

1) Permeability properties of the membrane toward these two ions
2) Presence of specific transport mechanisms resident in the membrane.

Question 30

Describe the differential expression of Beta 1 and Beta 2 subunits:

Answer 30

They are expressed differentially in skeletal muscle depending on contractile properties of the muscle.