Lecture 13: Ion Pumps

Question 1

What are the four types of ATP powered transport?

Answer 1

1. P-class pumps
2. V-class proton pump
3. F-class proton pump
4. ABC superfamily

Question 2

Where are P-class pumps found?

Answer 2

PM of plants, fungi, higher eukaryotes, sarcoplasmic reticulum

Question 3

What direction do P-type ATPases transport ions, small molecules?

Answer 3

Against the conc gradient

Question 4

What faces the cytosol?

Answer 4

The segments with ATP binding sites

Question 5

Where are all P-class pumps phosphorylated?

Answer 5

highly conserved aspartate residues

Question 6

what is the simplest type? What does P1B transport?

Answer 6

Type 1. transition metal ions important for bacterial resistance.

Question 7

What do Ca2+ ATPases allow?

Answer 7

intracellular conc of calcium low (10^-7M) to avoid interference with proteins

Question 8

What is Ca2+ release important for?

Answer 8

nerves and muscle contraction

Question 9

What is the Ca2+ pump in cardiac and skeletal systems?

Answer 9

SERCA

Question 10

In what direction do Ca2+ and protons move in the sarcoplasmic reticulum?

Answer 10

Ca into SR, protons out

Question 11

What is Ca2+ bound by in the SR?

Answer 11

Calreticulin and calsequestrin

Question 12

Describe how a skeletal muscle contraction works.

Answer 12

Action potential from the nervous system activates L type voltage dependent Ca2+ channels in the traverse tubule system. Ryanodine receptor releases Ca2+ from the SR into the cytoplasm which is sensed by troponin. This binds Ca causing a conf change which causes actin/myosin cross-bridging and muscle contraction. SERCA pumps the Ca excess back into the ER of muscle cells.

Question 13

What is the architecture of the P-type ATPases and where does Ca bind?

Answer 13

6TM-12 TM helices and three cytosolic domains called the phosphorylation, nucleotide binding and actuator domains. Ca binds the middle of the T domain.

Question 14

What is the homology of the M domain and what does this reflect?

Answer 14

18% and reflects evolution to accomadate different ion substrates.

Question 15

Describe the Nucleotide binding domain

Answer 15

Is a large insert within the P domain. Is linked to the P domain by a highly conserved hinge of antiparallel peptide strands. the size and sequence varies more than the other domains.

Question 16

Describe the actuator domain

Answer 16

smallest, highly conserved across P-type ATPases and doesn’t contain a cofactor binding site unlike other domains.

Question 17

What is the P domain, what is its signature sequence, what is its architecture, how conserved is it and what are the invariant residues?

Answer 17

The catalytic core of the SR Ca2+. DKTGTLT. spherical with two central b sheets flanked by 2 helices including the cytoplasmic end of M5. It is the most highly conserved domain and the invariant residues include Asp hinge which links the N and P domain and a central part of the b sheet important for folding.

Question 18

In the signature sequence, which part is reversibly phosphorylated?

Answer 18

the aspartate (D)

Question 19

What did all the members of P class ATPases evolve from and what do they do?

Answer 19

A common ancestor but transport different ions

Question 20

How many TM spanning regions are there? where are the highly conserved cytoplasmic domains, how many AAs are invariant?

Answer 20

10, inserted between M2+3 and M4+5. 87/900.

Question 21

Explain the E1-E2 reaction scheme

Answer 21

E1 conformation has high affinity ca2+ binding site exposed to the cytoplasmic side. E2 has low affinity Ca2+ binding site exposed to the lumen. For each ATP hydrolysed the ATPase exports 2Ca and imports 3 protons. Phosphorylation of Asp351 on P domain leads to formation of high energy intermediate occlusive of Ca. Conf change to low energy E2P intermediate allows release of ca into lumen in exchange for h+. Dephosphorylation to E2 allows enzyme to convert back to E1 and release protons into cytoplasm

Question 22

What is mg2+ and ATP needed for?

Answer 22

To form the conf change so the pump can recruit Ca2+. Mg thought ti maintain ATP in a particular conf for hydrolysis.

Question 23

What happens when the Ca2+ enters?

Answer 23

The Mg leave

Question 24

What is a difference between Na/K and Ca in SR?

Answer 24

Na/k is bidirectional, Ca isnt

Question 25

What is the E1/E2 scheme for the Na/K pump, in what direction are they transported in?

Answer 25

In the E1 state there is high affinity for Na+ so 3 are bound but low affinity for K+. In the E2 state there is low affinity for Na+ so none bound and high affinity for k+ SO TWO BOUND. na goes out, k goes in.

Question 26

What drugs block the ATP activity of Na/K pumps?

Answer 26

digoxin and ouabains

Question 27

Compare the structures of the alpha subunit of Na/K ATPase and SERCA.

Answer 27

very similar. ion binding site has A, P, N cytoplasmic domains, 10 TM helices like SERCA. M1-6 for transport, M7-10 for structural support like SERCA. Has phosphorylation site at Asp369 of DKTGT motif in P domain.

Question 28

What does binding of the substrate ion in Na/K pump trigger?

Answer 28

Formation of MG ion binding site which along with LYS691, offsets the electrostatic repulsion at the P site and allow transfer of the P to Asp369.

Question 29

What diseases are caused by P-type ATPase mutations?

Answer 29

SERCA1 -brody myopathy. SERCA2 - darier disease skin condition

Question 30

What happens in brody myopathy?

Answer 30

Skeletal muscles effected, muscle cramping and stiffening after exercise as don’t get reabsorption of Ca so contraction still triggered.

Question 31

How can P-type ATPases be drug targets?

Answer 31

SERCA can be overexpressed in prostate cancer and thapsigargin inhibits SERCA on prostate cancer cells specifically. na/K can cause heart failure and digoxin increase Na conc by inhibiting the pump.

Question 32

What speed do pumps transport ions?

Answer 32

10^0-10^3 as are more compex and need to undergo more conf changes.