Membrane transport Na+/K+ ATPase

Question 1

Na+/K+ ATPase

Answer 1

• antiporter on PM
• helps maintain resting potential, facilitate transport and regulate cellular volume, functions as signal transducer to regulate MAPK, ROS, intracellular calcium
• responsible for 1/5 of energy expenditure, in neurons 50%

Question 2

Structure of Na/K ATPase

Answer 2

• hydrophilic AA residue mainly in extra and intracellular loops
• sugar residues at extracellular hydrophilic loop
• hydrophobic AA residues mainly in transmembrane domain
• amino NH3 terminal at end of extracellular loop
• carboxyl terminal in intracellular loop
• alpha2, beta2 tetramer
• alpha subunit: 100kDa, 4 isoforms
• beta subunit: 55kDa, many isoforms, necessary for activity, S-S links glycosylated
• Steroid binding site, sugar units, ATP binding site

Question 3

Types of transport

Answer 3

• simple diffusion
• facilitated diffusion
• primary active transport
• secondary active transport
• ion channel
• ionophore mediated ion transport
  uniport/ symport / antiport

Question 4

P-type ATPases

Answer 4

• alpha-helical bundle primary transporters
• catalyse autophosphorylation of Asp residue
• appear in at least 2 conformations: E1 and E2
• mostly pump cations, also Flippases

Question 5

Examples of P-type ATPases

Answer 5

• Na+/K+ ATPase
• H+ ATPase (PM)
• H+/K+ ATPase (stomach parietal cell)
• sarco/ER Ca2+ ATPase

Question 6

Types of SERCA

Answer 6

• SERCA 1: striatal muscle
• SERCA 2: smooth and cardiac muscle, striatal m
• SERCA 3: platelets and endothelial cells, non muscle cells

Question 7

Types of Plasma membrane ATPases(PCMA)

Answer 7

• PCMA 1: general
• PCMA 2: neuronal
• PCMA 3: striatal muscle and brain
• PCMA 4: general

Question 8

Mechanism of Na/K ATPase pump

Answer 8

1. E1- ATP
2. E1- ATP- 3Na+
3. E1- P- 3Na+
4. E2- P- 2Na+
5. E2- P
6. E2- P- 2K+
7. E2- 2K+
8. E2- 2K+- ATP
9. E1- 2K+- ATP
10. E1- ATP again

Question 9

Mechanism of SERCA

Answer 9

1. Ca2+ binds to transmembrane (M) domain and ATP bind to nucleotide (N) domain
2. Phosphoric group transferred to Asp351 residue in P domain (requires Mg2+)
3. Ca2+ released from lumen
4. Activator (A) domain moves, ADP released
5. P domain dephosphorylated
6. A domain returns to original, releases Mg2+
7. P and M domain resets

Question 10

Ouabain (Strophantine)

Answer 10

• poisonous cardiac glycoside
• potent inhibitor of Na/K ATPase
• works especially in high conc. in vitro / intravenously
• Digoxin: same structure, more lipophilic cardiac glycoside, replaces Ouabain

Question 11

Alpha subunit isoforms

Answer 11

• alpha1: in most cells + epithelial cells + kidney outer medulla
• alpha2: striatal muscle + brain + heart + smooth m. + adipocytes
• alpha3: neurons + heart + ovary + leukocytes
• alpha4: testis
  sensitivity to ouabain:
  alpha2(0,1pM) - alpha3(>30nM) - alpha1(0,1mM)
  Km:
• K+e.c.: 0,5mM
• ATP: 0,15mM
• Na+i.c.: 10-20mM

Question 12

Gamma subunit

Answer 12

• regulator of Na/K ATPase in certain tissues.
• regulates transport kinetic of alpha subunit
• Tissue specific in kidney, pancreas and fetal liver
• 7,2kDa, 1transmembrane domain
• function: increase pump affinity to ATP
• important in anoxia
• 7 isoforms

Question 13

Regulation of Na+/K+ ATPase

Answer 13

1. Corticosteroids (aldosterone, dexamethazone)
2. Long term effect (increased expression of Na+ pump)
3. Mineralocorticoid type 1 receptor
4. Glucocorticoid type 2 receptor

Question 14

Mineralocorticoid type 1 receptor

Answer 14

• expressed in many tissues
• cytosolic receptor
• activated upon ligand binding
• receptor-ligand complex translocated into nucleus and binds to HREs in promoter region
• trans-repression: no ligand binding, receptor interacts with heat shock proteins and prevents transcription

Question 15

Hyperaldosterism

Answer 15

• generally from adrenal cancer
• hypertension and edema- excessive Na+ and H2O retention
• excretion of K+ - muscle weakness, paralysis

Question 16

Glucocorticoid type 2 receptor

Answer 16

• expressed in most cells
• regulates genes controlling development, metabolism, immune response
• pleiotropic
• receptor-GR complex:
• upregulates expression of anti-inflammatory proteins in nucleus
• represses expression of pro-inflammatory proteins in cytosol

Question 17

Aldosterone

Answer 17

• adaptation in kindney to decreased Na+ or increased K+ intake
• long term up regulation: isoform specific
• alpha1: vacular smooth m
• alpha2: heart
• alpha3: brain
• short term effect: increased activity of enzyme
• translocation of pump to PM and increase in affinity of enzyme to Na+

Question 18

Dopamine

Answer 18

• natriuretic effect
• synthesized in kindey proxiaml tubule
• paracrine + autocrine effects
• inhibits Na/K ATPase - decreased Na+ reabs.
• D1 and D2 receptors phosphorylated by PKA and PKC

Question 19

Epinephrine

Answer 19

• in skeletal muscle stimulates K+ uptake by Na/K ATPase
• important during extreme exercise - hyperkalemia - E counteracts this effect
• bind to beta2 adrenergic receptor- decrease K+ conc e.c.

Question 20

Norepinephrine

Answer 20

• in kidney antagonistic effect of dopamine

- in brain restores ion conc. of Na+ and K+ after nerve impulses fired

Question 21

Insulin

Answer 21

• short term effect: direct stimulation of Na/K ATPase
• in skeletal m.- only in oxidative slow twitch muscle - translocation of enzyme to PM
• long term effect: increased/ decreased expression

Question 22

Secondary active transports: Na+ co-transport

Answer 22

• glucose and AAs
• choline uptake into cholinergic nerve terminals
• epinephrine, norepinephrine, dopamine and serotonin repute by nerve terminal

Question 23

Na+/H+ exchanger

Answer 23

• not electrogenic- 1H+ for 1Na+
• highly dependent of pH
• 5 isoforms with 12 transmembrane domains:
• NHE1: general
• NHE3: epithelial cells, apical enterocytes
• NHE5: brain, testis

Question 24

Proton antiporter, VMATs

Answer 24

• 12 transmembrane segments
• broad selectivity
• isoforms:
• VMAT1: brain, neuroendocrine cells
• VMAT2: neurons, adrenal chromaffin cells
• VAChT: cholinergic synapses
• inhibited by H+ ionophores

Question 25

ABC transporters

Answer 25

• vitamin B12 importer
• homodimer, 10 transmembrane helical domains in each monomer + 2 nucleotide-binding domains
• ATP dependent
• transporter for AAs, peptides, metal ions, lipids, bile salts, drugs
• located in PM, mitochondrial membrane, ER
• transport agains conc. gradient
• mutations cause eg. cystic fibrosis, retinal degradation, anemia, Tangier disease

Question 26

Multi-drug transporter (MDR1)

Answer 26

• ABC transporter
• pumps hydrophobic compounds out of cell
• tumor resistance agains anti tumor drugs

Question 27

Tangier disease

Answer 27

• defect in gene for ABC transporter ABCA1
• reduced ability to transport cholesterol out of cell
• leads to accumulation of cholesterol
• reduced level of HDL - hypoalphalipoproteinemia

Question 28

Gamma subunit in heart (FXYD1, phospholemman)

Answer 28

• when dephosphorylated it decreases the Na+ affinity of the α-subunit
• adrenergic β1 receptor stimulation → PKA stimulation →
  phosphorylation of phospholemman → [Na+]i [Ca2+]i ↓ -prevention of arrythmia

Question 29

Gamma subunit in kidney (FXYD2)

Answer 29

• increases the affinity of the enzyme for ATP
• Kidney medulla is nearly anoxic under physiological conditions
• Some reabsorptions are under the control of the Na+-pump
• Moderate increase in the affinity for ATP → increased pump activity
  (Fine tuning! Large affinity increase would cause further ATP ↓ !)

Question 30

Endogenous cardiac glycosides

Answer 30

• sterioid structure
• synthesized in zona fasciculate from progesterone
• plasma conc.: 10-9 M
• role: regulation of vascular tone

Question 31

Glucose transporter in proximal tubule in kidney

Answer 31

• Glucose 160-180 g/day in the filtrate completely reabsorbed
• SGLT2 – low affinity (Kt 6 mM) large capacity transporter
• inhibitor in diabetes – glucosuria
• SGLT1 – high affinity low capacity transporter

Question 32

ABC transporters

Answer 32

ABCA1 – cholesterol, phopholipid transport - reverse cholesterol transport
ABCA3 – translocation of pulmonary surfactant lipids
mutation: respiratory distress syndrome
ABCB1 (MDR1 or P-glycoprotein, multidrug resistence transporter) - transport of lipophylic compounds – protection against toxins - increased expression in tumor cells: drug resistence
ABCB4 – canalicular membrane of hepatocytes
bile acid, phospholipid transport into the bile
ABCC1 (MRP-1, multidrug resistence protein)
- phospholipids, glutathione conjugates, anti-tumor drugs (also other than lipophylic!)
CFTR (ABCC7, cystic fibrosis transmembrane conductance regulator) - Cl- flux in apical membrane of epithelial cells
mutation: cystic fibrosis (thick mucus in the bronchi and pancreas
- blockage, infection, foetal ileus