L04 Flashcards
what happens when Sh gene is mutated in drosophila
it results in shaking legs
what is disrupted in Sh mutation
repolarization
what are the methods of studying ion channels
patch clamp Electrophysiology
- investigates the function of an ion channel
(Cryo) Electron Microscopy OR X-ray crystallography
- investigates the structure of an ion channel
what are gated ion channels
Ion channels open/close in response to stimuli
what are the types of gated ion channels
voltage gated
ligand gated (extracellular ligand) (neurotransmitter-gated)
ligand gated (intracellular ligand) (ion-gated nucleotide-gated)
mechanically gated
what is tetrodotoxin (TTX) produced by Pufferfish
sodium channel blocker that blocks the pore
what is Charybdotoxin (CTX) produced by Deathstalker Scorpion
potassium channel blocker that blocks the pore
what is ω-agatoxin produced by Funnel-web spider
Calcium channel blocker (blocks voltage sensor)
what does the use of ion channel toxins help us test
Substrate selectivity
- the separate pathways for the flux of different ions
gating
- Control ion channel opening/closing
- Dynamic control
- Voltage/ signal/ ligand/stretch gated
what is shaker also known as
KCNQ1 Potassium Channel
what subunits does KCNQ1 Potassium Channel have
4Alpha & 2beta subunits
how do you look at a protein structure
determine the structure experimentally e.g. X-ray crystallography
computational predictions
- accelerated by AI
what is the first thing to do when investigating the structure of an ion channel
1-Look at the amino acid sequence
2-using experimental methods
how does the amino acid sequence help predict the protein structure
Looking at the biochemical properties of specific amino acid side-chains
looking for conserved sequences
which part of the structure would have hydrophobic residues
transmembrane region
which part of the structure would have Polar/charged/hydrophilic residues
extramembrane, ligand binding etc.
what experimental methods are used in determining protein structures
X-ray crystallography
nuclear magnetic resonance
cryo-electron microscopy
what are the features of X-ray crystallography
sample must be crystallized in a solid frozen structure
any size macromolecule
atomic resolution but crystallization may take years and damage protein structure
what are the features of nuclear magnetic resonance
sample must be dissolved and radioactively labelled
small molecules
closer to real protein structure but larger proteins can not be resolved
what are the features of Cryo-Electron microscopy
sample is frozen in its native state
any size macromolecule
near-atomic resolution. fast sample preparation
what are the steps in X-ray crystallography
forming crystal of concentrated protein molecules when protein is combined with buffers and specific chaperones
crystal is exposed to X-rays creating diffraction patterns based on electron density
creation of electron density map
creation of atomic model
why is X-ray crystallography limited when it comes to ion channel structures
ion channels are
transmembrane proteins
Large proteins (high molecular weight)
Multiple conformations
Multiple subunits
Dynamic (open/close) & disordered
Not very soluble
what are the steps of Cryo-EM
protein purification (lowering the temperature)
taking ‘movie frame photos’
putting pictures in a model
viewing the protein in different positions
genetic and structural information allow scientists to input a sequence into Alphafold to predict structures using AI
true
how many domains does a typical alpha
subunit potassium channel have
4 domain
how many transmembrane helices does each alpha subunit domain have in a potassium channel
6
which transmembrane region of the alpha subunit domains is positively charged
4
what is the positive region (4th) in the alpha subunit domains linked to
gating the ion channel
what are regions 5 and 6 in the alpha subunit domains linked to
pore forming domain
what amino acids are commonly found in channels that regulate positive ions
amino acids with positively charged residues.
in which aspect of the ion channel are positively charged amino acids involved
gating
selectivity
give examples for positively charged amino acids
K lysine
R arginine
positive amino acids are conserved across different ion channels
True
how do Na ions differ to K ions
Na ions are smaller
what do positive amino acids do to the pore of an ion channel
select for narrow ions by making the pore narrow
how is ion channel selectivity removed
Site directed mutagenesis
the pore of the shaker is narrow
true
one substitution to shaker (W434F) results in a wider pore. how does this affect the membrane potential
loss of control due to increased leakage of ions.
what are the types of ion channel inactivation
N-type inactivation
C-type inactivation- hinged lid
what happens in N-type inactivation
Amino acids at N-terminus occlude the intracellular side of the channel pore
Leads to rapid inactivation
