PW - Potassium Channels Flashcards
What are Ligand Gated Ion Channels?
They are regulated by neurotransmitters/modulators and typically sense stimuli on the outside of the cell
- Example: nAChR
What are common properties of Ion Channels?
They facilitate ion diffusion down concentration gradients, are highly efficient with high selectivity, and respond to changes in pH, voltage, ions, ligands, and G-proteins
What are 2 common properties of potassium channels?
Highly efficient:
- 1000 fold selective for K+ over Na+
- 108 ions/sec – close to diffusion-limited
Found in many locations:
- Neurons – set resting potential
- Cardiac tissue – regulate heart beat
- Epithelial – regulate passage of salts and water
- Respond to changes in pH, voltage, ions, ligands, G-proteins
What are 4 features of potassium channels?
1) Highly homologous
2) Common pore domains
3) Two regulatory domains:
- N-terminus
- C-terminus – separate proteins
4) Appear to have evolved by addition of modular regulatory domains
What occurs during the structural studies of potassium channels?
Expression
- Bacterial channel – express in homologous host
- Remove weakly ordered domains
Purification
- Solubilized in decylmaltoside
- His tagged to aid purification
- C-terminus removed, gel filtered, exchanged into LDAO
Crystallized
- Many subsequent structures with Ab fragments to stabilize crystals
Structure
- X-ray crystallography
Describe the structure of KcsA (5)
- Protein cone shaped
- Constricted at the cytoplasmic face, open at extracellular face
- Each subunit has two transmembrane domains, and inner and an outer.
- In addition a shorter half helix forms fills the top of the channel, to make a pore
- The pore helix is oriented with the C-terminus inside, putting the dipolar towards the centre
What do crystal structures reveal about the ion conduction pathway? (2)
- K+ ions are desolvated
- A hydrated K+ ion is located in a cavity in the centre of the bilayer
Interestingly the ions are desolvated before transport, this is energetically unfavourable, and certainly not compatible with the rates of transport.
In addition, a fully hydrated ion appears in the central cavity
How is ion selectivity mantained in KcsA? (3)
Selectivity Filter – conduction pathway formed from a conserved TVGYG motif
- This motif allows the backbone carbonyl groups to point towards channel lining
The geometry of that inner sphere is matched almost perfectly by the position of the carbonyl groups
- Sodium is energetically unfavourable as the geometry is not suitable for sodium ions
The backbone is relatively rigid and the conduction is facilitated by fluctuations in the positioning of the CO groups
- Maintains selectivity of potassium ions
How are ions conducted in the KcsA channel?
Alternating states of which only two are ever occupied, 1 and 3 or 2 and 4.
- As carbonyl groups replace water, it pushes potassium ion from one state to another
- Adjacent sites experience electrostatic repulsion and so push the potassium along via a knock-on mechanism
In fact a water molecule is located between each K+ ion
If site 1 binds K+, then K+ at site 2 is pushed to site 3 and so on.
What is the regulatory domain of ligand gated channels (MthK) ? (5)
- RCK, Ca2+ sensing domain located on cytoplasmic face
- 4 copies per monomer, 2 attached to channel (2 additional copies)
- Make a gating ring of 8 RCK domains
- RCK domains have a a-b fold
- Cleft between two domains forms 2 Ca2+ binding sites
What occurs during MthK ligand regulated channel gating? (4)
- Bindings of Ca2+ causes changes in the relative orientations of the subunits (based on crystal structure of RCK domains alone.
- This cause the 2 domains to move in a rigid body rotation.
- This affects other RCK domains, and results in a force being applied at the end of the channel gate, forcing it to open.
- In such a way the energy associated with Ca2+ binding is linked to channel gating
What are 3 features of Kv channels?
- Voltage sensors composed of 4 helical TMD
- Sensitive to changes of a few mV
- S1-S4 contains the voltage sensing domain
Structure of the S1-S4 voltage sensing domain (2)
- Charged residues located on the S4 helix
- In this structure the first three Arg residues are exposed to water as they are peeled away from S1 and S2
Putative mechanism for voltage gating
May involve a conformational change in the R1 and R2 region into a 3_10 structure, and a movement into the bilayer complementing negative charges originally thought to interact with negative charges
