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
Mechanosensitive ion channels
Exist in multiple oligomeric forms - are diverse in sequence and structural architecture
Activated by mechanical stretch & cytoskeleton pulling & changes in bilayer tension
Ion permeation
Channels are often water-filled pores and ions must be dehydrated as they move through the selectivity filter
Channels end up allowing 10^8 to flow through per second
GYG motif
glycine-tyrosine-glycine motif defects have implications for disease
Quiz (True or False):
Potassium channels:
a) allow Potassium ions to pass through their pore and
restrict access to smaller Sodium ions
b) can be also mechanosensitive (multimodal function),
in particular the K2P dimeric channels
c) mostly form trimeric complexes from single subunit
6TM proteins
Fourth transmembrane protein - used as a voltage sensor
1-4 TM proteins used in voltage-gating
5-6 TM proteins used to form the filter and the pore
4 of these 6TM proteins are used to form a tetramer
