Voltage gated ion channels

Outline resting state (from Y1)

• At resting membrane potential ~70 mV

• Inside of cell negatively charged relative to outside

• K+ wants out but can’t leave due to electrostatic pressure

• Na+ wants in and can go in due to electrostatic pressure

Outline depolarisation (revision from Y1)

• Sodium channels open at ~55 mV (threshold of excitation)

  • sodium floods into neuron (Na+ attracted to -charge inside cell, also going with concentration gradient)

• membrane potential grows increasingly positive

• potassium channels are open

• inside of cell reaches 0, then turns positive

• potassium cations leave cell 

• sodium channels close at +40 mV

• potassium channels remain open 

Outline membrane repolarisation + hyperpolarisation (revision from Y1)

• potassium channels remain open

• potassium cations leave cell due to electrostatic pressure + concentration gradient

• even after membrane repolarised, potassium wants to leave, membrane hyperpolarises to lower than -70 mV

• potassium channels close after elapsed period, return to RMP

give an overview of voltage gated ion channels

• open in response to change in plasma membrane voltage

• Activation is voltage and time dependent

  • some channels close whilst voltage still present, some stay open

  • transient vs persistent (persistent = staying open while voltage persists, transient = closing when voltage still present)

• different channels selective to different ions (cations vs anions)

What is kinetics in the context of voltage gated ion channels?

• how fast to channels open when they get a voltage signa?

• how fast do channels close, if at all?

outline the structure of voltage gated sodium and calcium channels

• channel forms single polypeptide- alpha subunit

• 4 domains I-IV

• domains arranging to form pore

• domains contain 6 alpha helices

• regulatory beta subunits affect rate of act/inactivation

• similar across molecule type

• 4th alpha helices = voltage sensor (S4 segment)

outline the activation of Na channels 

• S4 segment contains +ve charged AA residues, positive charge caused by depolarisation interacts w/S4 segment + like repels like 

• depolarisation causes S4 to move outward 

• conformational change opens channel pore 

• channel can then conduct + move ions it’s selective for 

• rapid activation

outline the inactivation of Na channels

• Inactivation sequence: ball hanging off end of terminus

• inactivation sequence swings into pore + blocks channel

• even with voltage signal (during depolarisation), current not passing through channel due to this blockage

• allows for Na channels to inactivate very quickly

outline unidirectionality

• AP travels in one direction, left —> right from the site of initiation (close to the cell body)

• presynaptic cells activate postsynaptic cells- no back propogation causing signals to go back towards where they emanated from

how does the inactivation of sodium channels govern unidirectional conduction?

• for a period of time after channels activated, sodium channels remain inactive due to channel block

• during this period, channels are refractory and can no longer depolarise

• this refractory period means the AP cannot back propagate and ensures unidirectionality

outline the structure of potassium channels

• similar to Na channels EXCEPT: not a single polypeptide. Instead, 4 separate subunits forming a tetramer

• each subunit has 6 alpha helices 

• Some K+ channels don’t inactivate- unlike sodium channels

• For K+ channels that do activate, 4 N terminal inactivation segments, 1 for each subunit

• 1 inactivation segment can inactivate the channel 

How to voltage gated conductances drive the action potential?

• High densities of Na+ channels at the axon initial segment + nodes of Ranvier

• Voltage dependent activation ~ -55mV

• Na+ current flows into cell generating upstroke of AP

• Potassium lags behind sodium, channels open slower, slower kinetics

Outline how the delayed K+ current repolarises membrane potential after the AP

• K+ channels clustered next to nodes (juxtaparanodes)

• Voltage gated K+ channels open around 1ms after Na+ channels

• membrane permeability to K+ increases

• K+ ions move out across the membrane 

Label and talk through this diagram

This is a voltage clamp used to analyse ion channel function

The whole thing is a feedback circuit

We are recording voltage based on the reference electrode in the extracellular fluid and the recording electrode in the axon, as voltage is a potential difference

The dish has salt solutions replicating the ionic solution in animals’ nervous systems

We set the desired voltage at any voltage we like, then record the voltage to see which current we need to clamp our membrane at that voltage

Give a step-by-step run through of how voltage clamps work

1. set voltage into feedback amplifier

2. recording voltage of axon with membrane potential amplifier

3. this voltage is sent to feedback amplifier

4. feedback amplifier compares recorded voltage to desired voltage

5. if there’s a difference, feedback amplifier sends signal to send positive current into axon to try and depolarise it (compensatory current)

6. change in voltage read by first amplifier, feeds back to feedback amplifier

7. repeat until both amplifiers read same voltage

8. once amplifiers read same voltage- equilibrium, get a steady state current that is noted down 

9. measuring current required to reach one voltage is not the end of a voltage clamp experiment 

why do we do voltage clamps?

• voltage clamp activates VG ion channels

• Current flow through the channels can be detected

• we can explore what happens when we change voltage

Walk through this whole cell recording

• At the top- Vm = voltage clamp, clamped at 60 then membrane potential (depolarisation) to 0

• Im = membrane current, in this case compensatory current

• the waveform is in response of voltage gated channels

• yellow = potassium channel: going in positive deflection (upward, positive current) = potassium efflux- notice the lag

• blue = sodium channels: going in negative deflection (downwards, negative current) = sodium influx

• voltage clamp recordings are mirror opposite of what the cell is experiencing, as we are looking at the current compensating for what is occurring in channels

why are sodium currents always a downward waveform and potassium currents always an upward waveform?

Na: voltage clamp compensating to + charge caused by influx of sodium ions by injecting - charge

K: voltage clamp compensating to - charge caused by efflux of K ions by injecting + charge

how can we isolate channel currents through pharmacology?

• Tetrotodoxin (TTX) = sodium channel blocker, allows for isolation of potassium current

• Tetraethylammonium (TEA) = potassium channel blocker, allows for isolation of sodium current

• allow us to see current kinetics w/out contamination from other channel functions

what do we see when we isolate K+ and Na+ currents through blockers?

K+ channels = slower opening, do not inactivate

Na+ channels = fast opening, inactivate

What does I stand for? 

current: Im = membrane current, INa = sodium current, etc

what does V stand for?

Voltage- Vm = membrane voltage, VNa = sodium voltage, etc

What are families of voltage-clamp steps?

• When we change the voltage up and down in steps, progressively increasing the depolarisation of the voltage clamp

• Allows us to activate ion channels and see how much current flows through ion channels at different voltages

• persistent currents = currents that remain until voltage turned off

Walk through this K+ current family  

• Linear response- the more depolarised we get, the greater the current becomes

• Voltage plot: current in response to voltage, measure peak current at each voltage step and plot it 

• Ek at -80: equilibrium potential for K+: depolarisation and driving force directly balanced, zero current flow 

• Driving force increases as we depolarise, larger membrane current

outline how driving force relates to equilibrium potential

Driving force = Vm - Ek

Driving force = membrane potential - equilibrium potential

driving force determines membrane current, regulates how much current we can record

walk through this Na+ current family

• Immediately see that it’s more complex than K+ channel familiies

• -60 = no current

• Downward current at -30

• peak at 0

• reduction at +3-

• reversal to upward deflection at +60: current leaving cell

  • this is ENa - opposite end of spectrum to potassium

• biphasic response: current increases as we depolarise to a plateau, current then reduces to a point, then reverses over that

• driving force diminishes as we get closer to the equilibrium potential

• reduction in membrane current = due to decrease in driving force, not channels closing

• Ina reduces as driving force decreases

How do we measure channel conductance?

AKA ‘g’

Indirectly through membrane current- we can’t measure it experimentally 

Conductance = membrane current/driving force 

Example with potassium conductance: 

gK = Ik / (Vm - EK) 

potassium conductance = membrane current / (membrane potential - equilibrium potential)

How can we rearrange hodgkin and huxley’s equation for calculating current?

for potassium:

IK = gK(Vm-EK) (membrane current = conductance x driving force)

Ik should be zero at EK

As driving force decreases, no matter how big gK is, if you keep reducing driving force, membrane currents get smaller even if channels open

outline the difference between current and conductance

Linked in that one depends on the other: gate + passenger analogy 

Conductance is gates, current is passengers through gates

Conductance = gates open, passengers can get through (ions)

Gates closed = no conductance, no passengers getting through (resting membrane) 

Conductance can never be less than 0: all gates closed, cannot have less than 0 gates closed 

Membrane current can occupy + or - values, conductance can only be positive 

No current without conductance, but yes conductance without current- cannot have passengers passing through gates without gates being open, but gates =/= open in response to passengers waiting 

Outline A type potassium channels

• Regulate neuronal activity beyond repolarisation

• Control onset (how soon after) and frequency of firing of APs

• conduct K+ (efflux)- reduce excitability of neurons

• Voltage-gated

• Activate quickly unlike delayed rectifier

• Inactivate (like Ina, unlike delayed rectifier)

outline the in/activation of A-type K+ channels

AKA IA

• at RMP = mostly inactivated

• Inactivation removed by hyperpolarisation

• react instantaneously

• activate then deactivate quickly

Outline how A type potassium channels delay the initiation of AP firing 

neuron is sat at whatever MP it wants, current passed to depolarise it, triggers train of AP fires at such a high frequency that at first you can’t distinguish APs

• briefly hyperpolarising the same neuron before depolarising it = lag before it fires

• delay looks small but matters a lot at level of circuit function

• delaying firing = functional consequences across the circuit

• delay caused by the fact that we have IA in cell

• neuron depolarises, IAs open, rapid efflux of K+ causes slight negative balance against depolarisation, for a while depolarisation is happening while there is an outflow of K+

• IA channels help delay firing in the neuron