L7: Pumps Flashcards
4 types of ATP-powered pumps
- P-type ATPases
- ABC superfamily
- V-type proton pump
- F-type proton pump
P-type ATPases structure and function
- Large alpha catalytic subunit (up to 200 KDa). Some have associated beta subunits
- Pump cations
- All inhibited by micromolar amounts of orthovanadate
- During ATP hydrolysis, ATP donates its gamma-phosphate to a conserved aspartate during cation pumping forming a phosphorylated IM
- Members of the group have a diverse array of physiological functions
Na+/K+ ATPase in animal cells (struct., funct., location, inhibitors)
- Found in most PMs of animal cells (3 isoforms)
- 3Na+:2K+ per ATP hydrolysed
- House keeping functions:
a) Maintains high K+, low Na+ in cytosol (AP generation)
b) Maintains Na+ electrochemical gradient (Na+ coupled transport) - Composed of 2 alpha and 2 beta subunits
- Inhibited by ouabain
Fungal and Plant H+ ATPase (struct., funct., location)
- Found in all PMs of plant and fungal cells
- Composed of 1 alpha subunit
- 1H+ pumped per ATP hydrolysed
- Housekeeping functions…
a) Expelling excess H+ produced during metabolism
b) Gen. H+ electrochemical gradient which is used to drive H+-coupled transport
c) Maintain negative TM voltage (> -200mV)
d) Regulate cytosolic pH
e) (In plants) Acidifying extracellular medium - loosen cell walls
SERCA pump (struct., funct., location, inhibitors)
Sarcoplasmic endoreticulum Ca2+ ATPase
- Found in SR network of tubules in muscle cell cytoplasm (forming Ca2+ stores, 2 per ATP)
- Made up of 1 alpha subunit
- Restores low cytosolic Ca2+ after muscle contraction
- Inhibited by thapsigargin
PMCA ATPase (struct., funct., location) - Plasma membrane calcium ATPase
- Found in fungal, plant and animal PMs
- 1 or 2 Ca2+ per ATP, exchanges proton as well
- Maintains low cytosolic Ca2+ (high conc. is cytotoxic)
- Central role in cell signalling
- 1 alpha subunit
Gastric muscosal H+/K+ ATPase (struct., funct., location, inhibitors)
- PM of gastric epithelium cells/ stomach wall
- 2K+:2H+ per ATP hydrolysed
-> electroneutral - Maintain acidity (H+ secretion into lumen of stomach, 0.16 M HCl)
- Made up of 2 alpha, 2 beta subunits
Conserved structure for P-type ATPases
- N-terminus (4 TMS domains -> 4 stalk regions)
- C-terminus (6 TMS domains -> 5th stalk domain)
- Most of pump is in cytoplasm
- Region B - induces conformational change in ion binding site
- Region C - phosphorylation site and nucleotide binding site
- Region J - comprises a hinge to allow cytosolic regions to move and interact
Albert-Post model
E1-E2 model of P-type pump-mediated ion transport
- E1 facing cytosol has high affinity binding sites for Na+ (binds 3), low affinity for K+
- Binding of ATP phosphorylates aspartate, switches conformation to the other side -> E2
- E2 has low affinity for Na+, high affinity for K+ (binds 2)
- Na+ dissociate despite low conc. outside cell, hydrolysis of aspartyl phosphate returns it to E1 conformation
- K+ dissociate into cytosol (against gradient)
CPx pumps (describe, characterisations in bacteria vs humans, key differences to P-type pumps)
- Related to P-type; transport toxic and nutrient metal (e.g. Cu, Pb, Cd, Zn)
- Identified in Enterococcus, COPA and COPB
- Characterised in humans via Menkes disease and Wilsons disease
Key differences…
- N-terminus has 2 extra TMS domains, and has cysteine repeats
- Has a conserved CPx motif (x= cysteine, histidine or serine)
- C-region has a conserved histidine-proline
- C-terminus has fewer TMS domains
Menkes and Wilsons disease
- Menkes: Systemic copper deficiency caused by defect in C-region of CPx type pump
- Wilsons: excessive copper accumulation in the liver
V-type pumps
- Found in intracellular membranes of Euk cells, act as H+ ATPases
- Electroneutral
- Involved in waste/nutrient storage (plant vacuole) and neurotransmitter storage (synapse)
Rotational catalysis of V-type pump
- ATP hydrolysis by the As-Bs complex generates the torque in the D subunit to rotate the ring of 6 ‘c’ subunits
- Pumps by rotating H+ up
ATP Binding Cassette (ABC) transporters (function and clinical relevance x2)
- Use energy released from ATP hydrolysis to transport a wide variety of solutes in/out of cell
- Clinically important (cystic fibrosis, MDR transporters - pathogen and cancer cell drug resistance)
Structure of ABC transporters
- 2 ATP binding cassettes bordered by 6 TMS domains to the left
- Chamber forms in membrane between ATP-binding cassette for substrate binding
- Monomer
Flippase model for MDR1
- ABC transporter for amphipathic substrates
1) Lipid soluble molecule dissolves into cytosolic facing leaflet of PM, binds to chamber of MDR1
2) Powered by ATP hydrolysis, substrate molecule is flipped into exoplasmic leaflet of PM
3) Substrate molecule diffuses into EC space
