L12: Drugs Acting on Ion Channels

Question 1

What are the types of ion channels?

Answer 1

• voltage-gated (-sensitive, -activated)
• ligand-gated (e.g. nAChR, NMDAR, AMPAR, 5-HT3, GABAa, glycine)

Question 2

What is the importance of ion channels?

Answer 2

Ion channels are important in:
- neuronal excitability, e.g. membrane potential, action potential, transmitter release
- excitation-contraction coupling in muscle
- volume (cell size) control
- regulate cell cycle and division

Question 3

What is the conformation of voltage-gated Na+ channel in resting (closed), open and inactivated (closed) states?

Answer 3

• resting state: m-gates closed, h-gates open
• open state: m-gates and h-gates open
• inactivated state: m-gates open, h-gates closed

See L12, slide 4 for diagramm

Question 4

How is P-loop important in voltage-gated channels? Where is it located?

Answer 4

It acts as a selectivity filter. It has a signature sequence that only lets in specific ions. It connects 5-6 transmembrane domains on the outer side of the cell.

Question 5

How is inactivation gate important in voltage-gated channels? Where is it located?

Answer 5

It changes the shape after the channel has been open in order to block the pore.
It’s located on the inner side of the membrane between transmembrane domains 6-1 of subunits III and IV.

Question 6

What is the mechanism of ion selectivity in voltage-gated channels? Explain with an example of Na+ and K+ channels

Answer 6

For example, K+ and Na+ exist as hydrated molecules in water (with 8 H2O molecules).
1. When ion comes into the pore, water needs to be ripped off, however the ion is not stable anymore.
2. So it is stabilised by carbonyls, which come in conctact in the pore.
3. K+ ion is bigger than Na+ ion. This means that Na+ can go in the K pore, however this means that it doesn’t come into contact with carbonyls and is energetically unfavourable, so it doesn’t go through. On the other hand K+ becomes energetically favourable and goes through.

Question 7

How many binding sites does selectivity filter provide for permeating ions? How many of them are used?

Answer 7

4 stable binding sites for permeating ions, but only 2 used

Question 8

Why do ions move through the voltage-gated channel?

Answer 8

The ions are of the same charge, so they push each other like magnets. The difference in concentrations inside / outside the membrane drives the ions to go through the membrane.

Question 9

What is the mechanism of voltage-gated channels activation?

Answer 9

S4 is linked to S5 and S6 regions through linker region S4-S5. S4-S5 has a lot of charged amino acids that form non-covalent interactions with the inner part of S6. S6 has a proline-valine-proline region, which causes flexed structure. The interactions of S4-S5 region with S6 induce conformational change of PVP region, which leads to opening the pore.

Question 10

What mediates inactivation of voltage-gated channels?

Answer 10

Linker region between subunits III-IV, which has the IFM motif. This motif acts as a hinged lid and blocks the pore when it goes from open to inactivated state. Before the channel can open again it must go to resting (closed) state.

Question 11

What are the mutations of ion channels called? Which diseases are they responsible for?

Answer 11

Channelopathies, responsible for heart disease, epilepsy, deafness, cystic fibrosis

Question 12

What is the structure of voltage-gated Na+ channel?

Answer 12

• 4 subunits
• P loops between 5-6 TMs
• 6 transmembrane domains within each subunit

Question 13

What attracts K+ ions towards the selectivity fiilter regarding K+ channels?

Answer 13

The P-loop helices point their partial negative charge towards the cavity. This attracts K+ ions towards the selectivity filter.

Question 14

How are voltage-gated Na+ channels inactivated?

Answer 14

• Intracellular III-IV linker acts as a hinged lid and blocks pore
• Inactivation restored by pentapeptide containing IFM motif

Question 15

What’s IFM motif regarding voltage-gated Na+ channels?

Answer 15

Three key amino acids in the III-IV linker I1488, F1489, M1490 form the IFM motif.

Question 16

What’s the effect of tetrodotoxin on sodium channels?

Answer 16

Tetrodotoxin potently blocks the ability of neurons to conduct action potentials by blocking voltage-gated Na+ channels. It does this only from the extracellular side of the membrane and blocks open or closed channels equally well.

Question 17

How can tetrodotoxin affect Na+ channels?

Answer 17

TTX has guanidium moiety. Guanidium can permeate the Na+ channel and block the channel. TTX blocks from the extracellular side of the membrane only. TTX binds to closed or open channels equally effectively.

Question 18

How can Na+ channels be modified so that they become insensitive to tetrodotoxin?

Answer 18

Changing Glutamate at position 387 to Glutamine in the P-loop of Domain I makes the channel insensitive to TTX.

Question 19

What are local anaesthetics?

Answer 19

Local anaesthetics (LA’s) are a class of drugs that have their effects by preventing nerve conduction through blocking voltage-gated Na+ channels.

Question 20

What are local anaesthetics constituted of chemically? Are they basic or acidic?

Answer 20

Aromatic ring linked by either an ester bond or an amide bond to a basic side chain. Local anaesthetics (apart from benzocaine) are weak bases.

Question 21

How are the two classes of local anaesthetics structurally determined?

Answer 21

They fall into two structural classes either incorporating an amide (e.g. Lignocaine / lidocaine) or an ester (e.g. procaine, cocaine) bond

Question 22

Why is non-ionised form of a drug important?

Answer 22

Only non-ionised (non-polar) forms of a drug can cross the phospholipid bilayer which forms the cell membrane.

Question 23

What is the hydrophilic pathway of local anaesthetics action?

Answer 23

Local anaesthetics enter the cell through the membrane simply permeating it (NOT through the channel), then once the channel opens local anaesthetic goes inside and blocks the channel.

Question 24

What is the hydrophobic pathway of local anaesthetics action?

Answer 24

As the local anaesthetic is permeating the membrane, some of it can enter from the membrane phase. This means that even unopened channels can be blocked.

Question 25

What is the mechanism of sodium channel block by local anaesthetics?

Answer 25

A major determinant of local anaesthetic binding in hNav1.5 is a phenylalanine residue (F1759) located on the sixth transmembrane spanning region of domain 4 of the channel. This allows local anaesthetics to bind at a site in the channel pore, on the cytoplasmic side of the selectivity filter. Local anaesthetics may physically block Na+ permeation, however, studies suggest that a more important factor in the mechanism of block is the introduction of positive electrostatic potential that repels positively charged Na+, preventing permeation.

Question 26

What are the other drugs acting on sodium channels?

Answer 26

• anticonvulsants. Reducing the electrical excitability of cell membranes, possibly through use-dependent block of sodium channels. E.g. phenytoin, carbamazepine.
• Class I antidysrhythmic drugs. E.g. quinidine, lignocaine, flecainide.