Sodium - Potassium Pump

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- E 1 Form : E 1 has an inward –facing high affinity Na + binding site and reacts with ATP to form the activated product E 1 ~P only when Na + is bound. E 2 form : E 2 –P has an outward –facing high affinity K + binding site and hydrolyses to form Pi + E 2 only when K + is bound .

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

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

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

13. 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  G s -coupled GPCRs. In contrast, substances causing a decrease in cAMP downregulate the Na + / K + - ATPase. These include the ligands of the  G i -coupled GPCRs.

14. 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.

15. 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.

16. 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 .

17. 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 .

18. 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.

19. 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 .

20. 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.

21. Functions

22. 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. T hus , in total removing one positive charge carrier from the intracellular space   .

23. 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 .

24. 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 .

25. Functioning as signal transducer M embrane 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.

26. 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 .

27. 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.

28. 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.

29. 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.

30. 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 .

31. 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.

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