M&R 4.1 The (axonal) AP

Question 1

Describe some main features of an AP

Answer 1

1. A rapid change in MP from negative to positive and back again
2. Triggered once a threshold level of membrane depolarisation is reached
3. All or nothing (happens full or not at all - larger currents do not provoke larger APs)
4. Stimulus intensity is encoded by frequency of APs (not amplitude - amplitude always the same)
5. Propagated without loss of amplitude

Question 2

Sketch an axonal AP

Answer 2

x axis = time (ms)
Depolarisation phase should be ~0.5ms, total time ~1ms

y axis = membrane potential (mV)
RMP should be ~-70mV
Peak of depolarisation should be ~+30mV

Question 3

What causes the upstroke of the AP?

Answer 3

Depolarisation
Once membrane has reached threshold (~ -55mV) opening of voltage-gated sodium channels (VGNCs) causes rapid influx of Na+ ions into the cell down the Na+ conc gradient
MP depolarises (becomes more positive)
Positive feedback - more depolarisation causes more Na+ channels to open
MP reaches ~+30mV

Question 4

What is happening at the peak of the AP?

Answer 4

VGNCs become inactivated

Slower K+ channels are open

Question 5

What is happening during the downstroke of the AP?

Answer 5

Repolarisation
K+ efflux via VGKCs down its concentration gradient
Na+ influx is stopped due to inactivation of VGNCs
MP returns towards RMP (becomes more negative)
There is a slight overshoot where K+ channels remain open and MP becomes slightly more negative than the RMP = hyperpolarisation
Then VGKCs close and RMP is restored
(Inactivated VGNCs channels then enter closed state - become responsive to voltage again)

Question 6

What is the purpose of the hyperpolarisation phase of the AP?

Answer 6

Transiently makes the MP more negative and therefore further from the threshold potential
This assists in the directionality of the AP by making it harder for a portion of axon that just underwent an AP to undergo another one (therefore encourages forwards transmission)

Question 7

What method can be used for direct measurement of membrane currents, and how does it work?

Answer 7

Voltage clamping
Clamps the MP at a desired value
Measure the current required to keep the membrane at this value - because the current needed to keep the MP at that value is equal to the amount of current flowing across the membrane

Question 8

What is patch-clamping?

Answer 8

A contemporary version of voltage-clamping
It has higher resolution, so rather than looking at an area of membrane, can measure tiny currents flowing through single ion channels

Question 9

How do VGKCs react to maintained depolarisation?

Answer 9

They are voltage-gated so open in response to depolarisation
They open slowly
They stay open throughout depolarisation

Question 10

How do VGKCs react to repolarisation?

Answer 10

They close

But not immediately (because they cannot ‘inactivate’ like Na+ channels so their closure takes longer)

Question 11

How do VGNCs react to maintained depolarisation?

Answer 11

They activate quickly
They inactivate during maintained depolarisation
Therefore inward Na+ current eventually wanes to 0 even if depolarisation is maintained
They then need to enter the closed state before they can re-open (recovery time)

Question 12

What is the absolute refractory period? What causes it?

Answer 12

The period just after an AP when it is impossible to fire another one, no matter how strong the stimulus
Due to nearly all Na+ channels being in the inactivated state (cannot be opened - need to enter close state first)

Question 13

What is the relative refractory period? What causes it?

Answer 13

The period where Na+ channels are recovering from inactivation (and entering the closed state) so there is slow recovery of membrane excitability.

Can initiate an AP (because some Na+ channels are closed and therefore available to open) but requires a stronger than normal stimulus (because some K channels still open, so K+ efflux is opposing some of the depolarisation)

Question 14

What is accommodation?

Answer 14

Where a prolonged depolarisation causes an increase in the threshold potential.
This is because more Na+ channels are in the inactivated state, and K+ conductance increases
This makes the peak of the AP lower and the threshold higher (more positive)
Subsequent inputs won’t cause an AP to fire even if the original threshold is surpassed

Question 15

Describe the structure of a VG Na+ channel

Answer 15

1 channel is made up of 1 subunit (alpha)
The subunit can be split into 4 repeating quarters (I-IV)

Each quarter has 6 TM domains

• > 4th TM domain in each quarter detects voltage (contains lots of +ve AAs - voltage change causes conformational change so pore opens)
• > Between 5th & 6th TM domains in each quarter is a H5 pore-forming region (in 3D structure the 4 pore-forming regions come together to form the pore)

Between quarters III and IV is the inactivation particle (when pore is open it can swing into pore and block flow of Na+)

Question 16

Describe the structure of a VG K+ channel

Answer 16

1 channel is made of 4 subunits (alpha 1-4)
Each subunit is similar to one of the quarters of an Na+ channel

Each subunit has 6 TM domains

• > 4th TM domain in each subunit acts as voltage sensor
• > between 5th & 6th TM domain in each subunit is the pore/H5 region

Question 17

What are the main similarities and differences between the structure of VG sodium channels and VG potassium channels?

Answer 17

Overall size/structure of both is similar

Na+ channel is only 1 large subunit, made up of 4 repeating quarters, whereas K+ channel is 4 subunits (each of which resembles one quarter of the Na+ channel)

Each quarter of an Na+ channel / subunit of a K+ channel has:

• > 6TM domains
• > 4th TM domain is voltage-sensing
• > region between 5th & 6th TM domains forms pore

Na+ channel has an inactivation particle

Question 18

Which channels do local anaesthetics (e.g procaine) block?

Answer 18

Voltage-gated Na+ channels (VGNCs)

Question 19

How do local anaesthetics block VGNCs?

Answer 19

By interacting with the pore-forming regions on 6th TM domains to block channel pore.

Block channel from inside the cell (binding site is on cytoplasmic side)

Question 20

Do local anaesthetics block open or closed VGNCs better?

Answer 20

LA’s block open VGNCs better

Therefore the more a pain fibre is activated, the more the VGNCs open, and the more can get blocked

Question 21

Which kind of fibres do local anaesthetics affect best?

Answer 21

(in order)

1. Small myelinated axons
2. Unmyelinated axons
3. Large myelinated axons

Therefore affect pain fibres before motor fibres

Question 22

How does extracellular pH affect the ability of local anaesthetics to act?

Answer 22

LA’s act on the cytoplasmic side of the VGNC so have to cross the cell membrane to enter the cell.

Only unprotonated form can readily cross membrane. Can’t enter if protonated (so less able to enter at lower pH)
Therefore if outside of cell is more alkaline, the LA will be more easily able to enter the cell
If outside of cell more acidic (as in infection) the LA cannot enter the cell as readily