Lecture 8 - Studying Ion Channels & Structures of Ion Channels Flashcards
What experimental methods are there to study ion channels?
- Molecular biology tools
- Biochemistry tools
- Structural biology methods
- Cell biology methods
- Electrophysiology methods
Molecular biology tools
- Gene expression
- Cloning
- Sequencing
- Site-directed mutagenesis
Biochemistry tools
- Protein solubilization
- Protein isolation/purification
- Reconstitution into lipid bilayers
- Radioactive ion flux
Structural biology methods
- Cryo-Electron Microscopy (CryoEM)
- Atomic Force Microscroscopy (AFM)
- X-ray Crystallography
- Electron Paramagnetic Resonance (EPR)
Spectroscopy - Nuclear Magnetic Resonance (NMR) Spectroscopy
- Electrophysiology (Function)
Cell biology methods
- Protein tagging
- Western blotting
- Immunofluorescence
- Optical & Confocal microscopy
- PCR
- Flow cytometry
Electrophysiology methods
Patch-clamp
Patch-clamp: what cells can be used, what electro-recordings can be made, and what are the four types?
Isolated cells/tissue slices
- Patch-clamp macroscopic whole-cell current recordings
- Patch-clamp single-cell current recording
Inside-out patch
Outside-in patch
Whole-cell patch
On-cell patch
NMR spectroscopy: what does it do and what are the advantages and disadvantages?
Structure determination from protein samples
- Thermodynamics and kinetics
- Structural information (distances, angles, and coupling)
- 3D structure determination
- Protein dynamics
- Large sample amount required
- Labelling is expensive
- Requires complex data analysis
- Size limit of ~50kDa
EPR spectroscopy: what are its PELDOR/DEER advantages and what are the advantages and disadvantages?
PELDOR/DEER advantages:
* Condition flexibility (pH, buffer, lipid membranes)
* Small label (specificity, non-invasive)
* High resolution
* No large amount of protein is required
* Protein folding (state and oligomerization)
- Ensemble method
- Unlimited condition and flexibility
- Protein dynamics in vivo and in vitro
- Immediate results
- Labelling needed
- Specialised equipment and expertise required
- No real-time information, but time-resolved is possible (?)
- No 3D structure coordinates but can impose restraints
X-ray crystallography: what does it do and what are the advantages and disadvantages?
- High-resolution and single-particle
- Unlimited upper protein size
- Membrane environment and physiological conditions
- Labelling not required
- No real-time information
- Substantial computational resources required
- Equipment access and data collection
- Limited lower protein size
Cryo-electron microscopy: what does it do and what are the advantages and disadvantages?
- 3D structure determination
- High resolution
- Visualisation of molecular interactions
- No size limitation
- ‘static’ image
- No real-time information
- Non-membrane environment for membrane proteins
- Modifications required
Which methods (or combination of methods) are
most suited to study:
a) A small ion channel with disordered regions
b) A large ion channel in lipid environment
c) The dynamics of an intermediate-size ion channel
d) The 3D structure of an ion channel
e) The function and ion conductance properties of an ion
channel
K+ ion channels
~80 genes
- KV (voltage-gated)
- KCa (Ca-gated)
- Kir (inward rectifier)
- Twin Pore K channels
VGCaCs: what are the main two subfamilies?
High-voltage-activated (L-, Q-, N-, and R-type)
Low voltage-activated (T-type)
Auxillary proteins: what do they do?
Bind to core proteins in ion channels and act as regulators on the key structure
