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
