Lecture 2 - Neuronal ion channels

Question 1

What is the formula for Vm? (membrane potential) and what is the Vm of a resting cell?

Answer 1

Vin - V out

= -60 to - 70

Question 2

On a resting cell, in regards to the membrane potential (as the net charge on the inside and outside of a cell is relatively equal) - where is more negative, on the inside or outside of the cell?

Answer 2

The inside of the cell is more negative

Question 3

Define the Nernst potenitial

Answer 3

The equilibrium/Nernst potential of an ion is the membrane potential at which there is no net (overall) flow of that particular ion from one side of the membrane to the other

Question 4

What are some features of Resting membrane potential? (Bad question, just make sure to remember the facts of the answer)

Answer 4

• lipid bilayer is virtually impermeable to ions
• ions therefore can only cross the membrane through ion channels
• at rest, the passive ion fluxes are balanced so the membrane potential is constant - done by the Na/K-ATPase pump

Question 5

Why is the membrane potential in neurons almost the Nernst potential for K+ (-75mV)?

Answer 5

In neurons the membrane is highly permeable to K+ as K+ channels open easily (leading to passive potassium diffusion along a conc. gradient)
-the Nernst potential for K+ (-75mV) is however slightly offset by Na+ influx, to about -70mV

Question 6

What is the neuronal membrane potential?

Answer 6

-70mV

Question 7

What is the valence of K and when might this be used?

Answer 7

Valance of K = 58mV

When working out the Nernst potential for K+

Question 8

What is hyperpolarisation?

Answer 8

When the membrane becomes more polar (more negative)

Question 9

What is depolarisation?>

Answer 9

When the membrane becomes less negative, less polar

Question 10

How can the voltage of a neuron be altered experimentally?

Answer 10

changing the current

Question 11

What are electronic potentials?

Answer 11

Small depolarisation in a membrane voltage insufficient to pass the threshold of the sodium channel to open it

Question 12

What does the current clamp measure and how is this technique done?

Answer 12

Current clamp measures the Vm response to an known injected quantity of current into a cell

• Whole cells measurement
• Use a current generator and voltage amplifier

Question 13

What is investigated by a voltage clamp?

Answer 13

The amount of current necessary to achieve a defined membrane potential
E.g. Measure how much current necessary for repolarisation

Question 14

How is voltage clamping achieved?

Answer 14

• monitor the voltage across a cell membrane whilst injecting a metered amount of current to clamp the transmembrane voltage at a desired level
• Voltage clamp amplifier takes measurement of the voltage and adjusts current dependent on the difference between the set voltage and the measured voltage
• then depolarise the membrane and measure how much current is requires to repolarise

Question 15

What is the cell attached patch clamp technique for and how is it done? (Neher and Sakmann)

Answer 15

For measuring the activity of a single channel in the cell membrane

• place a glass electrode tip onto the membrane of the cell and apply a negative pressure to draw it up into the electrode
• this forms a high resistance seal between the walls of the tip and the membrane
• measure current required for repolarising and depolarising membrane (voltage clamp)
• can measure the activity of a single channel incorperated into the membrane and trapped in the tip of the electrode

Question 16

How was the action of single channels opening and closing illuatrated through the patch clamp technique?

Answer 16

Through patch clamp technique with voltage gating shown that channels gated the same level of current each time they were open
-gradient shows the open/closing kinetics

Question 17

What is the whole cell patch clamp technique and what does it allow?

Answer 17

Allows whole cell recording of Vm

• a pule of suction applied to a cell attached patch ruptures the membrane, and so that the membrane is continuous with the pipette
• must have v. well designed solution so the cell is not dialised
• allows properties of one type of channel to be studied by blocking the activity of other types with toxins/drugs/chemicals
• can study the behaviour of a population of channels by applying different currents and identify when the maximum current is flowing

Question 18

What conclusions were drawn about the properties of the activation and inactivation gates of the Na+ and K+ channels via voltage clamp analysis?

Answer 18

Na+
Activation gate
-Fast kinetics
-closed at resting potential, opens in response to depolarisation
Inactivation gate
-open at resting potential, closes in repose to depaolarisation
K+
Activation gate
-Slow kinetics
-closed at resting potential, open in response to depolarisation 
Inactivation gate
-not present

Question 19

What experiment did Hodekin and Katz do and what did this show?

Answer 19

• Altered the percentage of seawater/water a squid giant axon was bathed in
• showed different levels of action potential (mV levels) at different concentrations
• the action potential is slower and has less Mv the lower the % of salt concentration (less like sea water)

Question 20

Why are K+ and Na+ channels thought to have a common ancestor, what are there structures and which is thought to be more evolutionarily ancient?

Answer 20

-similar molecular structures
e.g.
Voltage gated K+ channel
-tetramer
-6 transmembrane spanning regions
-4 subunits
Voltaged gated Na+ channel
-monomer
-6 transmembrane spanning regions
-4 subunits

Na+ channel though to be more evolutionarily recent

Question 21

What structure is involved in the opening and closing of voltage gated ion channels?

Answer 21

• voltage sensor S4 helic
• opens/closes channel via a conformational change
• positively charged residues(lysine and arginine) in the S4 allow it to be sensitive to changes in membrane potential
• positively charged residues cause it to rotate as the membrane potential changes [at certain potentials (threshold) results in a conformational change]

Question 22

Why can’t the S4 helix be used to inactive voltage gated ion channels?

Answer 22

the S4 helix is sensitive to changed membrane potential

-therefore it is open when inactivation needs to occur

Question 23

What are the stability properties of the voltage gated Na+ channels?

Answer 23

open form = unstable, unenergectically favourable
blocked gate = stable
blocked gate and closed = unstable
closed resting = stable

Question 24

How are the Na+ voltage gated ion channels closed? (same principle for K+ channels but v. slow)

Answer 24

Through the action of two gates on the channel, the activation gate and the inactivation gate
1 - At resting membrane potential, the activation gate ‘m’ is closed and the inactivation ‘h’ gate on is open. This is the closed resting state and the channel is stabilised
2- Depolarisation leads to the open conformation (open ‘m’ gate, open ‘h’ gate) and Na+ influx
3 - Prolonged depolarisation sees the opening of the K+ channel (much slower)
4 - Open conformation is unstable, within a couple of ms protein blocks the inactivate gate (h) leading to a more stable, energectically favourable conformation
4 - As the mp drops during repolarisation, the ‘m’ gate closes, leading to a closed and inactivated state which is unstable
5 - ‘h’ inactivation gate opens, leading to the closed resting state which is stable
6 - ion channels then return to the resting membrane potential state

Question 25

What drugs have been shown to inhibit the NA+ channel, and what does this allow from an experimental point of view?

Answer 25

• TTX (tetrodotoxin from the puffer fish)
• BTX (Batrachotoxin from the poison dart frog)
• ScTx (Scorpian toxins)
• LA (Local anaesthetics)
• Saxitoxin (shellfish)

Blocking of Na+ channels allows the study of the action of K+ chanels

Question 26

How does the TTX toxin work?

Answer 26

Blocks the pore of the Na+ chanek

Question 27

Where does the BTX toxin act?

Answer 27

On the voltage gate of the Na+ channel

Question 28

Where do ScTx toxins and local anaesthesiacs act?

Answer 28

On top of the voltage gate of the Na+ channel

Question 29

What hereditory mutations in ion channels have been shown to cause disease through altering ion channel properties?

Answer 29

G1306V - glycine mutation in the loops
V1589M - valine mutation in the loops

lead to more spontaenous activity
-migranes, epilepsy, disorders of exitability
shown through expressing ion channels and WT in cells that don’t have voltage gated current e.g. HEK293 cells
-take whole cell recordings

Question 30

What does the resting membrane potential result from?

Answer 30

a balance in the permeability of Na+ (low) and K+ (high)

Question 31

How is the resting membrane potential maintained?

Answer 31

The action of the Na+/K+-ATPase

Question 32

Through what mechanism does the action potential arise?

Answer 32

from the membranes selective permeability to Na+ and K+ ions via the gating of voltage dependent ion channels

Question 33

What is the voltage-independent gate in sodium channels necessary for?

Answer 33

Fast inactivation linked to conformational stability

Question 34

How did Hodgkin increase knowledge of the NS?

Answer 34

-used the squid giant axon to make early electrophysiological recordings of the action potential and showed how it was composed of Na+ and K+ conductances

Question 35

What does Neher and Sakmanns patch clamp technique allow?

Answer 35

the study of the properties of individual ion channels

Question 36

How can the properties of channels be more easily studied?

Answer 36

Through expressing channels and their mutants in heterologous cell