L04

Question 1

what happens when Sh gene is mutated in drosophila

Answer 1

it results in shaking legs

Question 2

what is disrupted in Sh mutation

Answer 2

repolarization

Question 3

what are the methods of studying ion channels

Answer 3

patch clamp Electrophysiology
- investigates the function of an ion channel

(Cryo) Electron Microscopy OR X-ray crystallography
- investigates the structure of an ion channel

Question 4

what are gated ion channels

Answer 4

Ion channels open/close in response to stimuli

Question 5

what are the types of gated ion channels

Answer 5

voltage gated

ligand gated (extracellular ligand) (neurotransmitter-gated)

ligand gated (intracellular ligand) (ion-gated nucleotide-gated)

mechanically gated

Question 6

what is tetrodotoxin (TTX) produced by Pufferfish

Answer 6

sodium channel blocker that blocks the pore

Question 7

what is Charybdotoxin (CTX) produced by Deathstalker Scorpion

Answer 7

potassium channel blocker that blocks the pore

Question 8

what is ω-agatoxin produced by Funnel-web spider

Answer 8

Calcium channel blocker (blocks voltage sensor)

Question 9

what does the use of ion channel toxins help us test

Answer 9

Substrate selectivity
- the separate pathways for the flux of different ions

gating
- Control ion channel opening/closing
- Dynamic control
- Voltage/ signal/ ligand/stretch gated

Question 10

what is shaker also known as

Answer 10

KCNQ1 Potassium Channel

Question 11

what subunits does KCNQ1 Potassium Channel have

Answer 11

4Alpha & 2beta subunits

Question 12

how do you look at a protein structure

Answer 12

determine the structure experimentally e.g. X-ray crystallography

computational predictions
- accelerated by AI

Question 13

what is the first thing to do when investigating the structure of an ion channel

Answer 13

1-Look at the amino acid sequence

2-using experimental methods

Question 14

how does the amino acid sequence help predict the protein structure

Answer 14

Looking at the biochemical properties of specific amino acid side-chains

looking for conserved sequences

Question 15

which part of the structure would have hydrophobic residues

Answer 15

transmembrane region

Question 16

which part of the structure would have Polar/charged/hydrophilic residues

Answer 16

extramembrane, ligand binding etc.

Question 17

what experimental methods are used in determining protein structures

Answer 17

X-ray crystallography

nuclear magnetic resonance

cryo-electron microscopy

Question 18

what are the features of X-ray crystallography

Answer 18

sample must be crystallized in a solid frozen structure

any size macromolecule

atomic resolution but crystallization may take years and damage protein structure

Question 19

what are the features of nuclear magnetic resonance

Answer 19

sample must be dissolved and radioactively labelled

small molecules

closer to real protein structure but larger proteins can not be resolved

Question 20

what are the features of Cryo-Electron microscopy

Answer 20

sample is frozen in its native state

any size macromolecule

near-atomic resolution. fast sample preparation

Question 21

what are the steps in X-ray crystallography

Answer 21

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

Question 22

why is X-ray crystallography limited when it comes to ion channel structures

Answer 22

ion channels are

transmembrane proteins

Large proteins (high molecular weight)

Multiple conformations

Multiple subunits

Dynamic (open/close) & disordered

Not very soluble

Question 23

what are the steps of Cryo-EM

Answer 23

protein purification (lowering the temperature)

taking ‘movie frame photos’

putting pictures in a model

viewing the protein in different positions

Question 24

genetic and structural information allow scientists to input a sequence into Alphafold to predict structures using AI

Answer 24

true

Question 25

how many domains does a typical alpha subunit potassium channel have

Answer 25

4 domain

Question 26

how many transmembrane helices does each alpha subunit domain have in a potassium channel

Answer 26

6

Question 27

which transmembrane region of the alpha subunit domains is positively charged

Answer 27

4

Question 28

what is the positive region (4th) in the alpha subunit domains linked to

Answer 28

gating the ion channel

Question 29

what are regions 5 and 6 in the alpha subunit domains linked to

Answer 29

pore forming domain

Question 30

what amino acids are commonly found in channels that regulate positive ions

Answer 30

amino acids with positively charged residues.

Question 31

in which aspect of the ion channel are positively charged amino acids involved

Answer 31

gating selectivity

Question 32

give examples for positively charged amino acids

Answer 32

K lysine R arginine

Question 33

positive amino acids are conserved across different ion channels

Answer 33

True

Question 34

how do Na ions differ to K ions

Answer 34

Na ions are smaller

Question 35

what do positive amino acids do to the pore of an ion channel

Answer 35

select for narrow ions by making the pore narrow

Question 36

how is ion channel selectivity removed

Answer 36

Site directed mutagenesis

Question 37

the pore of the shaker is narrow

Answer 37

true

Question 38

one substitution to shaker (W434F) results in a wider pore. how does this affect the membrane potential

Answer 38

loss of control due to increased leakage of ions.

Question 39

what are the types of ion channel inactivation

Answer 39

N-type inactivation C-type inactivation- hinged lid

Question 40

what happens in N-type inactivation

Answer 40

Amino acids at N-terminus occlude the intracellular side of the channel pore Leads to rapid inactivation