Transporters & Ion Channels 2

Question 1

4 classes of ATP powered pumps

Answer 1

1) P-class pumps (ATPase)
2) V-class pumps (ATPase)
3) F-class pumps (ATPase)
4) ABC transporters

Question 2

Principal role of ATP-powered pumps

exceptions?

Answer 2

Use the energy from ATP hydrolysis to transport substrate uphill

Exception = F-class pumps that synthesise ATP using a proton-motive force
- In vivo, F-class ATPase cannot hydrolyse ATP (they aren't powered by ATP)

Question 3

V-class ATPases

Answer 3

Similar to F-class, just reversed mechanism (ATP hydrolysed to rotate the V1 unit to pump protons across membrane)

• Used to lower the pH in intracellular organelles (vacuole) but also in plasma membrane of some animals (e.g. insect gut cells)
• To prevent build up of electrochemical potentials, vacuoles also contain ClCs

Question 4

P-class ATPases

Answer 4

NOT in Bacteria. Found in fungi, plants and eukaryotes functioning primarily to pump cations (Na/K, Ca)

Contain one or two identical alpha-subunits containing:

• Transmembrane pump
• Phosphorylation site
• ATPase domain

Question 5

P-class Ca+ ATPase pump distribution and role

Answer 5

In all eukaryotic cells (and Sarcoplasmic reticulum in muscle cells)

• Excitation triggers depolarization in muscle and opening of Ca channels so the Ca can flow out of SR into cell
• P-class pumps Ca back into the SR

Question 6

P-class Ca+ pump’s mechanism of function in SR

Answer 6

Pumping Ca+ from cytosol to SR:

1) E1 conformation: channel opens towards cytosolic side to expose 2x Ca+ binding sites (ATP binds to ATP binding site)
2) ATP hydrolysis occurs and a phosphate (Pi) is bound to the phosphorylation site (E1P)
3) Phosphate binding provides energy for a conformational change to E2 (E2P): channel opens towards SR lumen and calcium gets released (released as calcium binding site changes conformation to reduce Ca binding affinity)
4) Dephosphorylation of phosphorylation site provides energy for conformational change back to E1

Question 7

P-class Na/K ATPase

Answer 7

Essential for neuronal function (generating resting potential) and identical to Ca+ pump

• still 1 ATP per cycle of E1/E2
• Only difference: when open to cytosol, 3x Na+ enter channel; when open to exterior, 2x K+ bind

Question 8

ABC transporters distribution and structure

Answer 8

Found in bacteria (active nutrient uptake, some export) and higher eukaryotes (export toxins and drugs)

All have 2 Nucelotide Binding Domains (NBDs)

4 domains typically expressed (varying number of peptides depending on organism/role)

4 subfamilies:

• B-family ABC transporters: export polar and amphipathic substrates
• Type 1&2 ABC importers: Bacterial import

Question 9

ABC transporter:

Multidrug Resistance Transporter 1 (MDR1, ABCB1)

Answer 9

ATP binding & hydrolysis gives rise to large conformational changes transmitted to TM domains although sequence of ATP binding, hydrolysis and ADP release is unclear

• Uses ‘occluded state’ principle

Question 10

Type 1 ABC importer

Answer 10

Found in G-ve bacteria, uses a chaperone for molecule to interact with transporter (ATP binding + chaperone = conformational change)

e. g. Vitamin B12 uptake
- BtuB - specific Beta-barrel coupled to TonB
- BtuF - Periplasmic binding protein
- BtuCD - ABC transporter

Question 11

CFTR

Answer 11

Belongs to ABC family but is a Chloride channel (not pump)

ATP hydrolysis @ 2 NBDs promotes opening of a ClC

• no occluded state
• ATP isn’t the regulation mechanism, the R-domain is

Question 12

CFTR’s R (regulatory) domain

Answer 12

Must be phosphorylated by a kinase before ATP is able to open the channel
- Upon phosphorylation, R domain becomes unstructured and no longer blocks the 2 NBDs from binding

Cholera = constitutive phosphorylation of R-domain
- therefore ClCs constantly open and osmotic balance is disrupted