Lecture 4 - ATP powered pumps

Question 1

Describe the structure of P-type ATPases

Answer 1

They have a conserved structure with:
N-terminus - 4 TMS
C-terminus 6 TMS
Region B - which induces conformational changes in ion binding site
Region C - phosphorylation site and nucleotide binding site
Region J - makes a hinge which allows cytosolic regions to move and interact
* They consist of large α catalytic subunits although some have associated β subunits
* They all pump cations such as K+, Na+ and Mg2+
* They are all inhibited by micromolar amounts of orthovanadate which works by competing with the phosphorylation site.
* During ATP hydrolysis ATP donates its phosphate to a conserved aspartate during cation pumping forming a phosphorylated intermediate

Question 2

Give examples of P-type ATPases

Answer 2

The Na+/K+ ATPase in animal cells - Important in the generation of action potentials it is composed of 2α and 2β subunits. The transport of Na and K are closely coupled so if there is an absence of one there will be very little activity. It is inhibited by ouabain

Fungal and plant H+ ATPase - The pump is important in generating a H+ electrochemical gradient and removing excess H+ generated during metabolism. It is composed of 1α subunit

Sarcoplasmic reticulum Ca2+ ATPase - It functions to restore low cytosolic calcium after muscle contraction and is inhibited by thapsigargin. It is composed of 1α subunit however it has 3 isoforms.

Plasma membrane Ca2+ ATPase - It maintains the low cytosolic calcium ion concentration needed for cell signalling it is made of 1α subunit and the calcium is exchanged with a H+ ion.

Gastric mucosal H+K+ ATPase - Composed of 2α and 2β subunits it pumps 2K+:2H+ per ATP hydrolysed (electroneutral overall) this increases the H+ conc in the lumen of the stomach leading to the acidic environment

Question 3

What is the effect of conformational changes on P-type ATPases

Answer 3

Conformational changes induce changes in cation binding which causes changes in affinity allowing:
* Binding of ions at low concentration
* Dissociation of ions at high concentrations
ATP hydrolysis and phosphorylation of the aspartate results in the movement of the head regions and this conformational change exposes the binding site for the substrate

Question 4

Describe the structure of CPx pumps and name disorders associated with their dysfunction.

Answer 4

Similar to P-type pumps however they transport toxic and nutrient metals. They were first identified in the bacterium Enterococcus. They have:
* 2 Extra TMS domains on the N terminus
* Cystine repeats on the N terminus
* Conserved CPx motive hence the name where x can be a cysteine, histidine or serine
* C region - conserved histidine-proline
* C-terminus - Fewer TMS domains
Menkes disease - systemic copper deficiency
Wilsons disease - Excessive copper accumulation in the liver

Question 5

What are V-type pumps

Answer 5

Operate almost exclusively as H+ ATPases on intracellular membrane son eucaryotic cells. ATP hydrolysis by the A3B3 complex generates the torque in the D subunit to rotate the ring of 6c subunits. Establishing H+ electrochemical gradients

Question 6

What are ABC transporters

Answer 6

ATP Binding Cassette (ABC transporters)
sections of transmembrane spanning domains and 2 ATP binding domains. Have a range of physiological functions.