Human Physiology

1. Na+- K+ ATPases
PRADEEP SINGH, SHALU SINGH
M.Sc. Medical Biochemistry
HIMSR, Jamia Hamdard

2. Introduction
Na+-K+-ATPase is an integral
membrane protein found in the cells of
all higher eukaryotes.
It is responsible for translocating
sodium and potassium ions across the
cell membrane utilizing ATP as the
driving force.

3. For every three sodium ions pumped out of the cell, two potassium ions are
pumped in.
This transport produces both a chemical and an electrical gradient across
the cell membrane.
The electrical gradient is essential for maintaining the resting potential of
cells and for the excitable activity of muscle and nerve tissue.
The sodium gradient is used to drive numerous transport processes,
including the translocation of glucose, amino acids, and other nutrients into
cells.

4. Physiologically, Na+ - K+ ATPase present in organs such as the intestines and
the kidney regulates fluid reabsorption and electrolyte movement by
establishing an ionic gradient across epithelial membranes.
It is estimated that approximately 23% of the ATP consumed in humans at
rest is utilized by the sodium pump.
The Na+-K+-ATPase is a member of the P-type class of active cation
transport proteins of ATPases.

5. History
The sodium-potassium pump was discovered in 1957 by the Danish
scientist Jens Christian Skou.
He was awarded a Nobel Prize for his work in
1997.

6. Structure
The Na+ - K+-ATPase can function as an α β dimer.
Consists of –
- a catalytic α subunit with ten trans-membrane segments
(8 transmembrane α-helical segments and two large cytoplasmic
inclusions, and
- a single trans-membrane glycosylated β subunit (single
transmembrane helix and a large extracellular domain), required for
stabilization.
There are four isoforms of α(1-4) and three isoforms of β expressed in a
tissue-specific fashion.

7. The α subunit contains the ATP binding site, the phosphorylation site,
and amino acids essential for the binding of cations and cardiac
glycosides, which suggests that this subunit plays a major role in the
catalytic function of the enzyme.
The β subunit appears to be involved in maturation of the enzyme,
localization of the ATPase to the plasma membrane, and stabilization of a
K+-bound intermediate form of the protein.

8. Electrogenic pump
Drives 3 positively charged ions (Na+) out of the cell for every 2 it pumps in (K+).
It drives a net electrical charge across the membrane ,tending to create an
electrical potential, with cell’s inside being negative relative to outside.
This electrogenic effect of the pump ,however seldom directly contributes more
than 10% of the membrane potential.

9. Forms of sodium - potassium pump
Na+ - K+ ATPase exists in two forms-
E1 Form: E1 has an inward –facing high affinity Na+ binding site and reacts
with ATP to form the activated product E1~P only when Na+ is bound.
E2 form : E2 –P has an outward –facing high affinity K+ binding site and
hydrolyses to form Pi + E2 only when K+ is bound .

10. Mechanism
The (Na+-K+)-ATPase operates in accordance with
the following ordered sequencial reaction scheme:
1)E1.3Na+,which acquired its Na+ inside the cell, binds
ATP to yeild the tertiary complex E1.ATP.Na+
2)The tertiary complex reacts to form the high energy
aspartyl phosphate intermediate E1~P.3Na+
3)This high energy intermediate relaxes to its low
energy conformation ,E2-P.3Na+ and relaxes its bound
sodium outside the cell .

11. 4) E2-P binds 2K+ from outside the cell to form E2-P.2k+ .
5) The phosphate group is hydrolysed ,yeiding E2.2K+
6) E2.2K+ changes conformation , releases its 2K+ inside the cell
,and replaces its Na+ , thereby completing the transport cycle.

13. Regulation
Na+ -K+ ATPase are regulated in two ways :-
Exogenous
Endogenous

14. Exogenous
The Na/K+-ATPase is upregulated by cAMP. Thus, substances causing an increase in
cAMP upregulate the Na+/K+-ATPase.
These include the ligands of the Gs-coupled GPCRs.
In contrast, substances causing a decrease in cAMP downregulate the Na+/K+-ATPase.
These include the ligands of the Gi-coupled GPCRs.

15. Endogenous
The Na+-K+-ATPase can be pharmacologically modified by administrating
drugs exogenously.
For instance, Na+-K+-ATPase found in the membrane of heart cells is an
important target of cardiac glycosides ( for example digoxin and ouabain)
,inotropic drugs used to improve heart performance by increasing its force
of contraction.

16. Rate
In order to maintain a reasonable rate of transport, the free energies
of all its intermediates must be roughly equal.
If some intermediates were much more stable than others ,the stable
intermediates would accumulate, thereby severely reducing the overall
transport rate.

17. INHIBITION
The pump requires binding by Na+ , K+ and ATP for its operation. Therefore , if the
concentration of any of these substances is too low , the pump does not function.
When the temperature is reduced.
During oxygen lack.
Metabolic Poisons e.g. 2,4 DNP that prevents the formation of ATP.

18. Clinical Significance
Certain steroids derived from plants are potent inhibitors (Ki < 10 nM) of the Na+–K+
pump.
 Digitoxigenin and ouabain are members of this class of inhibitors, which are known
as cardiotonic steroids because of their strong effects on the heart .
 These compounds inhibit the dephosphorylation of the E2-P form of the ATPase
when applied on the extracellular face of the membrane.

19. Digitalis
Digitalis is a mixture of cardiotonic steroids derived from the dried leaf of the foxglove plant
(Digitalis purpurea).
The compound increases the force of contraction of heart muscle and is consequently a choice
drug in the treatment of congestive heart failure.
Inhibition of the Na1–K1 pump by digitalis leads to a higher level of Na1 inside the cell.
The diminished Na1 gradient results in slower extrusion of Ca21 by the sodium–calcium
exchanger.
The subsequent increase in the intracellular level of Ca21 enhances the ability of cardiac
muscle to contract.

21. Ouabain
Ouabain is a cardiac glycoside that acts by inhibiting the Na+/K+-ATPase(but it is
not selective).
Once ouabain binds to this enzyme, the enzyme ceases to function, leading to
an increase of intracellular sodium.
This increase in intracellular sodium reduces the activity of the sodium-calcium
exchanger (NCX), which pumps one calcium ion out of the cell and three sodium
ions into the cell down their concentration gradient.

22. Therefore, the decrease in the concentration gradient of sodium into the
cell which occurs when the Na/K-ATPase is inhibited reduces the ability of
the NCX to function.
This in turn elevates intracellular calcium.
This results in higher cardiac contractility and an increase in cardiac vagal
tone. The change in ionic gradients caused by ouabain can also affect the
membrane voltage of the cell and result in cardiac arrhythmias.

23. Functions

24. Resting Potential
In order to maintain the cell membrane potential, cells keep a low
concentration of sodium ions and high levels of potassium ions within the
cell (intracellular).
The sodium-potassium pump mechanism moves 3 sodium ions out and
moves 2 potassium ions in.
Thus, in total removing one positive charge carrier from the intracellular
space .

25. Transport
Export of sodium from the cell provides the driving force for several
secondary active transporters membrane transport proteins, which
import glucose, amino acids, and other nutrients into the cell by use of the
sodium gradient.

26. Controlling Cell Volume
Failure of the Na+-K+pumps can result in swelling of the cell.
A cell's osmolarity is the sum of the concentrations of the various ion species
and many proteins and other organic compounds inside the cell.
 When this is higher than the osmolarity outside of the cell, water flows into
the cell through osmosis. This can cause the cell to swell up and lyse.
The Na+-K+pump helps to maintain the right concentrations of ions.
Furthermore, when the cell begins to swell, this automatically activates
the Na+-K+ pump.

27. Functioning as signal transducer
Membrane protein can also relay extracellular ouabain - binding signalling
into the cell through regulation of protein tyrosine phosphorylation.
 The downstream signals through ouabain-triggered protein
phosphorylation events include activation of the mitogen-activated protein
kinase (MAPK) signal cascades, mitochondrial reactive oxygen species (ROS)
production, as well as activation of phospholipase C (PLC) and inositol
triphosphate (IP3) receptor (IP3R) in different intracellular compartments.
Protein-protein interactions play a very important role in Na+-K+ pump-
mediated signal transduction.

28. For example, Na+-K+ pump interacts directly with Src, a non-receptor
tyrosine kinase, to form a signaling receptor complex.
Src kinase is inhibited by Na+-K+ pump, while, upon ouabain binding, the
Src kinase domain will be released and then activated.
Based on this scenario, NaKtide, a peptide Src inhibitor derived from Na+-
K+ pump, was developed as a functional ouabain-Na+-K+pump-mediated
signal transduction.
 Na+-K+pump also interacts with ankyrin, IP3R, PI3K, PLC-gamma
and cofilin.

29. Controlling Neuron activity states
The Na+-K+ pump has been shown to control and set the intrinsic activity mode
of cerebellar Purkinje neurons, accessory olfactory bulb mitral cells and probably
other neuron types.
This suggests that the pump might not simply be a homeostatic, "housekeeping"
molecule for ionic gradients; but could be a computation element in
the cerebellum and the brain .
Indeed, a mutation in the Na+-K+ pump causes rapid onset dystonia parkinsonism,
which has symptoms to indicate that it is a pathology of cerebellar computation.

30. Furthermore, an ouabain block of Na+-K+pumps in the cerebellum of a live mouse
results in it displaying ataxia and dystonia.
Alcohol inhibits sodium-potassium pumps in the cerebellum and this is likely how
it corrupts cerebellar computation and body co-ordination.
The distribution of the Na+-K+ pump on myelinated axons, in human brain, was
demonstrated to be along the internodal axolemma, and not within the nodal
axolemma as previously thought.

31. Hormones
Major hormonal controls over pumps can be summarized as follows :-
Thyroid Hormones appear to a major player in maintaining steady state
concentrations of pumps in most tissues. This effect appears to result from
stimulation of subunit gene transcription.
Aldosterone is a steroid hormone with major effects on sodium homeostasis
.It stimulates both rapid and sustained increases in pump numbers within several
tissues. The sustained effect is due to enhanced transcription of genes for both
subunits.

32. Catecholamines have varied depending on specific hormones and tissue.
For example , dopamine inhibits Na+- K ATPase activity in kidney , while
epinephrine stimulates pump activity in skeletal muscles . These effects
seem to be mediated via phosphorylation or dephosphorylation of the
pumps.
Insulin is a major regulator of potassium homeostasis and has multiple
effects on sodium pump activity.
Within minutes of elevated insulin secretion, pumps containing alpha1 and
2 isoforms have increased affinity for sodium and increased turnover rate .

33. Sustained elevations in insulin causes upregulation of alpha-2 synthesis . In skeletal
muscle ,insulin may also recruit pumps stored in cytoplasm or recruit latent pumps
already present in membrane.

35. THANK YOU