Lecture 13: Ion Pumps Flashcards
What are the four types of ATP powered transport?
- P-class pumps
- V-class proton pump
- F-class proton pump
- ABC superfamily
Where are P-class pumps found?
PM of plants, fungi, higher eukaryotes, sarcoplasmic reticulum
What direction do P-type ATPases transport ions, small molecules?
Against the conc gradient
What faces the cytosol?
The segments with ATP binding sites
Where are all P-class pumps phosphorylated?
highly conserved aspartate residues
what is the simplest type? What does P1B transport?
Type 1. transition metal ions important for bacterial resistance.
What do Ca2+ ATPases allow?
intracellular conc of calcium low (10^-7M) to avoid interference with proteins
What is Ca2+ release important for?
nerves and muscle contraction
What is the Ca2+ pump in cardiac and skeletal systems?
SERCA
In what direction do Ca2+ and protons move in the sarcoplasmic reticulum?
Ca into SR, protons out
What is Ca2+ bound by in the SR?
Calreticulin and calsequestrin
Describe how a skeletal muscle contraction works.
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.
What is the architecture of the P-type ATPases and where does Ca bind?
6TM-12 TM helices and three cytosolic domains called the phosphorylation, nucleotide binding and actuator domains. Ca binds the middle of the T domain.
What is the homology of the M domain and what does this reflect?
18% and reflects evolution to accomadate different ion substrates.
Describe the Nucleotide binding domain
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.
Describe the actuator domain
smallest, highly conserved across P-type ATPases and doesn’t contain a cofactor binding site unlike other domains.
What is the P domain, what is its signature sequence, what is its architecture, how conserved is it and what are the invariant residues?
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.
In the signature sequence, which part is reversibly phosphorylated?
the aspartate (D)
What did all the members of P class ATPases evolve from and what do they do?
A common ancestor but transport different ions
How many TM spanning regions are there? where are the highly conserved cytoplasmic domains, how many AAs are invariant?
10, inserted between M2+3 and M4+5. 87/900.
Explain the E1-E2 reaction scheme
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
What is mg2+ and ATP needed for?
To form the conf change so the pump can recruit Ca2+. Mg thought ti maintain ATP in a particular conf for hydrolysis.
What happens when the Ca2+ enters?
The Mg leave
What is a difference between Na/K and Ca in SR?
Na/k is bidirectional, Ca isnt
